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Abstract

Introduction

The spore-forming bacterial species Bacillus velezensis is commonly utilized in feed for livestock and aquaculture. In recent years, there has been increased interest in introducing B. velezensis into human supplements and food. Before it can be safely administered in humans, the safety of each B. velezensis strain needs to be established. The objective of this study was to evaluate the in vivo safety of Bacillus velezensis strain BV379 by high-dose oral administration to rats in a 28-day subchronic toxicity study.

Methods

In this study, 80 animals were assigned to four groups: vehicle control, 1 × 10¹⁰, 4 × 10¹⁰, or 10 × 10¹⁰ CFU/kg bw/day by gavage. The following toxicological assessments were performed: ophthalmological examinations; observations for viability, signs of gross toxicity, and behavioral changes; in-life parameters, including body weight and food consumption; urinalysis, hematology, clinical chemistry, and coagulation assessments; macroscopic and microscopic tissue assessments; and bacterial enumeration in selected tissues.

Results

Under the conditions of this study, no adverse clinical endpoints were attributed to the administration of Bacillus velezensis strain BV379, which was well-tolerated up to the highest dose of 10 × 10¹⁰ CFU/kg bw/day.

Conclusion

These results support the in vivo pre-clinical safety of Bacillus velezensis strain BV379 for use in food and supplements.

Keywords

BV379 safety toxicity Bacillus velezensis

Introduction

Strains of the spore-forming bacterial species B. velezensis have become a popular additive in the aquaculture and animal feed industries due to their production of potentially beneficial metabolites.¹ Since 2017, B. velezensis has had Qualified Presumption of Safety (QPS) status by the European Food Safety Authority (EFSA) with two qualifications: (1) lack of any acquired antimicrobial resistance genes to clinically relevant antimicrobials; and (2) absence of toxigenic activity.^2,3 Accordingly, several commercial B. velezensis probiotic strains have been incorporated into feed for poultry (e.g., chickens and turkeys), swine, ornamental fish, and dogs.^4–13

In the past few years, there has been increased interest in the implementation of B. velezensis strains into human dietary supplements and food, but few preclinical and clinical studies have been initiated to demonstrate B. velezensis safety. Two B. velezensis strains that were classified as different Bacillus species at the initiation of the clinical trials,^14,15 B. velezensis SCD (Bispan) and B. velezensis C-3102 (also known as DSM 15544 or Calsporin®), were shown to be safe for oral consumption of up to 4.8 × 10¹⁰ CFU/day throughout four human clinical trials.^16–19 Further, four B. velezensis strains have been demonstrated to be safe in healthy mice administered oral doses ranging from 3.3 × 10⁹ – 60 × 10⁹ CFU/kg initial body weight/day for a duration of 6 days – 8 weeks.^20–24 To date, no studies on the oral toxicity and safety of B. velezensis in rat models have been published.

The aim of the current study was to evaluate the oral safety of the recently discovered soil isolate B. velezensis strain BV379 (BV379) in Sprague-Dawley rats in order to determine its suitability as a human oral supplement and for food applications. Using a series of in silico and in vitro assays, BV379 was previously shown to tolerate conditions similar to the gastrointestinal tract (low pH conditions and bile acid exposure) and high-heat conditions that may be used in food manufacturing processes.²⁵ More importantly, BV379 lysates do not adversely impact human intestinal epithelial cell viability and monolayer permeability.²⁵ However, BV379 has not been tested for safety in a mammalian species. In order to assess the no-observed-adverse-effect-level (NOAEL) for the oral administration of Bacillus velezensis BV379, a 28-day subchronic oral toxicity study in rats was performed at three oral doses of BV379 (≤10 × 10¹⁰ CFU/kg bw/day). Based on the results of the previous in silico and in vitro safety studies, we hypothesized that BV379 would be well-tolerated without treatment-related toxicity or pathology at the doses administered.

Methods

Study design

The study was performed at Product Safety Labs (PSL; Dayton, New Jersey, USA). PSL is AAALAC (Association for Assessment and Accreditation of Laboratory Animal Care) accredited and certified. The study based on Organization for Economic Co-operation and Development (OECD) and U.S. Food and Drug Administration (FDA) Guidelines.^26,27 The study also met the requirements of U.S. FDA GLP: 21 CFR Part 58, 1978, and all subsequent updated versions.

BV379 neat test substance and dose preparation

Bacillus velezensis strain BV379 spore powder was prepared by culturing BV379 in a yeast-based medium for 42-46 h at 37°C. Per proprietary food grade manufacturing protocols, the culture was then adjusted to a pH < 5, pelleted, spray-dried, and stored under ambient storage conditions until use. The BV379 spore powder was suspended in distilled water to make three different dose concentrations: 2 × 10⁹, 8 ×10⁹, and 2 × 10¹⁰ CFU/mL (10, 40, and 100 mg/mL; w/v). A vehicle control (distilled water, 0 CFU/mL) was used for control groups.

Animals

All animal care and experimental protocols followed the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals and were fully approved by the PSL Institutional Animal Care and Use Committee (National Research Council of the National Academies).²⁸ Male and female Sprague-Dawley rats (Charles River Laboratories, Raleigh, NC) were used. Prior to the initiation of testing, the rats were acclimated to laboratory conditions for eight days. Following acclimation, adult male and female animals (n = 80) were selected for the study based on adequate body weight gain, freedom from clinical signs of disease or injury, and body weight within ±20% of the mean. Selected rats were randomly distributed by body weight using a stratified randomization procedure. The animals weighed 238-278 g (males) and 177-213 g (females) and were approximately seven to eight weeks of age at initiation of dosing.

The animals were group-housed with two animals of the same sex per cage under standard laboratory conditions (i.e., adequate fresh air supply, room temperature (19-22°C), relative humidity of 46%–78%, and 12 h light and 12 h dark cycle), and the room was kept clean and vermin free. The animals were fed (ad libitum) with 2016 Certified Teklad Global Rodent Diet^® (Inotiv, West Lafayette, IN, USA) throughout the acclimation and experimental periods.

Experiment

Eighty (80) adult rats (40 males and 40 females) were randomly distributed into four test groups (10/sex/group). Through preliminary methodology work with the neat test material, it was determined that concentrations of 10, 40, and 100 mg/mL, when prepared daily with distilled water as a vehicle, would provide CFU concentrations of 2 × 10⁹, 8 × 10⁹, and 2 × 10¹⁰ CFU/mL. When these concentrations are administered at a dose volume of 5 mL/kg of bodyweight, the dose levels are calculated to be 1 × 10¹⁰, 4 × 10¹⁰, and 10 × 10¹⁰ CFU/kg bw/day. These dose levels are equivalent to 50, 200, and 500 mg/kg bw/day. Dose levels of 0 (vehicle control), 1 × 10¹⁰ (low dose), 4 × 10¹⁰ (medium dose), and 10 × 10¹⁰ (high dose) CFU/kg bw/day of BV379 were administered to all animals within their respective treatment group. These particular dose ranges have been previously tested with other Bacillus strains in rats.^29,30

Each animal was dosed by oral intubation using a stainless-steel ball-tipped gavage needle attached to an appropriate syringe. Dose administration occurred once daily (7 days/week) for a period of at least 28 days (31 days for males and 32 days for females). The first day of administration was considered Day 1 of the study. Dosing was at approximately the same time each day (±2 h).

BV379 neat test substance and dose stability and homogeneity

Samples of the neat BV379 spore powder were collected at the beginning (Day 1) and at the end of the dosing phase of the study (Day 29) and were analyzed to evaluate stability of the neat material under storage conditions at the testing facility. Furthermore, prior to initial dosing on Day 1, samples from the BV379 dose preparations were collected by using a pipette to aspirate liquid from the top, middle, and bottom of the mixing container for each concentration. The vehicle control was sampled from the middle only. Dose preparations were also sampled at the end of the study for verification of dose concentration, also from the middle only. The dosing preparations sampled for homogeneity and concentration verification were analyzed for viable CFU content. Briefly, samples were diluted, plated onto trypticase soy agar (TSA) plates, and incubated overnight at 37°C. An automated plate counter was utilized to quantify colonies.

Clinical and ophthalmological examination, body weight, and feed consumption

During the acclimation period, both eyes of each animal were examined by focal illumination, indirect ophthalmoscopy, and slit-lamp microscopy. Mydriatic eye drops were administered before ophthalmoscopy, and the eyes were examined in subdued light. The ophthalmoscopy examination was repeated on all animals at Day 29.

All animals were observed at least twice daily for viability and overt clinical signs. In addition, detailed cage-side observations were performed daily. Abnormal findings were recorded. Before the first treatment on Day 1 and approximately weekly thereafter, detailed observations were conducted while handling the animals. These observations were also generally performed on the same days the animals were weighed and feed consumption measurements were taken. Physical assessments included but were not limited to changes in skin, fur, eyes, and mucous membranes, the occurrence of secretions and excretions, and autonomic activity (e.g., lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, and response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backward) were recorded. The date and time of all observations and/or mortality checks were recorded.

Individual body weights were recorded at least two times during acclimation. All animals were weighed on Day 1 and approximately weekly after that (intervals of seven days ±1). Body weight gain was calculated for each interval. Feed consumption was measured and recorded to coincide with body weight measurements.

Clinical pathology

At their respective points of termination, all surviving animals were subjected to clinical pathology tests, including hematology, clinical (serum) chemistry, coagulation, and urinalysis. Animals were fasted overnight (at least 15 h) prior to the initiation of blood collection and necropsy. Blood samples were collected from the sublingual vein or vena cava/abdominal aorta (coagulation) under isoflurane anesthesia. Any clinical parameter that was significantly different in BV379-treated groups as compared to controls was compared against the testing facility’s historical controls (animals at 11-22 weeks of age; September 2018 – June 2019; Tables S1-S3).

Hematology

Approximately 500 µL of blood was collected in a pre-calibrated tube containing K₂EDTA for hematological assessments. The hematological parameters included erythrocyte count (RBC), hemoglobin concentration (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), red cell distribution width (RDW), absolute reticulocyte count (ARET), platelet count (PLT), total white blood cell (WBC), absolute neutrophil (ANEU), absolute lymphocyte (ALYM), absolute monocyte (AMON), absolute eosinophil (AEOS), absolute basophil (ABAS), and absolute large unstained cell (ALUC). Mean corpuscular hemoglobin concentration (MCHC) was calculated. In case they were needed to substantiate or clarify any hematology findings, separate blood smears were prepared from each animal undergoing hematological evaluation and stained with New Methylene Blue or Wright-Giemsa stain.

Coagulation assessment

Approximately 1.8 mL of blood was collected in pre-calibrated tubes containing 3.2% sodium citrate. These samples were centrifuged in a refrigerated centrifuge, and the plasma was transferred to labeled tubes. Plasma samples were stored at −80°C until analysis. Prothrombin time (PT) and activated partial thromboplastin time (APTT) were evaluated.

Clinical chemistry

Approximately 1000 µL of blood was collected into a tube containing no preservatives for clinical chemistry assessments. The measured serum chemistry parameters included aspartate aminotransferase (AST), alanine aminotransferase (ALT), sorbitol dehydrogenase (SDH), alkaline phosphatase (ALKP), total bilirubin (BILI), urea nitrogen (BUN), blood creatinine (CREA), total cholesterol (CHOL), triglycerides (TG), fasting glucose (GLUC), total serum protein (TP), albumin (ALB), globulin (GLOB), calcium (Ca), inorganic phosphorus (IPHS), sodium (Na), potassium (K), and chloride (Cl).

Urinalysis

Animals were placed in metabolism cages the day before termination to collect urine samples. Urine samples were refrigerated until analysis. The urinalysis parameters included volume, pH, glucose concentration, ketone concentration, protein concentration, urobilinogen concentration, specific gravity, volume, and microscopic urine sediment examination.

Terminal necropsy and tissue collection

At the conclusion of the experimental phase of the study, all surviving animals were euthanized by exsanguination from the abdominal aorta under isoflurane anesthesia. All animals were subjected to a full necropsy, which included examination of the external surface of the body, all orifices, and the thoracic, abdominal, and cranial cavities and their contents. The tissues were weighed wet as soon as possible after dissection to avoid drying. The tissues included adrenals, kidneys (combined), spleen, brain, liver, thymus, epididymides (combined), testes (combined), uterus, heart, and ovaries with oviducts (combined).

The following tissues were weighed at least 24 h after preservation in 10% neutral buffered formalin: prostate, seminal vesicles with coagulating gland (combined), thyroid/parathyroid, and pituitary. The following organs/tissues were preserved in 10% neutral buffered formalin: accessory genital organs (prostate and seminal vesicles), ileum with Peyer’s patches, rectum, salivary glands (sublingual adrenals, submandibular and parotid glands), larynx, aorta, kidneys, liver, skeletal muscle, femur, lungs, skin, bone marrow from femur and sternum, mesenteric and mandibular lymph node, spinal cord (cervical, midthoracic, and lumbar), brain (sections to include medulla/pons/cerebellar/cerebral cortex), mammary gland, cecum, nose, sternum, ovaries, cervix, oviducts, stomach, colon, pancreas, thyroid, parathyroid, duodenum, thymus, duodenum, trachea, esophagus, peripheral nerve (sciatic), urinary bladder, Harderian gland, pharynx, uterus, heart, pituitary gland, and vagina.

The following organs/tissues were preserved in a modified Davidson’s fixative and stored in ethanol: eyes, optic nerves, epididymides, and testes.

Histopathology

Histopathological examinations were performed on preserved organs and tissues of all animals from both the control and high-dose groups. All tissues were processed, trimmed, embedded in paraffin, sectioned at 5 microns, and stained with hematoxylin and eosin (H&E). Microscopic evaluation of the slides for each tissue was performed by a board-certified veterinary pathologist. Microscopic findings were qualitatively scored as indicated below³¹ and the incidence of any microscopic findings were identified with gross correlations and comparisons to corresponding clinical pathology, in-life, or other collected necropsy endpoints, including but not limited to absolute and relative organ weights:

1 = Minimal (subtle to trivial change; affecting up to 5% of the tissue presented)

2 = Mild (an easily identifiable change affecting 6–25% of the tissue presented)

3 = Moderate (an obvious change affecting 26–50% of the tissue presented)

4 = Marked (an extensive change affecting 51%–75% of the tissue presented)

5 = Severe (a maximal change affecting greater than 75% of the tissue)

Tissue bacterial isolate enumeration and taxonomic analysis

In order to determine if BV379 was capable of translocation through the gut barrier and into the bloodstream and external tissues, previously published methods were followed.³² Briefly, three fresh (unfixed) tissue samples were assessed from a subset of four randomly selected rats from the vehicle control group and from subsets of five rats from each treatment group (1 × 10¹⁰, 4 × 10¹⁰, and 1 × 10¹⁰ BV379 CFU/kg bw/day) from each sex (male and female) for a total of 38 sets of animal tissues. Samples of whole blood were collected, and sections of liver and mesenteric lymph nodes were excised and maintained on ice. Samples of liver were taken after the liver was weighed. Approximate sample sizes were 0.5 mL of blood and 0.2-0.5 grams of tissue, without compromising any other possible study endpoints. Aseptic and other appropriate methods were utilized in an effort to avoid contamination of samples (e.g., the use of alcohol to clean instruments and surfaces). Each tissue sample was homogenized and plated onto three TSA plates for a total of nine TSA plates per animal. Blood was plated directly on plates with 0.1 mL per plate. Liver and mesentery tissues were diluted 1:10 (weight to volume) in sterile buffer, homogenized, and 0.1 mL of the homogenate was added to each plate. Plates were incubated at approximately 38°C for 72 h or until colony growth was adequate for counting. Bacterial counts were tallied across the three plates that were incubated per tissue. The mean CFU/gram of tissue was calculated based on the amount of sample collected and dilution factors for each specific sample (0.3 mL of plated blood and 0.03 g of liver and mesentery).

All plates were visually inspected for colony growth after incubation and individual colonies were counted. An automated plate counter was utilized to assist in enumerating highly populated plates. The mean CFU/gram of tissue or mL of blood was calculated based on the amount of respective sample evaluated, plate colony count, and dilution factor.

The sensitivity of the method was determined for each sample type using the average amount of tissue in each preparation, assuming a minimum detection limit of 1 CFU/plate, and factoring for sample dilution. Dilutions of the sample preparations either prior to inoculation or prior to plating were accounted for in the calculations by additional multiplication of the dilution factor (DF) by the appropriate factor.

Sensitivity (CFU / g or mL) = \frac{Minimum Detectable CFU / plate (1 CFU)}{Average amount of sample (g or mL)} \times DF

For enumeration and translocation, colonies were counted and morphology was evaluated based on seven characteristics (i.e., size, form/shape, elevation, margin, surface, opacity, and color). For each distinct colony morphology identified, five representative colonies were picked, re-streaked onto TSA plates, and incubated for 20 h at 38⁰C. These identified colonies were used for DNA extraction and 16S rRNA variable region 4 (V4) PCR amplification.³³ 16S amplicon libraries were then sequenced on an Illumina MiSeq. The consensus sequence generated for each isolate was produced and a taxonomic designation was assigned by comparison to the Genbank database.^34,35 Assignments based on the V4 region of the 16S rRNA gene are reliable at the genus level, but may not differentiate among species of the same genus or strains of the same species.³⁶

In order to further distinguish BV379 from other Bacillus species, random amplified polymorphic DNA (RAPD) analysis was performed using the S30 primer.^37,38 The RAPD profile of B. velezensis BV379 was distinguished from other Bacillus species using this method by analyzing two preparations of BV379, Bacillus licheniformis, Bacillus thuringiensis, Bacillus cereus, and Bacillus amyloliquefaciens (Figure S1). This RAPD protocol does not, however, distinguish BV379 from other B. velezensis strains (Figure S2). It was presumed that any B. velezensis isolate identified in the tissue samples in this study likely originated from BV379.

Individual clinical assessments of study animals with high tissue bacterial concentrations

Results of the translocation study showed that B. velezensis was found in tissues of some animals, including controls. To determine whether animals with high tissue concentrations of bacteria (arbitrarily chosen as ≥ 50 colonies per tissue sample analyzed, which is equivalent to ≥167 CFU/mL blood or ≥1,667 CFU/g liver or mesentery tissue) had any evidence of toxicity, the organ weights, hematology, clinical chemistry, urinalysis, and coagulation data of animals which met this criterion (n = 7) were compared to historical controls at the PSL facility (Sprague-Dawley rats at 11-22 weeks of age from September 2018-June 2019; Tables S1-S3). Further, individual histopathological assessments of the control animal and three high-dose animals with ≥50 colonies per tissue sample were performed. These analyses would help determine any irregularities about the animals that could lead to increased risk of harboring bacterial isolates in extraintestinal tissues.

Statistical analyses

Mean and standard deviations were calculated for all quantitative data. Male and female rats were evaluated separately. Statistical analyses were conducted using Provantis^TM (version 10), Tables and Statistics, Instem LSS, Staffordshire, UK.

For in-life endpoints identified as multiple measurements of continuous data over time (e.g., body weight, feed consumption), treatment and control groups were compared using one-way repeated measures analysis of variance (RMANOVA), testing the effect of treatment over discrete time intervals. Significant changes observed for treatment were further analyzed by a post hoc, multiple comparisons test of the individual treated groups to the control group (e.g., Dunnett’s test).

All endpoints with single measurements of continuous data within groups (e.g., organ weight, relative organ weight, translocation data, etc.) were evaluated for homogeneity of variance (Bartlett’s test) and normality (Shapiro-Wilk test). Where homogeneous variances and normal distribution were observed, treated and control groups were compared using a one-way ANOVA. When one-way ANOVA was significant, a comparison of treated groups to control was performed with a multiple comparisons test (e.g., Dunnett’s test).

Where variance was considered significantly different, groups were compared using a non-parametric method (Kruskal-Wallis). When non-parametric ANOVA was significant, a comparison of treated groups to control was performed (Dunn’s test). Clinical pathology data was initially evaluated by Levene’s test for homogeneity and the Shapiro-Wilk test for normality. When the preliminary test was not significant, one-way ANOVA followed by Dunnett’s test was used, while in the case of significance, one-way ANOVA followed by Dunnett’s test. Transformations of the data were used to achieve normality and variance homogeneity. The order of transforms attempted was log, square-root, and rank-order. If the log and square-root transformations failed, the rank order was used.

Data collected during in-life as well as organ weight data and clinical pathology results were judged for statistical difference from controls at a probability value of p < .05.

Results

BV379 neat test substance and dose stability and homogeneity

The CFU viability and concentrations of the B. velezensis strain BV379 neat test substance showed that the strain was stable under the ambient storage conditions and over the course of the study. The concentrations of neat BV379 test substance were 2.98 × 10¹¹ CFU/mL on Day 1 (initial) and 2.84 × 10¹¹ CFU/mL on Day 29 (final). The difference over the course of the study was −4.7%, and the overall stability was determined to be 95.3%.

The BV379 dose preparations for Day 1 were also found to be homogeneously distributed, stable, and met or exceeded target dosing concentrations (i.e., 2 × 10⁹, 8 × 10⁹, and 2 × 10¹⁰ CFU/mL). The relative standard deviation (RSD) values of the CFU concentrations throughout the sampling locations in the low-, medium-, and high-dose preparations were 1.18%, 1.11%, and 1.42%, respectively (Table S4).

The concentrations of the BV379 dose preparations for Day 29 of the trial were also confirmed to have met or exceeded the target BV379 CFU concentrations (Table S5).

Survival, clinical observations, ophthalmological examination

There were no BV379-related mortalities or clinical observations over the course of this study. One medium-dose (4 × 10¹⁰ CFU/kg bw/day) male was found dead on Day 26. Gross observations of this animal were consistent with expected findings in a deceased animal (such as hypostatic congestion), not indicative of a pathologic process. Additional observations included marked acute hemorrhage with moderate inflammation surrounding the pharynx, with an apparent perforation, indicative of accidental trauma, associated with the gavage procedure. Furthermore, this unscheduled-death incident was unrelated to BV379 exposure. There were no adverse clinical signs noted prior to death. All clinical observations were considered incidental, and of no toxicological significance.

For male rats, incidental in-life clinical signs included slight alopecia on the left/right forelimb or head in two high-dose (10 × 10¹⁰ CFU/kg bw/day) animals, and superficial to deep eschar and a deep wound on the head of a single medium-dose (4 × 10¹⁰ CFU/kg bw/day) animal. For the female rats, incidental in-life clinical signs included slight alopecia on the neck or head of a single control group animal, and one medium-dose (4 × 10¹⁰ CFU/kg bw/day) animal. In addition, one medium-dose female was observed to exhibit low activity/flaccid behavior.

All animals included in the study were normal upon ophthalmic exam, with the exception of one male receiving 1 × 10¹⁰ CFU/kg bw/day, that had a chorioretinal scar in the right eye on Day 29. The finding was not attributed to BV379 administration, therefore, the BV379 was not considered to be an ocular toxicant.

Body weights, feed, and water consumption

There were no changes in body weight, body weight gain, or feed consumption in male and female rats attributable to the administration of B. velezensis strain BV379 (Figure 1, Tables S6–S9). Mean weekly body weights, daily body weight gain, and feed consumption for male and female rats for all BV379-treated groups were comparable to the vehicle control.

Figure 1.

Weekly body weights of (a) male and (b) female rats across oral administration of BV379 for 31 and 32 days, respectively. Between-group differences were tested via one-way repeated measured ANOVA and Dunnett test (no statistically significant differences were observed).

Hematology

There were no statistically significant BV379-related changes in hematology parameters at all dose levels tested (Table 1).

Table 1.

Hematology and Coagulation parameters in male and female rats after oral administration of BV379 for 31 and 32 days, respectively^a.

BV379 dose (CFU/kg bw/d)	Males				Females
BV379 dose (CFU/kg bw/d)	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰
	n = 10	n = 10	n = 9	n = 9	n = 9	n = 10	n = 10	n = 10
ABAS (x 10³/uL)^b	0.05 ± 0.02	0.05 ± 0.03	0.05 ± 0.02	0.05 ± 0.03	0.05 ± 0.02	0.03 ± 0.02	0.05 ± 0.03	0.06 ± 0.04
AEOS (x 10³/uL)^c,d	0.09 ± 0.03	0.11 ± 0.05	0.11 ± 0.04	0.10 ± 0.40	0.08 ± 0.03	0.08 ± 0.02	0.08 ± 0.03	0.12 ± 0.10
ALUC (x 10³/uL)^c	0.09 ± 0.04	0.12 ± 0.06	0.13 ± 0.05	0.11 ± 0.04	0.09 ± 0.04	0.08 ± 0.03	0.10 ± 0.04	0.09 ± 0.04
ALYM (x 10³/uL)^c,b	8.02 ± 1.57	8.96 ± 2.49	8.65 ± 1.94	7.77 ± 2.21	6.98 ± 2.18	4.95 ± 1.30	7.76 ± 1.94	7.3 ± 3.00
AMON (x 10³/uL)^b	0.40 ± 0.15	0.46 ± 0.22	0.47 ± 0.10	0.38 ± 0.10	0.28 ± 0.10	0.27 ± 0.12	0.27 ± 0.14	0.30 ± 0.12
ANEU (x 10³/uL)^b,c	1.74 ± 0.41	1.80 ± 0.72	2.42 ± 1.20	1.44 ± 0.63	0.87 ± 0.26	0.74 ± 0.20	0.93 ± 0.42	0.95 ± 0.38
ARET (x 10³/uL)^c,b	222.71 ± 26.73	255.79 ± 46.38	233.49 ± 37.44	236.31 ± 37.62	210.96 ± 41.57	197.73 ± 31.08	193.30 ± 37.83	181.18 ± 41.98
HCT (%)^c	50.71 ± 2.69	48.88 ± 2.86	50.10 ± 2.87	52.38 ± 3.32	49.34 ± 2.11	49.20 ± 2.09	50.05 ± 1.94	50.79 ± 2.13
HGB (g/dL)^c	16.53 ± 0.76	15.96 ± 0.74	16.29 ± 0.87	17.04 ± 0.97	16.13 ± 0.65	15.83 ± 0.46	16.25 ± 0.51	16.47 ± 0.68
MCV (fL)^c	59.84 ± 2.23	59.99 ± 1.16	59.52 ± 1.13	59.38 ± 1.76	58.70 ± 1.51	59.78 ± 1.69	59.76 ± 2.03	59.63 ± 1.81
MCH (pg)^c	19.51 ± 0.84	19.61 ± 0.58	19.34 ± 0.44	19.32 ± 0.46	19.17 ± 0.59	19.23 ± 0.50	19.41 ± 0.56	19.33 ± 0.65
MCHC (g/dL)^c	32.58 ± 0.44	32.67 ± 0.62	32.50 ± 0.50	32.54 ± 0.32	32.68 ± 0.38	32.17 ± 0.57	32.47 ± 0.46	32.43 ± 0.28
PLT (x 10³/uL)^d	1194.60 ± 85.96	1176.60 ± 110.63	1083.56 ± 181.79	1137.33 ± 98.04	1130.33 ± 134.92	1039.90 ± 138.38	993.00 ± 371.08	1070.20 ± 228.09
RBC ( ×10⁶/uL)^c	8.48 ± 0.45	8.16 ± 0.53	8.42 ± 0.43	8.82 ± 0.49	8.42 ± 0.50	8.23 ± 0.34	8.38 ± 0.33	8.52 ± 0.33
RDW (%)^c	12.32 ± 0.30	12.66 ± 0.70	12.36 ± 0.49	12.46 ± 0.56	11.94 ± 0.63	11.92 ± 0.36	11.70 ± 0.36	11.87 ± 0.38
WBC ( ×10³/uL)^c	10.39 ± 2.00	11.51 ± 3.36	11.66 ± 1.72	9.85 ± 2.87	8.34 ± 2.40	6.15 ± 1.45	9.22 ± 1.99	8.80 ± 3.40
APTT (seconds)^d	15.59 ± 1.18	15.09 ± 1.58	16.49 ± 2.55	15.34 ± 0.75	13.46 ± 1.50	14.25 ± 1.28	15.77 ± 1.61**	16.57 ± 1.43***
PT (seconds)^c	10.01 ± 0.36	9.76 ± 0.39	9.95 ± 0.36	9.87 ± 0.25	9.10 ± 0.32	9.06 ± 0.20	9.32 ± 0.24	9.27 ± 0.19

^aData are means ± standard deviation; where two subscripts are listed, two different statistical analyses were performed for the male and female animal groups;^*p < .05;^**p < .01;^***p < .001. BW, body weight; ABAS, absolute basophils; AEOS, absolute eosinophils; APTT, activated partial thromboplastin clotting time; ALUC, absolute large unstained cells; ALYM, absolute lymphocytes; AMON, absolute monocytes; ANEU, absolute neutrophils; ARET, absolute reticulocytes; HCT, hematocrit; HGB, hemoglobin; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count; PT, prothrombin time test; RBC, red blood cell; RDW, red cell distribution width; WBC, white blood cell count.

^bBetween-group differences for each sex were tested via ANOVA and Dunnett tests with log-transformed data.

^cBetween-group differences for each sex were tested via ANOVA and Dunnett tests.

^dBetween-group differences for each sex were tested via Kruskal-Wallis one-way ANOVA and Dunn’s tests.

Coagulation assessment

Statistically significant increases were noted for activated partial thromboplastin clotting time (APTT) in medium-dose and high-dose females compared to controls (Table 1). The APTT values in the medium-dose and high-dose females (15.77s and 16.57s) were within the range of facility historical APTT values (11.2-30.5s) for female control animals 11-22 weeks of age (Table S1). There were no other BV379-associated changes in coagulation parameters at all dose levels tested (Table 1).

Clinical chemistry

Some serum chemistry endpoints were significantly different in BV379-treated animals as compared to controls (Table 2). More specifically, all male BV379-treated groups exhibited decreased blood sodium (Na) levels that marginally decreased with increase in dose. Furthermore, both medium-dose and high-dose male treatment groups exhibited decreased blood alanine aminotransferase (ALT), albumin (ALB), calcium (Ca), and triglyceride (TG) levels, which also appeared to incrementally decrease with dose. Blood chloride (Cl) levels were also lower in the high-dose males. All clinical chemistry parameters that were significantly different in BV379-treated males were within the range of historical control values for 11–22-week-old male Sprague-Dawley rats at the testing facility (Table S2): ALT, 16-161 U/L; ALB, 2.8-4.6 g/dL; Ca, 9.1-13.4 mg/dL; Cl, 93.1-112.8 mmol/L; Na, 131-157; TG, 18-376 mg/dL. There were no significantly different serum chemistry parameters in BV379-treated females as compared to female controls.

Table 2.

Clinical chemistry parameters in male and female rats after oral administration of BV379 for 31 and 32 days, respectively^a.

BV379 dose (CFU/kg bw/d)	Males				Females
BV379 dose (CFU/kg bw/d)	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰
	n = 10	n = 10	n = 9	n = 10	n = 10	n = 10	n = 10	n = 10
ALT (U/L)^b,c	33.2 ± 6.2	28.9 ± 5.0	27.2 ± 3.4*	26.9 ± 4.8*	27.0 ± 3.3	27.2 ± 3.9	33.8 ± 19.0	26.8 ± 8.6
ALB (g/dL)^b	4.31 ± 0.24	4.23 ± 0.19	3.97 ± 0.25**	3.96 ± 0.16**	5.17 ± 0.22	5.20 ± 0.48	5.18 ± 0.28	5.08 ± 0.32
ALKP (U/L)^b	140.2 ± 29.9	149.7 ± 30.8	137.6 ± 30.7	152.9 ± 29.4	72.1 ± 10.8	68.7 ± 18.5	78.4 ± 16.4	69.0 ± 20.2
AST (U/L)^b,d	112.0 ± 29.5	99.6 ± 14.6	110.1 ± 23.9	108.3 ± 17.6	89.0 ± 23.7	77.0 ± 16.8	96.6 ± 22.0	87.9 ± 25.9
Ca (mg/dL)^b	12.07 ± 0.74	11.69 ± 0.73	11.34 ± 0.46*	11.26 ± 0.57*	12.83 ± 1.01	13.13 ± 1.13	13.34 ± 1.03	12.91 ± 0.88
Cl (mmol/L)^b	107.07 ± 2.00	106.52 ± 2.12	105.36 ± 1.47	104.61 ± 1.39*	105.56 ± 1.64	105.25 ± 2.49	107.07 ± 1.72	105.51 ± 1.97
CHOL (mg/dL)^b	69.1 ± 20.5	58.0 ± 9.9	58.0 ± 9.6	57.8 ± 11.7	92.3 ± 22.0	90.2 ± 9.2	85.3 ± 12.5	91.5 ± 18.1
CREA (mg/dL)^c,b	0.20 ± 0.02	0.19 ± 0.02	0.20 ± 0.04	0.20 ± 0.03	0.24 ± 0.03	0.23 ± 0.03	0.24 ± 0.04	0.24 ± 0.05
GGT (U/L)	ND	ND	ND	ND	ND	ND	ND	ND
GLOB (g/dL)^c,b	2.15 ± 0.17	2.05 ± 0.21	2.17 ± 0.19	2.26 ± 0.18	1.51 ± 0.24	1.52 ± 0.28	1.38 ± 0.19	1.40 ± 0.15
GLUC (mg/dL)^b,c	152.6 ± 30.9	141.9 ± 33.7	119.7 ± 21.6	123.1 ± 25.4	126.0 ± 27.8	168.8 ± 54.4	109.6 ± 9.3	107.9 ± 21.9
HDL (mmol/L)^d,b	1.08 ± 0.37	0.88 ± 0.16	0.89 ± 0.16	0.89 ± 0.19	1.92 ± 0.42	1.90 ± 0.23	1.79 ± 0.25	1.89 ± 0.36
IPHS (mg/dL)^b	11.40 ± 0.98	10.79 ± 1.37	10.61 ± 1.03	10.69 ± 1.25	10.92 ± 0.76	10.52 ± 0.84	11.18 ± 0.79	11.08 ± 1.13
K (mmol/L)^b	6.91 ± 1.13	6.90 ± 1.24	7.18 ± 1.36	7.21 ± 1.31	7.42 ± 1.64	7.34 ± 0.90	7.98 ± 1.57	7.93 ± 1.62
LDL (mmol/L)^c,d	0.24 ± 0.12	0.21 ± 0.06	0.26 ± 0.09	0.31 ± 0.09	0.23 ± 0.08	0.22 ± 0.06	0.20 ± 0.05	0.24 ± 0.07
Na (mmol/L)^c,b	146.50 ± 2.42	144.10 ± 2.51*	141.89 ± 1.76***	141.80 ± 1.03***	142.00 ± 2.62	141.50 ± 1.58	143.00 ± 2.40	141.50 ± 2.01
SDH (U/L)^d,c	10.43 ± 3.33	11.80 ± 7.81	6.51 ± 2.64	8.55 ± 4.75	7.43 ± 4.59	8.48 ± 2.20	10.37 ± 7.10	9.22 ± 5.58
BILI (mg/dL)^b,d	0.06 ± 0.02	0.07 ± 0.02	0.06 ± 0.02	0.08 ± 0.01	0.09 ± 0.03	0.09 ± 0.02	0.10 ± 0.04	0.09 ± 0.02
TP (g/dL)^c,b	6.46 ± 0.21	6.28 ± 0.28	6.13 ± 0.29	6.22 ± 0.33	6.68 ± 0.29	6.72 ± 0.60	6.56 ± 0.34	6.48 ± 0.36
TG (mg/dL)^d,b	50.8 ± 19.0	49.4 ± 22.1	28.8 ± 7.0***	24.9 ± 3.2***	35.0 ± 7.2	44.1 ± 17.9	37.9 ± 10.3	33.50 ± 11.0
BUN (mg/dL)^b	14.6 ± 1.6	13.8 ± 1.4	13.4 ± 1.7	14.7 ± 1.8	16.5 ± 1.6	16.0 ± 3.5	16.5 ± 2.4	16.7 ± 2.1

^aData are means ± standard deviation; where two subscripts are listed, two different statistical analyses were performed for the male and female animal groups;^*p < .05;^**p < .01;^***p < .001. BW, body weight; ALT, alanine aminotransferase; ALB, albumin; ALKP, alkaline phosphatase; AST, aspartate aminotransferase; Ca, calcium; Cl, chloride; CHOL, total cholesterol; CREA, creatinine; GGT, gamma-glutamyl transferase; GLOB, globulin; GLUC, glucose; HDL, high-density lipoprotein; IPHS; inorganic phosphorus; K, potassium; LDL, low-density lipoprotein; Na, sodium; ND, not detected as values were below the detection limit; SDH, sorbitol dehydrogenase; BILI, total bilirubin; TP, total protein; TG, triglycerides; BUN, blood urea nitrogen.

^bBetween-group differences for each sex were tested via ANOVA and Dunnett tests.

^cBetween-group differences for each sex were tested via Kruskal-Wallis one-way ANOVA and Dunn’s tests.

Urinalysis

There were no statistically significant BV379-related changes in urinalysis parameters at all dose levels tested, with the exception of increased urine volume (22.75 ± 11.98 mL) and decreased specific gravity (1.01 ± 0.01) in the high-dose males (Table 3). These values were within the range of test facility historical control values for 11–22-week-old males (Table S3): urine volume, 0.5-30.0 mL; specific gravity, 0.503-1.030.

Table 3.

Urinalysis parameters in male and female rats after oral administration of BV379 for 31 and 32 days, respectively^a.

BV379 dose (CFU/kg bw/d)	Males				Females
BV379 dose (CFU/kg bw/d)	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰
	n = 10	n = 10	n = 9	n = 10	n = 9	n = 9	n = 10	n = 9
Urine (mL)^b,c	8.05 ± 10.13	15.55 ± 10.61	9.94 ± 8.32	22.75 ± 11.98**	2.94 ± 2.14	2.67 ± 3.04	2.80 ± 1.69	2.94 ± 1.45
pH^c,c	6.95 ± 0.60	7.05 ± 0.55	6.78 ± 0.62	7.10 ± 0.52	6.11 ± 0.22	6.17 ± 0.35	6.30 ± 0.42	6.06 ± 0.17
Urine glucose (mg/dL)	ND	ND	ND	ND	ND	ND	ND	ND
Urine ketone (mmol/L)^d,c	25.5 ± 15.7	19.5 ± 14.8	19.4 ± 12.1	11.0 ± 12.2	3.9 ± 4.9	2.2 ± 2.6	0.0 ± 0.0	0.6 ± 1.7
Urine protein (mg/dL)^d,c	39.5 ± 32.6	26.5 ± 27.5	31.1 ± 26.9	22.0 ± 27.8	16.7 ± 11.7	18.3 ± 10.0	28.0 ± 26.3	13.3 ± 9.0
Specific gravity^d,c	1.02 ± 0.01	1.02 ± 0.01	1.02 ± 0.01	1.01 ± 0.01*	1.03 ± 0.00	1.03 ± 0.00	1.03 ± 0.00	1.03 ± 0.00
Urobilinogen (EU/dL)^d,c	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00	0.20 ± 0.00

^bBetween-group differences for each sex were tested via ANOVA and Dunnett tests with log-transformed data.

^cBetween-group differences for each sex were tested via ANOVA and Dunnett tests.

^dBetween-group differences for each sex were tested via Kruskal-Wallis one-way ANOVA and Dunn’s tests.

Organ weights

All absolute and relative organ weights for the BV379-treated males and females were comparable to the respective control group values (Tables 4 and S10–S15).

Table 4.

Terminal body weight and organ weights and weight ratios of male and female rats after oral administration of BV379 for 31 and 32 days, respectively^a.

BV379 dose (CFU/kg bw/d)	Males				Females
BV379 dose (CFU/kg bw/d)	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰	0	1 × 10¹⁰	4 × 10¹⁰	10 × 10¹⁰
Terminal BW (g)^b	398.5 ± 20.0	414.9 ± 41.7	400.0 ± 33.4	399.4 ± 31.9	241.8 ± 12.3	247.5 ± 15.0	252.8 ± 21.3	249.0 ± 22.7
Adrenal glands (g)^b	0.07 ± 0.01	0.06 ± 0.02	0.06 ± 0.02	0.07 ± 0.02	0.08 ± 0.02	0.07 ± 0.03	0.15 ± 0.22	0.08 ± 0.01
Brain (g)^b,c	2.18 ± 0.17	2.15 ± 0.09	2.20 ± 0.11	2.18 ± 0.11	2.05 ± 0.08	2.06 ± 0.06	2.04 ± 0.10	2.00 ± 0.08
Heart (g)^b,c	1.29 ± 0.09	1.34 ± 0.12	1.27 ± 0.10	1.33 ± 0.12	0.84 ± 0.05	0.90 ± 0.06	0.89 ± 0.05	0.87 ± 0.05
Kidneys (g)^b,c	2.83 ± 0.28	2.92 ± 0.31	2.88 ± 0.31	3.06 ± 0.51	1.85 ± 0.16	1.87 ± 0.18	1.83 ± 0.18	1.89 ± 0.21
Liver (g)^b,c	12.98 ± 1.51	12.38 ± 2.17	11.78 ± 1.86	11.98 ± 2.01	7.64 ± 0.79	7.94 ± 0.81	8.17 ± 1.02	8.28 ± 1.18
Pituitary gland (g)^c	0.02 ± 0.00	0.02 ± 0.01	0.02 ± 0.00	0.01 ± 0.00	0.02 ± 0.01	0.02 ± 0.00	0.02 ± 0.01	0.02 ± 0.00
Spleen (g)^b,c	0.79 ± 0.14	0.88 ± 0.18	0.80 ± 0.11	0.81 ± 0.14	0.55 ± 0.08	0.53 ± 0.06	0.60 ± 0.08	0.56 ± 0.09
Thymus (g)^b,c	0.40 ± 0.08	0.37 ± 0.09	0.38 ± 0.08	0.38 ± 0.06	0.40 ± 0.10	0.38 ± 0.10	0.44 ± 0.08	0.40 ± 0.06
Epididymides (g)^b	1.20 ± 0.07	1.19 ± 0.17	1.27 ± 0.15	1.19 ± 0.12	NA	NA	NA	NA
Testes (g)^b	3.42 ± 0.41	3.51 ± 0.38	3.68 ± 0.45	3.50 ± 0.20	NA	NA	NA	NA
Ovaries & oviducts (g)^c	NA	NA	NA	NA	0.13 ± 0.02	0.15 ± 0.02	0.14 ± 0.02	0.14 ± 0.01
Uterus (g)^b	NA	NA	NA	NA	0.64 ± 0.25	0.69 ± 0.20	0.61 ± 0.15	0.56 ± 0.12
Adrenal/TBW (Ratio)^d	0.17 ± 0.02	0.16 ± 0.04	0.16 ± 0.04	0.18 ± 0.05	0.32 ± 0.08	0.2 ± 0.09	0.60 ± 0.86	0.32 ± 0.05
Brain/TBW (Ratio)^b	5.48 ± 0.50	5.23 ± 0.56	5.54 ± 0.42	5.48 ± 0.45	8.51 ± 0.57	8.33 ± 0.45	8.09 ± 0.46	8.13 ± 0.76
Heart/TBW (Ratio)^c	3.24 ± 0.22	3.23 ± 0.20	3.17 ± 0.16	3.34 ± 0.40	3.48 ± 0.24	3.64 ± 0.24	3.52 ± 0.23	3.49 ± 0.22
Kidneys/TBW (Ratio)^b	7.10 ± 0.52	7.05 ± 0.44	7.21 ± 0.62	7.66 ± 1.01	7.66 ± 0.66	7.57 ± 0.67	7.25 ± 0.43	7.60 ± 0.67
Liver/TBW (Ratio)^b	32.39 ± 2.63	29.82 ± 4.06	29.42 ± 3.89	29.94 ± 4.16	31.62 ± 3.28	32.07 ± 2.47	32.36 ± 3.50	33.24 ± 3.37
Pituitary/TBW (Ratio)^b	0.004 ± 0.001	0.004 ± 0.001	0.004 ± 0.001	0.004 ± 0.001	0.007 ± 0.002	0.007 ± 0.001	0.007 ± 0.002	0.008 ± 0.001
Spleen/TBW (Ratio)^c,b	1.98 ± 0.26	2.12 ± 0.39	2.03 ± 0.34	2.02 ± 0.24	2.28 ± 0.38	2.14 ± 0.26	2.36 ± 0.23	2.26 ± 0.31
Thymus/TBW (Ratio)^c,d	1.00 ± 0.19	0.90 ± 0.23	0.97 ± 0.21	0.95 ± 0.17	1.67 ± 0.42	1.55 ± 0.45	1.73 ± 0.28	1.61 ± 0.17
Epididymides/TBW (Ratio)^d	3.01 ± 0.24	2.92 ± 0.60	3.19 ± 0.33	3.00 ± 0.36	N/A	N/A	N/A	N/A
Testes/TBW (Ratio)^b	8.61 ± 1.16	8.57 ± 1.52	9.21 ± 0.87	8.81 ± 0.93	N/A	N/A	N/A	N/A
Ovaries with oviducts/TBW (Ratio)^b	N/A	N/A	N/A	N/A	0.54 ± 0.09	0.62 ± 0.11	0.57 ± 0.10	0.58 ± 0.05
Uterus/TBW (Ratio)^d	N/A	N/A	N/A	N/A	2.64 ± 0.98	2.76 ± 0.78	2.42 ± 0.52	2.25 ± 0.47
Adrenal/BrW (Ratio)^b,d	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01	0.03 ± 0.01	0.04 ± 0.01	0.03 ± 0.01	0.06 ± 0.11	0.04 ± 0.01
Heart/BrW (Ratio)^b	0.59 ± 0.05	0.62 ± 0.06	0.57 ± 0.04	0.61 ± 0.07	0.41 ± 0.03	0.44 ± 0.03	0.44 ± 0.03	0.44 ± 0.03
Kidneys/BrW (Ratio)^b	1.31 ± 0.20	1.36 ± 0.15	1.31 ± 0.11	1.41 ± 0.22	0.90 ± 0.08	0.91 ± 0.09	0.90 ± 0.07	0.94 ± 0.14
Liver/BrW (Ratio)^b	6.02 ± 0.81	5.76 ± 0.98	5.34 ± 0.78	5.51 ± 0.98	3.73 ± 0.43	3.86 ± 0.38	4.02 ± 0.53	4.16 ± 0.66
Pituitary/BrW (Ratio)^b	8.21 ± 1.76	7.17 ± 2.65	8.08 ± 1.76	6.56 ± 1.65	8.42 ± 2.60	8.33 ± 1.85	9.05 ± 2.15	9.45 ± 1.41
Spleen/BrW (Ratio)^c,b	0.36 ± 0.07	0.41 ± 0.09	0.36 ± 0.05	0.37 ± 0.07	0.27 ± 0.04	0.26 ± 0.03	0.29 ± 0.04	0.27 ± 0.04
Thymus/BrW (Ratio)^b,c	0.18 ± 0.03	0.17 ± 0.04	0.17 ± 0.03	0.17 ± 0.03	0.20 ± 0.04	0.18 ± 0.05	0.22 ± 0.04	0.20 ± 0.03
Epididymides/BrW (Ratio)^b	0.55 ± 0.05	0.55 ± 0.07	0.58 ± 0.06	0.55 ± 0.05	N/A	N/A	N/A	N/A
Testes/BrW (Ratio)^b	1.56 ± 0.21	1.63 ± 0.16	1.67 ± 0.22	1.61 ± 0.10	N/A	N/A	N/A	N/A
Ovaries with oviducts/BrW (Ratio)^b	N/A	N/A	N/A	N/A	0.06 ± 0.01	0.08 ± 0.01	0.07 ± 0.01	0.07 ± 0.01
Uterus/BrW (Ratio)^d	N/A	N/A	N/A	N/A	0.31 ± 0.12	0.34 ± 0.10	0.30 ± 0.07	0.27 ± 0.06

^aData are means ± standard deviation (n = 9-10 per group; see Tables S10–15 for details). CFU, colony-forming unit; NA, not applicable; TBW, total body weight.

^bBetween-group differences for each sex were tested via ANOVA and Dunnett tests.

^cBetween-group differences for each sex were tested via ANOVA and Dunnett tests with log-transformed data.

^dBetween-group differences for each sex were tested via ranked ANOVA and Dunn’s tests.

Macroscopic findings

There were no gross lesions found related to the administration of B. velezensis strain BV379 in animals terminated at the study conclusion (Table S16). There were only three macroscopically observed findings in these terminally sacrificed animals:

• One vehicle control (0 CFU/kg bw/day) male: This animal exhibited a small left testis that was microscopically confirmed to be marked unilateral atrophy of the seminiferous tubular epithelium. This was related to a same-side epididymal malformation found microscopically that resulted in blockage of the efferent ducts from the testis to the epididymis.

• One low-dose (1 × 10¹⁰ CFU/kg bw/day) male. This animal exhibited a small left testis noted at necropsy. Microscopically, this testis also had atrophy of the seminiferous epithelium.

• One low-dose (1 × 10¹⁰ CFU/kg bw/day) female: For this animal, the uterus was noted to be “fluid-filled” at necropsy. Microscopically, this correlated with simple dilation, which represents a normal phase of the estrus cycle in the rat.

Histopathological findings

Histopathological examinations were performed on preserved organs and tissues of animals from both control and high-dose groups (n = 10). Microscopic findings did not increase in incidence in BV379-treated animals compared to controls (Tables S17–18). Most observations were minimal cellular infiltrates (score of 1), usually consisting of foci of a few lymphocytes or other mononuclear cells, in various tissues such as liver, lung, kidney, heart, or prostate across all control and treatment animals. More specifically, the majority of male and female animals from both treatment and control groups had minor cellular infiltrates in their liver samples, with one BV379-treated female being the only animal to be free of liver cellular infiltrates or any other finding (Tables S17-S18). Mononuclear cell infiltrates and minimal signs of chronic progressive nephropathy in kidney samples were observed in two to five animals in each examined group. Other minimal findings were noted for epididymis, Harderian gland, thyroid, nasal turbinate, pancreas, and Peyer’s Patch samples. However, the incidences of these findings were mostly limited to 0—3 animals per group (where applicable by sex). In regards to more severe findings, one control male was observed with marked atrophy of the seminiferous tubule (score of 4) and three females (two control and one high-dose) had mild dilation in samples collected from the uterus (scores of 2).

Tissue bacterial isolate enumeration and taxonomic analysis

A total of 430 isolates derived from blood, liver, and mesentery tissue were subjected to molecular characterization. Of these, 322 (75%) were not differentiable from the BV379 test strain by 16S rRNA (V4) analysis or RAPD fingerprinting (Table S19). The presumptive BV379 bacterial isolates were detected in tissue samples across all treatment and vehicle control groups (Table S20).

For blood-plated samples, bacterial isolates were detected from all groups including the vehicle control (Table S21). All blood-derived isolates, including isolates from the vehicle control group, were identified as Bacillus velezensis and could not be differentiated from the BV379 test substance by 16S rRNA (V4) analysis or RAPD fingerprinting. The median bacterial concentrations across the control and treatment groups ranged from 0-3 CFU/mL blood, with no statistically significant differences in blood plate counts between BV379 treated groups and controls (males: p = .80; females: p = .34). Liver bacterial isolates were detected from the majority (90%) of animals across each sex and dose group, including vehicle controls (Table S22). The median liver bacterial concentrations were 67-533 CFU/g tissue, with no statistically significant treatment effects (males: p = .80; females: p = .46). Mesentery bacterial isolates were detected from all samples including vehicle controls (Table S23). Median mesentery bacterial concentrations ranged from 66-1,433 CFU/g tissue. Similar to the blood and liver samples, there were no statistically significant treatment effects (males: p = .70; females: p = .09).

Individual clinical assessments of study animals with high tissue bacterial concentrations

To determine if animals with high tissue concentrations of bacteria (≥167 CFU/mL blood or ≥1,667 CFU/g liver or mesentery tissue) had any evidence of toxicity, the organ weights, hematology, clinical chemistry, urinalysis, and coagulation data of a subset of seven animals were compared to historical controls within the testing facility (PSL). The subset of seven animals included one male control, one low-dose female, two medium-dose males, one high-dose male, and two high-dose females.

Most animals exhibited organ weights, hematology, clinical chemistry, urinalysis, and coagulation values that did not deviate from facility historical control ranges. One exception includes the male control rat, which exhibited a slightly higher MCV value than the historical control range (62.8 fL vs. 51.2-61.5 fL). This same control rat also had lower urinary protein than the historical control range (15 mg/dL vs. 30-300 mg/dL). In addition, one medium-dose male rat had a slightly higher alkaline phosphate value than historical controls (146 U/L vs. 36-143 U/L) and one high-dose male rat had slightly higher inorganic phosphorus values than the historical control range (12 mg/dL vs. 4.6-11.7 mg/dL).

Individual histopathological results of the control male and three high-dose animals with high tissue concentrations of bacteria were assessed. All histopathological findings were minimal in severity (scores ≤1) with the exception of the male control:

• Vehicle control (0 CFU/kg bw/day) male: Testicular atrophy that was marked and unilateral. The epididymis failed to develop properly and was atrophic to the point of appearing not to be patent. Presence of minimal chronic nephropathy, minimal mononuclear cell infiltrate in kidney and liver, and minimal focal acinar atrophy.

• High-dose (10 × 10¹⁰ CFU/kg bw/day) male: Minimal chronic progressive nephropathy and minimal mononuclear cell infiltrate in liver.

• High-dose (10 × 10¹⁰ CFU/kg bw/day) female: Minimal mononuclear cell infiltrate in liver and minimal respiratory epithelium infiltrate in nasal turbinates.

• High-dose (10 × 10¹⁰ CFU/kg bw/day) female: Minimal chronic progressive nephropathy in kidney and minimal mononuclear cell infiltrate in liver and kidney.

Discussion

At least eight commercial feed additives containing B. velezensis strains (e.g., EnzaPro, PB6, and Calsporin) are presumed safe for use in a variety of animals (e.g., swine, dogs, cattle, and poultry) by the EFSA Panel on Additives and Products or Substances used in Animal Feed.^5–13 For example, the B. velezensis feed additive Calsporin® (previously classified as B. subtilis¹⁴) was previously administered to sows and suckling piglets in doses of up to 3 × 10⁸ CFU/g feed for up to 170 days with no reported adverse effects.¹³ Calsporin has also been safely administered to dogs in concentrations as high as 1 × 10¹⁰ CFU/g feed for up to 37 days.⁴

However, there is sparse preclinical and clinical data on the oral safety of B. velezensis in healthy subjects. A few preclinical studies have examined the effects of oral supplementation with B. velezensis strains isolated from yak intestines, Tibetan sheep feces, and the soybean fermentation starter meju on healthy mice.^20–24 No adverse effects were observed when the strains were administered in 3.3 × 10⁹ – 6 × 10¹⁰ CFU/kg initial body weight/day for six days to eight weeks. Clinical studies on the oral safety of B. velezensis have been limited to two strains, Calsporin and Bispan (formally classified as B. polyfermenticus) .^15–19 Throughout the Calsporin studies, participants consumed 2.2 × 10⁹ – 4.8 × 10¹⁰ CFU/day for four to eight weeks. Health was assessed by physical examination, urinalysis, hematology, and clinical chemistry, for which there were no noted deviations. The single B. velezensis Bispan clinical trial demonstrated that supplementation of 3.1 × 10⁸ CFU/day for eight weeks did not adversely impact blood immune markers and anthropometric measurements in healthy male volunteers .¹⁹ While these clinical studies support the oral safety of Calsporin and Bispan, future studies with additional strains and doses are needed to more fully establish the preclinical and clinical safety of the B. velezensis species as a whole.

The objective of this study was to further strengthen the current body of work on the oral safety of B. velezensis in healthy subjects and to support the safe use of B. velezensis strain BV379 for use in dietary supplements and food. There were no treatment-related macroscopic, microscopic, or organ weight findings resulting from the oral administration of B. velezensis strain BV379 to male and female Sprague-Dawley rats at dose levels of 1 × 10¹⁰, 4 × 10¹⁰, and 10 × 10¹⁰ CFU/kg bw/day for at least 28 days. All remarkable histological findings (e.g., cellular infiltrates in liver, kidney, and lungs) were likely incidental, spontaneous, or developmental changes, typically seen in rats of this age and strain, and they did not occur more frequently in BV379-treated animals.³⁹ There were also no changes in body weight, feed consumption, clinical observations, or mortalities that were associated with BV379 oral administration. A single mortality was noted for a medium-dose male, just prior to the scheduled study termination, which was unrelated to BV379 exposure. In this animal, pathologic observations of acute hemorrhage and moderate inflammation surrounding the pharynx, with apparent perforation that was indicative of inadvertent trauma, were attributed to the oral gavage procedure.

A number of clinical pathology endpoints, including serum chemistry, coagulation, and urinalysis parameters were statistically significantly different in BV379-treated animals as compared to controls. More specifically, APTT was increased across groups in a dose-dependent manner, reaching statistical significance in medium and high-dose females. However, there were no changes in mean PT time that corresponded to the increases in APTT. In addition, there were no clinical, macroscopic, or microscopic findings that would support a specific diagnosis attributable to effects on clotting factors within the coagulation cascade. Statistically significant serum chemistry changes included decreases in blood Na levels across all male treatment groups. Both medium-dose and high-dose males also exhibited statistically significant decreases in ALT, ALB, Ca, and triglyceride levels in a dose-dependent manner. High-dose males alone exhibited significantly lower blood Cl levels. Statistically significant increases in urine volume and decreased specific gravity in the high-dose male group as compared to controls were also noted. However, there were no correlating clinical, macroscopic, or microscopic changes that would support a hepatotoxic effect or other adverse physiological condition throughout any of the BV379-treated males. Further, the physiological differences associated with BV379 administration can be considered non-adverse in nature, as none of the measured parameters deviated outside the range of values that have been previously observed in the testing facility’s historical controls (Tables S1-S3).

In addition, the above clinical pathology results are not concerning in regards to human supplement applications. The smallest dose (1 × 10¹⁰ CFU/kg BW/day), which only resulted in decreased Na levels in male animals, is substantially larger than the typical intended dose for spore-forming probiotic supplements (≤2 × 10⁹ CFU/day).^40–43 A dose of 2 × 10⁹ CFU/day equals approximately 2.86 × 10⁷ CFU/kg BW/day for a 70 kg individual. Even when accounting for inter- and intra-species differences using a 100-fold safety factor (approximately 2.86 × 10⁹ CFU/kg BW/day),⁴⁴ the intended dose is still lower than the lowest dose tested in this study.

At the end of the study, bacteria were isolated from blood, liver, and mesentery tissues from both males and females throughout all groups, including vehicle control animals. The vast majority of these bacterial isolates were undifferentiable from the BV379 test substance based on 16S rRNA sequence and RAPD analysis. There were no significant treatment-related differences in the number of isolates from any of the three tissues examined. Isolation of bacteria from blood plates is typically rare in translocation studies. Since bacterial isolates indistinguishable from BV379 were detected throughout all tissues and treatment groups, including controls, it is likely that post-euthanasia contamination of the samples had occurred. This is also substantiated by the fact that none of the study animals exhibited clinical signs of bacteremia.

To further assess the potential for tissue sample contamination, a review was conducted to determine if a subset of seven animals with high tissue levels of B. velezensis strain BV379 exhibited any evidence of toxicity when compared to historical test facility control values. In general, most control and treated animals that had tissue samples with high levels of BV379 were not observed to be abnormal with regards to organ weights, hematology, clinical chemistry, urinalysis, and coagulation values. Out of the subset of seven animals, one male control rat exhibited a slightly higher MCV value than the historical control range (62.8 fL vs. 51.2-61.5 fL) and lower urinary protein than the historical control range (15 mg/dL vs. 30-300 mg/dL). These excursions were not considered to be toxicologically relevant, nor related, since all other organ weight, hematology, clinical chemistry, urinalysis, and coagulation values for this animal were within range of historical controls. In addition, one medium-dose male rat had higher alkaline phosphate value than historical controls (146 U/L vs. 36-143 U/L) and one high-dose male rat had slightly higher inorganic phosphorus values than the historical control range (12 mg/dL vs. 4.6-11.7 mg/dL). Since these deviations from the historical control ranges were so minimal, they are likely not toxicologically relevant. No other serum chemistry values for these two animals were out of historical control range. Furthermore, the individual histopathological results of the control male and three high-dose animals with high tissue concentrations of bacteria were assessed. All three high-dose animals exhibited histopathological traits that are typical for rats of this age.³⁹ The control male exhibited a malformation in the epididymis and testicular atrophy, but these findings were likely congenital in nature.

The results of the historical control comparisons and individual histological assessments of animals with high levels of BV379 in tissue samples further support that BV379 likely did not translocate from the gut into extraintestinal tissue in the study rats. Rather, it is most likely that post-euthanasia contamination occurred. The conclusion that sample contamination occurred in lieu of in vivo translocation is in alignment with previous in vitro work demonstrating that BV379 lysates do not negatively impact intestinal epithelial cell (Caco-2) transepithelial electrical resistance (TEER) (i.e., monolayer permeability) or cell viability.²⁵

Furthermore, previous genomic analyses have shown that the BV379 genome does not encode for any Bacillus-associated toxins, virulence factors, or enzymes involved in toxin production .²⁵ While BV379 does encode five antimicrobial resistance genes, additional in vitro work has also shown that BV379 is susceptible to eight medically-relevant antibiotics based on EFSA-established minimum inhibitory concentration assay guidelines for Bacillus strains.^25,45 Altogether, the preclinical in vivo safety results presented herein, together with previously published in silico and in vitro safety data, support the preclinical safety of BV379.

Conclusion

The objective of this study was to evaluate the in vivo safety of Bacillus velezensis strain BV379 by high-dose oral administration to rats in a 28-day subchronic toxicity study. Under the conditions of this study and based on the toxicological endpoints evaluated, the no-observed-adverse-effect-level (NOAEL) for the oral administration of Bacillus velezensis BV379 was determined to be 10 × 10¹⁰ CFU/kg bw/day for male and female Sprague-Dawley rats. The results of this study, along with recently published in silico and in vitro safety assessments of BV379, demonstrate that the strain is safe for use in food and dietary supplements and provide an important foundation of safety for future clinical work and efficacy studies.

Supplemental Material

Supplemental Material - Subchronic oral toxicity assessment of Bacillus velezensis strain BV379 in sprague-dawley rats

Supplemental Material for Subchronic oral toxicity assessment of Bacillus velezensis strain BV379 in sprague-dawley rats by Mark R. Bauter, Laura M. Brutscher, Laurie C. Dolan and Jessica L. Spears in Human & Experimental Toxicolog.

Supplemental Material

Supplemental Material - Subchronic oral toxicity assessment of Bacillus velezensis strain BV379 in sprague-dawley rats

Footnotes

Declaration of conflicting interests

The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: M.R.B. is an employee of Product Safety Labs, which received compensation for conducting the rat toxicity study. L.M.B and J.L.S. are employees of BIO-CAT Microbials, LLC, which provided funding and manufactured the Bacillus velezensis BV379 spores for this study. BIO-CAT Microbials, LLC is the assignee of a patent application describing compositions and methods of use related to the microbial strain described herein (Patent Application No. 63/512,678). The funders had input into the final study design and interpretation of the data. The funders were also involved in the writing and editing of the manuscript. L.C.D is an employee of GRAS Associates, LLC, a consulting firm that received compensation from BIO-CAT Microbials, LLC for assisting L.M.B and J.L.S. The authors declare no other conflicts of interest.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was funded by BIO-CAT Microbials, LLC and BIO-CAT, Inc.

Ethical Approval

All animal experiments described in this study were conducted at Product Safety Labs (PSL), an AAALAC-accredited facility, in compliance with the U.S. Animal Welfare Act Regulations (9 CFR) and the Guide for the Care and Use of Laboratory Animals (8th edition, National Research Council, 2011). Study protocols were reviewed and approved by the PSL Institutional Animal Care and Use Committee (IACUC) prior to study initiation P713.01 BCM (A 28-Day Oral Gavage Study in Rats with MPCA Assessment (IACUC Protocol Approved September 17, 2019). Humane care and use of laboratory animals were ensured throughout the experimental phases of this research.

ORCID iD

Mark R. Bauter

Data availability statement

Data not presented within the article or Supplementary Materials are available upon reasonable request from the corresponding author.

Supplemental Material

Supplemental material for this article is available online.

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