Genetic, acute and subchronic toxicity studies of matured hop extract produced by extraction from heat-treated hops

Abstract

It has been demonstrated that successive ingestion of matured hop extract (MHE), produced by extraction from heat-treated hops, results in body fat reduction in animals and humans; however, preclinical safety studies have not been reported. In this study, we conducted in vitro and in vivo safety studies for MHE. Genotoxicity was evaluated using the Ames test, in vitro chromosomal aberration test, and in vivo micronucleus test. To assess acute safety, a single, oral administration of MHE to rats was monitored. Subchronic safety was assessed by repeated feeding with MHE for 90 days. The in vitro chromosomal aberration test was positive at 3,330 μg/mL and 5,000 μg/mL without metabolic activation. However, MHE did not induce any reverse mutation with or without metabolic activation in the Ames test, and no abnormalities were observed at a dose of 2,000 mg/kg body weight in the rat micronucleus test. In the acute and subchronic safety studies, no deaths or toxicological signs were recorded during the observation period. In addition, no changes in body weights, feed/water consumption, clinical signs, ophthalmoscopy, urinalysis, hematology, blood biochemistry, organ weights, or histopathology were observed after repeated administration of MHE. Therefore, the no-observed-adverse-effect-level (NOAEL) of MHE was considered to be over 3,484 and 4,022 mg/kg body weight/day in males and females, respectively. These results indicate that there is no safety concern for MHE in the present preclinical safety study.

INTRODUCTION

Hop (Humulus lupulus L.) is a species of perennial plant in the Cannabaceae family. Since early times, hops have been used as a medicinal plant in Europe (DeLyser and Kasper, 1994) and are widely used to provide beer with its peculiar bitterness and flavor (Steenackers et al., 2015). The components of bitterness in hops are the bitter acids, α- and β-acids, derived from phloroglucinol. Isomerization of α-acids during the beer brewing process results in iso-α-acids, which are the main cause of bitterness in beer and have a variety of bioactivities (Yajima et al., 2004, 2005; Nozawa et al., 2005; Miura et al., 2005, Obara et al., 2009, Ano et al., 2017). In contrast, oxidation of α- and β-acids produces bitter acid oxides, such as matured hop bitter acids (MHBA). All of the structurally elucidated compounds of MHBA possess a common β-tricarbonyl moiety similar to α-, β-, and iso-α-acids (Taniguchi et al., 2013, 2014a, 2014b, 2015). We demonstrated that MHBA suppresses diet-induced body fat accumulation in animals (Morimoto-Kobayashi et al., 2015). It was also recently demonstrated in a human clinical trial that ingestion of matured hop extract (MHE), containing MHBA as active components, decreases body fat (Morimoto-Kobayashi et al., 2016). In addition to the efficacy, the safety of MHE was confirmed in the clinical trial (Morimoto-Kobayashi et al., 2016). Thus, MHE is beneficial to overweight and obese individuals; however, the toxicity of MHE in a preclinical study has not been reported. In this report, we investigated the genotoxicity, and acute and subchronic safety of MHE using a bacterial reverse mutation test, in vitro chromosomal aberration test, and in vivo micronucleus test, in addition to single and repeated administration of MHE.

MATERIALS AND METHODS

All the experiments in this study, mutagenicity, genotoxicity, acute and subchronic toxicity tests, were performed at BoZo Research Center Inc. (Tokyo, Japan) under the Good Laboratory Practice (GLP) conditions. At the commencement of the animal study, the study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of BoZo Research Center Inc., according to Guidelines for Animal Studies.

Chemicals and reagents

The following positive controls used in the Ames test were dissolved in dimethyl sulfoxide (DMSO, Wako Pure Chemical Industries, Ltd., Osaka, Japan): 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2, Wako); sodium azide (SAZ, Wako); 2-aminoanthracene (2AA, Wako); 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine HCl (ICR-191, Polyscience, Inc., Warrington, PA, USA); benzo[α]pyrene (B[α]P, AccuStandard, Inc., New Haven, CT, USA). The following positive controls for the in vitro chromosomal aberration test and in vivo micronucleus test were dissolved in sterilized water (Otsuka Pharmaceutical Co., Ltd., Tokyo, Japan): mitomycin C (MMC, Kyowa Hakko Kirin Co., Ltd., Tokyo, Japan); cyclophosphamide (CP, Wako). S9 mix was prepared by mixing S9 (Kikkoman Biochemifa Co., Ltd., Tokyo, Japan) with cofactor-I or -C (Oriental Yeast Co., Ltd., Tokyo, Japan) reconstituted with distilled water and sterilized by filtration.

Preparation of test substance

MHE was prepared as previously described (Morimoto-Kobayashi et al., 2016). In brief, hop pellets (approximately 400 kg) were purchased from HopSteiner (Mainburg, Germany) and incubated at 60°C for 120 hr to oxidize α- and β-acids. The oxidized pellets were then extracted with deionized water (7,600 kg) at 55°C for 30 min. An aliquot of extract was filtered to remove the debris, concentrated to about 12° Brix, heated at 90°C for 4 hr, and cooled to room temperature. A brown liquid (approximately 228 kg) was yielded and an aliquot of the liquid was lyophilized. Finally, MHE was yielded as brown powder (approximately 7.6 kg). HPLC analysis confirmed that the MHE contained 20.5% of MHBA, as previously described (Taniguchi et al., 2015).

Genotoxicity study

Bacterial reverse mutation (Ames) test

The bacterial reverse mutation (Ames) test was conducted in accordance with the pre-incubation method (Maron and Ames, 1983). Salmonella typhimurium strains TA 100, TA1535 and Escherichia coli strain WP2uvrA were used to detect base-pair substitutions; S. typhimurium TA98 and TA1537 were used to detect frameshift mutations.

Aliquots (0.1 mL) of the negative (DMSO), and positive control solutions, and test solutions were added to 0.5 mL of 100 mM phosphate buffer (pH 7.4) for the direct method or S9 mix for the metabolic activation method, followed by the addition of 0.1 mL of bacterial suspension. The mixtures were pre-incubated at 37°C for 20 min with shaking at 80 rpm, before 2 mL of top agar, pre-warmed at 45°C, was added: the tests were performed in triplicate. The mixtures were poured onto minimal glucose medium agar plates and incubated at 37°C for 48 hr. The series of procedures above were performed under a fluorescent light coated with an ultraviolet absorber. After incubation, neither precipitation nor coloration induced by the test substance was observed on any of the plates. The numbers of revertant colonies were counted using an automatic colony counter (CA-11D, System Science Co., Ltd., Tokyo, Japan). The results were considered positive when the average number of revertant colonies was twice higher than that of the negative control plate, and when revertant colonies were induced in a dose-dependent manner. The dose of the positive controls and the test substance were as follows: positive controls (AF-2, 0.01 and 0.1 μg/plate; SAZ, 0.5 μg/plate; ICR-191, 1.0 μg/plate; 2AA, 2 and 10 μg/plate; B[α]P, 5 μg/plate); test substance (313, 625, 1,250, 2,500 and 5,000 μg/plate).

In vitro chromosomal aberration test

Chinese hamster lung fibroblasts (CHL/IU) were used in this study. The test was carried out in accordance with the established protocol (Ishidate, 1989). First, the cell-growth inhibition test was carried out with and without S9 mix to determine the correct dose for the chromosomal aberration test. The maximum dose of the test substance was set to 5,000 μg/mL, which was provided by “Guidelines for the designation of food additives and revision of standards for use of food additives” (Ministry of Health, Labour and Welfare in Japan, 1996). The final concentrations were selected as follows: 5,000, 2,500, 1,250, 625, 313, 156, 78.1 and 39.1 μg/mL. Since cytotoxicity in more than 50% of cells was observed, the following doses were selected for the chromosomal aberration test: short-term treatments, 5,000, 2,500, 1,250, and 625 μg/mL with S9 mix, and 5,000, 3,330, 2,220, and 1,480 μg/mL without S9 mix. Negative and positive controls were also prepared.

In the short-term treatment, 2 × 10⁴ cells were inoculated on each plate (60 mm diameter) and cultured for 3 days at 37°C in 5% CO₂ air. The cells were then exposed to the controls and test substances for 6 hr with or without S9 mix. The medium was then replaced with fresh culture medium and cells were incubated for a further 18 hr. The cells were then treated with 0.1 mL of colcemid (10 μg/mL demecolcin solution) for the last 2 hr of the incubation period. The treated cells were collected by trypsinization and centrifuge, treated with 75 mM KCl for 15 min, and then fixed with a methanol-acetic acid mixture (3:1). The fixed cell suspension was applied onto two sites on a glass slide. Two glass slides per plate were prepared, and cells in metaphase (100 chromosomes/plate) were microscopically observed. After air-drying for about 1 day, the preparations were stained with 2% Giemsa solution for 15 min for chromosome observation. The results were judged according to the guidelines (Ishidate, 1987). Chromosome preparations in the continuous treatment were not examined because the results of the short-term treatment were positive.

In vivo Micronucleus test

A micronucleus test was carried out in accordance with a previous protocol (Hayashi et al., 1983). Eight-week old male Sprague-Dawley (Cr1: CD) rats (SD rats) were purchased from Charles River Laboratory Japan, Inc. (Kanagawa, Japan). The body weights of 25 SD rats ranged from 289 to 310 g, and the rats were divided into five groups (n = 5) so that the average body weight between the groups was as equal as possible. MHE were dissolved in sterilized water and orally administered at 2,000 (high dose group), 1,000 (middle dose group), and 500 (low dose group) mg/kg body weight/day. Sterilized water was orally administered to the negative control group. MMC was dissolved in sterilized water and intraperitoneally injected at 2 mg/kg body weight in the positive control group. The doses were administered once-daily for two days with an interval of about 24 hr for the negative and test substance groups. Only one injection was given in the positive control group. Bone marrow cells were collected approximately 24 hr after finishing the administration. Bone marrow cell smears were prepared and stained with acridine orange. Numbers of polychromatic erythrocytes (PCEs) per 200 erythrocytes, and micronucleated polychromatic erythrocytes (MNPCEs) per 2,000 PCEs were counted.

Acute oral toxicity test

Administration

The body weights of 10 male and 10 female SD rats at 6-week old (Charles River Japan Inc.) ranged from 147 to 157 g (male) and from 125 to 136 g (female). The rats were divided into two groups (n = 5) so that the average body weight among the groups was as equal as possible. A single, oral administration of MHE, dissolved in sterilized water, was given at a dose of 2,000 mg/kg body weight. Sterilized water was orally administered to the negative control group. The administration volume was set at 10 mL/kg body weight. Before administration, all rats were fasted for 16 hr from the previous day. Diet and tap water were fed to rats ad libitum 4 hr after administration.

Clinical observations, body weight, and pathological examination

The rats were observed for 14 days after administration. Clinical signs, such as external abnormalities on the body surface, nutritional condition, posture, behavior and feces, were observed before administration, immediately after, then 5, 15 and 30 min, 1, 2, 4 and 6 hr after administration, and then once-daily. Body weights were measured on days 0, 1, 2, 3, 7, 10 and 14 after administration. After the end of the 14-day observation period, all rats were euthanized by cutting the abdominal aorta under isoflurane anesthesia and autopsied.

Subchronic oral toxicity test

Administration

The body weights of 40 male and 40 female SD rats at 6-week old (Charles River Japan Inc.) ranged from 183 to 206 g (male) and 148 to 178 g (female). The rats were divided into five groups (n = 10) so that the average body weight among the groups was as equal as possible. MHE was mixed with the standard powder diet, CR-LPF (Oriental Yeast Co., Ltd.) at a concentration of 1.25, 2.5, or 5.0% for low, middle or high dose groups, respectively. CR-LPF without MHE was given to the control group. Diets and tap water were fed to the rats ad libitum for 90 days.

Clinical observation, body weight, feed consumption and water intake

Clinical signs, such as external abnormalities on the body surface, nutritional condition, posture, behavior and feces, were observed twice daily. Body weight was measured on day 1 and day 7, and once a week thereafter. Body weights were measured after fasting for 16 hr from the previous day on the day of necropsy; internal organ weights were then evaluated. Feed and water consumption were measured once per week throughout the test period.

Ophthalmoscopy

Animals were examined prior to the start of administration (during quarantine and acclimation) and in week 13 (day 90). First, mydriasis was induced by applying Mydrin P ophthalmic solution (Santen Pharmaceutical Co., Ltd., Osaka, Japan) onto the eyes. The anterior ocular segment, optic media, and fundus of the eye were then examined using an indirect ophthalmoscope (Omega 200, HEINE Optotechnik GmbH & Co., KG, Herrsching, Germany).

Hematology

On the day of necropsy, the day after the end of the administration period, all rats were fasted overnight for 16-21 hr from the previous day. A blood sample was then collected from the abdominal aorta of each animal, under isoflurane anesthesis. Analyzed hematological parameters were as follows: erythrocytes, hemoglobin, hematocrit, mean cell volume (MCV), mean cell hemoglobin (MCH), mean cell hemoglobin concentration (MCHC), reticulocytes, platelets, leukocytes, differential leukocyte count, prothrombin time (PT), activated partial thromboplastin time (APTT), and fibrinogen level (FIB).

Blood biochemistry

Blood samples (approximately 4 mL) were collected into heparinized test tubes, and centrifuged twice to obtain plasma (1,690 xg, 10 min, 4°C). The plasma was used for the analysis of: aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), γ-GTP, alkaline phosphatase (ALP), total cholesterol, triglycerides, phospholipids, total bilirubin, glucose, blood urea nitrogen (BUN), creatinine, Na⁺, K⁺, Cl^-, Ca²⁺, inorganic phosphorous (IP), total protein, A/G ratio, protein fractions.

Urinalysis

In week 13 (days 86 and 87), all rats were placed in cages equipped with a urine collector, and urine samples were collected for 4 hr under conditions of fasting and free access to water, and then 20 hr under conditions of free access to diet and water. The collected urine samples were examined to analyze: pH, protein, ketone body, glucose occult blood, bilirubin, urobilinogen, specific gravity, color tone, sediment (urine collected for 4 hr); urinary volume, Na⁺, K⁺ and Cl^- levels (urine collected for 24 hr).

Necropsy and organ weight

All animals were subjected to euthanasia by cutting the abdominal aorta after collecting blood samples under isoflurane anesthesia. Detailed necropsy was performed by macroscopic observation of the systemic organs and tissues, including external body surface, head, chest and abdomen. The absolute weights of the following organs were measured: brain, pituitary, thyroid, salivary gland, thymus, heart, lung, liver, spleen, kidney, adrenal gland, testis, prostate, seminal vesicle, ovary, and uterus. For paired organs, the weight of the right and left organs was measured separately and summed thereafter.

Histopathology

The following organs and tissues were collected for histopathology: cerebrum, cerebellum, thoracic spinal cord, sciatic nerve, ocular bulbs, optic nerve, Harderian gland, hypothalamus, thyroid gland, parathyroid gland, adrenal gland, thymus, spleen, submandibular lymph node, mesenteric lymph node, heart, thoracic aorta, trachea, lung (including bronchus), tongue, esophagus, stomach, duodenum, jejunum, ileum (including Peyer’s patch), cecum, colon, rectum, submandibular gland, sublingual gland, liver, pancreas, kidney, bladder, testis, ovary, epididymis, uterus, prostate gland, vagina, seminal vesicle, oviduct, mammary gland (groin), sternum (including bone marrow), thigh bone (including bone marrow), skeletal muscle of the thigh, skin (groin), and nasal cavity and Zimbal’s gland. Tissues and organs were fixed in 10% phosphate-buffered formalin solution with the following exceptions: the ocular bulbs and optic nerve were fixed with 3% glutaraldehyde / 2.5% formalin mixture; testis and epididymis were fixed with Bouin’s solution. These organs and tissues were then preserved in 10% phosphate-buffered formalin solution. All fixed tissue and organs were embedded in paraffin, and sections were stained with hematoxylin-eosin. Only sections prepared from rats in the control and high dose (5.0%) groups were microscopically examined. Only one organ from a pair was microscopically examined. The parathyroid glands from both sides were mounted with the corresponding thyroid glands and specimens from one side only were examined because there was a possibility that the parathyroid glands alone might be too small to prepare the specimens.

Statistical analysis

Mean and S.D. of the values in each group were calculated. The frequencies of MNPCEs among the negative control and test substance groups were statistically evaluated by Kastenbaum-Bowman test (Kastenbaum and Bowman, 1970) and Cochran-Armitage trend test (Yoshimura, 1987). Data variance within the PCE, urinalysis, hematological and blood biochemical examinations, and organ weight measurements were examined using Bartlett’s test (significance level: 1%) (Snedecor and Cochran, 1989). The differences in mean values among the control and test substance groups were analyzed using the following methods: Dunnett’s test when the variance was equal (Dunnett, 1955, 1964); Steel’s test when the variance was unequal (Steel, 1959) among the groups (two-tailed test). SAS Release 9.1.3 (SAS Institute Inc., Cary, NC, USA) was used for the analysis. In each of the statistical tests, P < 0.05 was considered as statistically significant.

RESULTS AND DISCUSSION

Genotoxicity test

To examine the mutagenicity and genotoxicity potential of MHE, we conducted an Ames test, in vitro chromosomal aberration test, and in vivo micronucleus test in accordance with previous reports.

To verify the mutagenic potential of MHE, a reverse mutation test was carried out using S. typhimurium TA100, TA1535, TA98 and TA1537, and E. coli WP2uvrA in conditions with and without metabolic activation. Precipitation and coloration due to the test substance were not found at any dose under the test conditions either with or without metabolic activation. The number of revertant colonies was counted: no increase was observed under both conditions (Table 1). The number of revertant colonies in the test samples was less than two-fold higher than that in the negative control. Dose dependency was not observed. In addition, the test substance did not cause growth inhibition to any of the bacterial strains at any dose, with or without metabolic activation (data not shown). Thus, MHE has no potential to reverse mutation in the bacteria.

Table 1. Effect of matured hop extract on revertant colonies in the Ames test.

S9 mix	Test substance	Dose (μg/plate)	Number of revertants (number of colonies/plate)
			Base pair substitution type										Frameshift type
			TA100			TA1535			WP2uvrA				TA98			TA1537
-	MHE	0a	100	±	8	7	±	2	19	±	4		19	±	3	7	±	2
		313	108	±	15	10	±	2	14	±	4		22	±	5	7	±	3
		625	92	±	4	10	±	4	17	±	3		21	±	3	9	±	4
		1,250	116	±	6	11	±	2	14	±	4		19	±	6	12	±	3
		2,500	112	±	15	8	±	6	16	±	4		20	±	6	10	±	2
		5,000	133	±	9	9	±	2	14	±	2		24	±	3	8	±	2
	AF-2	0.01	569	±	22		-		79	±	5			-			-
		0.1		-			-			-			432	±	42		-
	SAZ	0.5		-		263	±	3		-				-			-
	ICR-191	1.0		-			-			-				-		1,587	±	89
+	MHE	0a	138	±	20	11	±	6	19	±	3		36	±	2	10	±	4
		313	116	±	10	10	±	4	23	±	6		32	±	3	10	±	2
		625	132	±	11	12	±	6	21	±	7		34	±	2	14	±	5
		1,250	123	±	13	11	±	4	18	±	2		34	±	5	13	±	2
		2,500	126	±	26	13	±	2	18	±	3		31	±	6	8	±	2
		5,000	159	±	9	12	±	4	17	±	6		34	±	6	12	±	2
	B[α]P	5.0	911	±	43		-			-			338	±	25	92	±	11
	2AA	2.0		-		390	±	23		-				-			-
		10.0		-			-		1,047	±	127			-			-

Values are expressed as means ± S.D. of 3 plates. The used strains are as follows: TA100, Salmonella typhimurium TA100; TA1535, S. typhimurium TA1535; WP2uvrA, Escherichia coli WP2uvrA; TA98, S. typhimurium TA98; TA1537, S. typhimurium TA1537. MHE, matured hop extract. Positive controls: AF-2, 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide; SAZ, sodium azide; ICR-191, 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine HCl; 2AA, 2-aminoanthracene; B[α]P, benzo[α]pyrene.

^a Negative control (sterilized water)

The in vitro chromosomal aberration test was carried out using CHL/IU cells to evaluate whether MHE induces chromosomal aberrations. Initially, a cell growth inhibition test with and without metabolic activation was performed to determine the dose required for the chromosomal aberration test. Since cytotoxicity in more than 50% of cells was observed (data not shown), the following doses were selected for the chromosomal aberration test: 5,000, 2,500, 1,250, and 625 μg/mL with S9 mix; 5,000, 3,330, 2,220, and 1,480 μg/mL without S9 mix. In the short-term treatment, the structural chromosomal aberration ratio was 6% at 5,000 μg/mL with metabolic activation, indicating false positive (Table 2). Without metabolic activation, the structural chromosomal aberration ratio was 10.5% at 3,330 μg/mL and 20.5% at 5,000 μg/mL, indicating positives (Table 2). Precipitation occurred at all doses of MHE in the absence or presence of S9 mix. Chromosome preparations in the continuous treatment were not examined because the results of the short-term treatment were positive. In contrast, the frequency of polyploids, an index of numerical chromosomal aberration, was lower than 5% at all doses with and without metabolic activation, indicating no numerical chromosomal aberration (Table 2). Overall, these results suggest that MHE has a potential for clastogenic potential.

Table 2. Chromosome aberration in cultured Chinese hamster lung fibroblasts (CHL/IU) treated with matured hop extract.

Time (hr)	S9 mix	Test substance	Dose (μg/mL)	Cells observedb	Cell growth rate (%)c	Structural aberration (%)d	Judgemente		Polyploid cells (%)	Judgemente
6-18	-	MHE	0a	200	100	1.0	-		0	-
			1,480	200f	113	0.5	-		0.5	-
			2,220	200f	100	1.5	-		0	-
			3,330	200f	78	10.5	+		0.5	-
			5,000	200f	24	20.5	+		0.5	-
		MMC	0.075	200	-	36.0	+		1.0	-
	+	MHE	0a	200	100	2.0	-		0.5	-
			625	200f	96	0	-		0	-
			1,250	200f	89	0.5	-		1.5	-
			2,500	200f	80	1.5	-		0.5	-
			5,000	200f	50	6.0	±		2.0	-
		CP	14	200	-	43.0	+		0	-

MHE, matured hop extract. Positive controls: CP, cyclophosphamide; MMC, mitomycin C.

^a Negative control (sterilized water)

^b Combination of data from two slides (100 cells/slide).

^c Cell growth rate (%): cell counts from test substance treatment group against the negative control.

^d Total number of cells with aberration including gap

^e < 5%, - (negative); 5-10%, ± (false positive); ≧10%, + (positive)

^f Precipitations were observed.

The results of the in vivo micronucleus test in rat bone marrow are shown in Table 3. The frequencies of MNPCEs in PCEs were not changed in the test substance groups whereas there was marked elevation in the positive control group (Table 3). There was no significant difference in the test substance groups compared to the negative control group. The frequencies of PCEs in total erythrocytes in the test substance and positive control groups were not significantly changed compared to the negative control group (Table 3). There was no dose dependency in the frequencies of MNPCEs and PCEs in the test substance groups. Moreover, the frequencies of MNPCEs in the negative and positive control group were within the normal range of physiological background data. From the results mentioned above, it is judged that MHE does not have clastogenic potential in rats at the maximum dose, which is recommended by OECD guidelines (OECD, 1997).

Table 3. In vivo micronucleus test of matured hop extract in rats.

Group	Test substance	Dose (mg/kg body weight/day)	MNPCE / PCE							PCE / Total erythrocyte
			(%)c			(Min / Max)				(%)d			(Min / Max)
Negative control	MHE	0b	0.09	±	0.04	(0.05	/	0.15)		64.5	±	5.9	(57.0	/	72.5)
Low		500	0.14	±	0.04	(0.10	/	0.20)		65.1	±	7.2	(54.5	/	74.5)
Middle		1,000	0.11	±	0.07	(0.00	/	0.15)		58.7	±	2.9	(54.0	/	61.5)
High		2,000	0.11	±	0.07	(0.05	/	0.20)		64.3	±	4.9	(58.0	/	71.0)
Positive controla	MMC	2	1.90	±	0.54	(1.35	/	2.70)		50.4	±	5.2	(46.5	/	59.5)

Male SD rats were used (n = 5/group). Bone marrow smears were observed about 24 hr after the 2nd administration. Data are expressed as means ± S.D. No significant difference was observed among the groups. MHE, matured hop extract; MMC, mitomycin C; MNPCE, micronucleated polychromatic erythrocyte; PCE, polychromatic erythrocyte.

^a Single administration only for the positive control group.

^b Sterilized water was administered.

^c Proportion (%) of MNPCE per 2,000 PCEs.

^d Proportion (%) of PCE, including MNPCE, per 200 erythrocytes.

In summary, MHE has slight potential to induce structural chromosomal aberration under in vitro conditions; however, the present study suggests there is no genotoxic concern under the in vivo conditions.

Acute and subchronic toxicity test

We conducted single and repeated oral administrations of MHE to male and female SD rats to investigate acute and subchronic toxicities.

Clinical signs and body weights were observed for 14 days after a single administration, and all rats were autopsied. No adverse clinical signs or effects, including death were observed after administration of MHE at a dose of 2,000 mg/kg body weight during the observation period. Body weight gain was not affected by MHE administration and no abnormal changes in any animal were observed at autopsy. There were no noticeable toxic signs caused by MHE at 2,000 mg/kg body weight, indicating that an acute toxic dose of MHE would be higher than 2,000 mg/kg body weight in rats.

Successive administration of MHE was performed for 90 days and clinical observations, ophthalmoscopy, body weights, feed and water intakes were recorded. In addition, urine, blood, organ and tissue samples were collected and analyzed to evaluate the safety. The intake of test substance was calculated based on feed consumption. The mean amounts of MHE intake from the mixed diet containing 1.25, 2.5 and 5.0% of MHE were 871/998, 1,706/1,936 and 3,484/4,022 mg/kg body weight/day (for males/females), respectively.

No adverse clinical signs or effects, including death were observed after successive MHE administration. Throughout the test period, body weight gain, and water and feed consumption were unaffected by MHE intake. Whereas MHE is effective on reduction in body fat in overweight or obese individuals (Morimoto-Kobayashi et al., 2016) and diet-induced obese mice (Morimoto-Kobayashi et al., 2015), the final body weight was not affected in this study using normal rats. No abnormalities in the ophthalmoscopy observations were observed in any animal. There was no significant change in any of the urinalysis measurements, except for potassium excretion and urinary protein: potassium excretion was significantly higher in male and female rats in the high (5.0%) dose group compared to the corresponding control groups (Table 4). Urinary protein analysis showed that 1/10 male rats in the middle (2.5%) dose group and 3/10 male rats in the high (5.0%) dose group had ≧ 100 mg/dL protein present in their urine (Table 5). These values were within the normal range of physiological background data from oral administration studies using rats of the same age. Analysis of blood biochemistry, renal function parameters, BUN and creatinine clearance showed no significant change (Table 7). In addition, no abnormal histopathological findings in the kidneys were revealed. Because of these data, the change in potassium excretion and urinary protein levels is considered to be physiological fluctuation.

Table 4. Urinary tests in rats after 90 days subchronic oral administration of matured hop extract.

	Male													Female
	Control			1.25% MHE			2.5% MHE			5% MHE				Control			1.25% MHE			2.5% MHE			5% MHE
Urine volume (mL/24 hr)	15.7	±	6.3	14.7	±	11.7	13.4	±	3.7	18.1	±	5.8		8.3	±	3.9	11.5	±	5.1	12.4	±	9.7	13.4	±	7.0
Na+ (mmol/24 hr)	1.8	±	0.4	1.6	±	0.6	1.6	±	0.5	1.7	±	0.5		0.9	±	0.3	1.2	±	0.3	1.0	±	0.4	1.0	±	0.3
K+ (mmol/24 hr)	4.3	±	0.9	3.7	±	1.3	4.6	±	1.1	5.7	±	1.1*		2.2	±	1.0	2.9	±	0.7	2.6	±	0.7	3.2	±	1.0*
Cl- (mmol/24 hr)	2.5	±	0.5	2.2	±	0.9	2.5	±	0.6	2.7	±	0.6		1.3	±	0.5	1.7	±	0.4	1.4	±	0.4	1.5	±	0.5

Male and female SD rats were used (n = 10/group). Data are expressed as mean ± S.D. MHE, matured hop extract.

* P < 0.05 using Dunnett's test (two-side) and compared to the control.

Table 5. Urinary protein in rats after 90 days subchronic oral administration of matured hop extract.

	Male					Female
	Control	1.25% MHE	2.5% MHE	5% MHE		Control	1.25% MHE	2.5% MHE	5% MHE
Urinary proteina
- or ±	8	5	6	5		8	10	9	10
1+	2	5	3	2		2	0	1	0
2+	0	0	1	3		0	0	0	0

Male and female SD rats were used (n=10/group). Data are expressed as numbers of rat. MHE, matured hop extract.

^a -, negative; ±, ≧ 15 mg/dL; 1+, ≧ 30 mg/dL; 2+, ≧ 100 mg/dL.

Table 6. Hematological tests in rats after 90 days subchronic oral administration of matured hop extract.

	Male													Female
	Control			1.25% MHE			2.5% MHE			5% MHE				Control			1.25% MHE			2.5% MHE			5% MHE
Erythrocytes (104/µL)	908	±	48	887	±	29	932	±	40	914	±	41		817	±	24	803	±	27	803	±	35	797	±	34
Hemoglobin (g/dL)	16.1	±	0.4	15.9	±	0.5	16.1	±	0.5	16.2	±	0.5		15.3	±	0.4	15.3	±	0.4	15.3	±	0.7	15.1	±	0.5
Hematocrit (%)	44.7	±	1.5	43.9	±	1.3	44.8	±	1.7	45.0	±	1.8		41.7	±	1.2	41.5	±	1.0	41.3	±	2.0	40.8	±	1.5
MCV (fL)	49.3	±	1.6	49.6	±	1.4	48.1	±	1.0	49.3	±	1.5		51.0	±	1.0	51.7	±	2.0	51.4	±	1.0	51.2	±	1.7
MCH (pg)	17.8	±	0.6	17.9	±	0.4	17.3	±	0.5	17.7	±	0.4		18.8	±	0.4	19.1	±	0.6	19.1	±	0.5	18.9	±	0.5
MCHC (g/dL)	36.0	±	0.4	36.1	±	0.5	36.0	±	0.8	35.9	±	0.6		36.8	±	0.5	36.9	±	0.5	37.1	±	0.5	36.9	±	0.6
Reticulocyte (%)	2.0	±	0.4	2.1	±	0.3	1.8	±	0.3	1.9	±	0.2		1.8	±	0.2	1.8	±	0.3	1.7	±	0.4	1.7	±	0.3
Platelet (104/µL)	114.3	±	9.6	121.4	±	15.9	106.3	±	10.4	109.5	±	10.2		119.8	±	11.2	113.3	±	12.1	120.0	±	12.9	127.7	±	16.0
Leukocytes (102/µL)	87.1	±	21.8	96.4	±	20.0	93.3	±	12.2	77.7	±	13.3		55.3	±	15.1	50.3	±	14.9	46.5	±	7.8	55.3	±	14.0
Differential leukocyte count
Lymphocytes (102/µL)	67.3	±	20.0	70.5	±	13.7	66.8	±	8.8	53.4	±	9.7		39.6	±	13.4	36.6	±	12.2	34.9	±	6.1	40.9	±	11.0
Neutrophils (102/µL)	14.9	±	2.9	20.4	±	12.0	21.7	±	9.1	20.5	±	6.4a		12.3	±	4.9	10.4	±	3.7	9.1	±	4.1	11.5	±	5.6
Eosinophils (102/µL)	1.6	±	0.6	1.5	±	0.5	1.4	±	0.4	1.2	±	0.3		1.0	±	0.3	1.0	±	0.4	0.8	±	0.2	0.8	±	0.2
Basophils (102/µL)	0.3	±	0.2	0.2	±	0.1	0.3	±	0.1	0.2	±	0.0		0.1	±	0.1	0.1	±	0.1	0.1	±	0.0	0.1	±	0.0
Monocytes (102/µL)	2.3	±	0.8	2.6	±	1.3	2.3	±	0.7	1.9	±	0.6		1.7	±	0.7	1.8	±	1.0	1.2	±	0.4	1.5	±	0.4
LUC (102/µL)	0.8	±	0.3	1.1	±	0.7	0.9	±	0.5	0.5	±	0.2		0.6	±	0.4	0.5	±	0.3	0.4	±	0.1	0.5	±	0.3
PT (sec)	13.0	±	1.1	13.2	±	1.3	13.1	±	1.1	12.9	±	0.9		11.9	±	0.6	11.3	±	0.4b	11.5	±	0.6	11.3	±	0.4b
APTT (sec)	16.7	±	1.2	16.9	±	2.0	17.2	±	1.9	16.5	±	1.7		14.0	±	0.9	13.3	±	1.4	13.5	±	1.0	13.6	±	0.6
FIB (mg/dL)	291	±	34	299	±	29	283	±	18	282	±	28		176	±	10	184	±	17	185	±	20	189	±	18

Male and female SD rats were used (n = 10/group). Data are expressed as mean ± S.D. MHE, matured hop extract; MCV, mean cell volume; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; APTT, activated partial thromboplastin time; PT, prothrombin time; LUC, large unstained cells.

^a P < 0.05 using Steel test (two-side) and compared to the control.

^b P < 0.05 using Dunnett's test (two-side) and compared to the control.

Table 7. Blood biochemistry tests in rats after 90 days subchronic oral administration of matured hop extract.

	Male													Female
	Control			1.25% MHE			2.5% MHE			5% MHE				Control			1.25% MHE			2.5% MHE			5% MHE
AST (IU/L)	71	±	24	57	±	5	60	±	8	60	±	6		57	±	6	62	±	15	57	±	10	57	±	8
ALT (IU/L)	35	±	17	30	±	4	29	±	3	30	±	5		26	±	4	31	±	7	28	±	6	27	±	5
LDH (IU/L)	45	±	17	35	±	11	31	±	5**	34	±	5		39	±	12	42	±	15	43	±	20	41	±	12
ALP (IU/L)	314	±	72	302	±	48	323	±	67	315	±	27		130	±	33	152	±	55	141	±	34	151	±	55
γ-GTP (IU/L)	1	±	0	1	±	0	1	±	1	1	±	0		1	±	0	1	±	0	1	±	1	1	±	1
Total cholesterol (mg/dL)	80	±	18	78	±	13	67	±	11	71	±	16		76	±	12	83	±	10	91	±	26	84	±	20
Triglycerides (mg/dL)	57	±	21	54	±	23	49	±	21	48	±	18		21	±	15	22	±	7	25	±	16	21	±	14
Phospholipids (mg/dL)	114	±	19	113	±	16	101	±	13	102	±	16		137	±	18	149	±	14	154	±	45	139	±	32
Total bilirubin (mg/dL)	0.1	±	0.0	0.1	±	0.0	0.1	±	0.0	0.1	±	0.0		0.1	±	0.0	0.1	±	0.0	0.1	±	0.0	0.1	±	0.0
Glucose (mg/dL)	121	±	12	121	±	10	118	±	7	120	±	11		129	±	17	132	±	13	127	±	13	124	±	12
BUN (mg/dL)	13	±	1	13	±	1	14	±	2	13	±	1		15	±	2	16	±	2	16	±	2	16	±	1
Creatinine (mg/dL)	0.22	±	0.02	0.23	±	0.04	0.24	±	0.04	0.24	±	0.04		0.27	±	0.02	0.28	±	0.03	0.27	±	0.04	0.28	±	0.04
Na+ (mmol/L)	146	±	1	146	±	1	146	±	1	145	±	1		142	±	1	143	±	1	143	±	1	143	±	2
K+ (mmol/L)	3.5	±	0.1	3.5	±	0.2	3.5	±	0.2	3.4	±	0.2		3.4	±	0.1	3.3	±	0.2	3.4	±	0.3	3.3	±	0.1
Cl- (mmol/L)	107	±	1	107	±	1	107	±	1	106	±	1		108	±	1	107	±	2	107	±	2	107	±	2
Ca2+ (mg/dL)	10.4	±	0.4	10.2	±	0.3	10.2	±	0.2	10.2	±	0.2		10.2	±	0.3	10.3	±	0.2	10.3	±	0.4	10.4	±	0.3
IP (mg/dL)	5.6	±	0.7	5.4	±	0.3	5.5	±	0.6	5.4	±	0.5		4.5	±	0.7	4.6	±	0.6	4.2	±	1.2	4.6	±	0.6
Total protein (g/dL)	6.5	±	0.3	6.4	±	0.3	6.4	±	0.2	6.4	±	0.3		6.6	±	0.2	6.8	±	0.2	6.7	±	0.5	6.7	±	0.3
A/G ratio	0.8	±	0.1	0.9	±	0.1	0.8	±	0.1	0.8	±	0.1		1.2	±	0.1	1.2	±	0.1	1.3	±	0.2	1.2	±	0.2
ALB-E (g/dL)	2.9	±	0.1	3.0	±	0.2	2.9	±	0.2	2.9	±	0.1		3.6	±	0.2	3.7	±	0.2	3.8	±	0.5	3.6	±	0.4
α1-Globulin (g/dL)	1.3	±	0.2	1.2	±	0.2	1.2	±	0.2	1.2	±	0.1		0.9	±	0.1	0.9	±	0.1	0.9	±	0.2	0.9	±	0.1
α2-Globulin (g/dL)	0.6	±	0.1	0.6	±	0.1	0.7	±	0.1	0.7	±	0.1		0.6	±	0.1	0.6	±	0.1	0.6	±	0.1	0.7	±	0.1
β-Globulin (g/dL)	1.2	±	0.1	1.2	±	0.1	1.2	±	0.1	1.2	±	0.1		1.1	±	0.1	1.1	±	0.1	1.1	±	0.1	1.1	±	0.1
γ-Globulin (g/dL)	0.4	±	0.1	0.4	±	0.2	0.5	±	0.1	0.4	±	0.1		0.4	±	0.1	0.5	±	0.1	0.4	±	0.1	0.5	±	0.1

Male and female SD rats were used (n=10/group). Data are expressed as mean ± S.D. MHE, matured hop extract; AST, aspartate aminotransferase; ALT, alanine aminotransferase; ALP, alkaline phosphatase; LDH, lactate dehydrogenase; BUN, blood urea nitrogen; IP, inorganic phosphorous.

** P < 0.01 using Steel test (two-side) and compared to the control.

Hematological tests showed that the number of neutrophils were significantly higher in males in the high (5.0%) dose group compared with the control group (Table 6). PT was significantly shorter in females in the low (1.25%) and high (5%) dose groups compared with the control group (Table 6). Blood biochemistry analysis showed LDH activity was significantly lower in male rats in the middle (2.5%) dose group (Table 7). These changes were considered to be within the normal range of physiological background.

Analysis of relative organ weights showed that the following organs were significantly heavier: thyroid gland (males in the 2.5% and 5.0% groups and females in the 5.0% group), salivary gland (females in the 5.0% group), heart (females in the 5.0% group), and liver (females in the 2.5% and the 5.0% groups) (Table 8). However, there were no significant changes in the absolute organ weights (Table 8) and no abnormal histopathological findings were observed in any of the analyzed organs, suggesting these changes were not toxicologically significant.

Table 8. Absolute and relative organ weights in rats after 90 days subchronic oral administration of matured hop extract.

	Male													Female
	Control			1.25% MHE			2.5% MHE			5% MHE				Control			1.25% MHE			2.5% MHE			5% MHE
Absolute organ weights
Final body weight (g)	516	±	34	488	±	49	481	±	43	500	±	48		297	±	20	293	±	25	280	±	30	275	±	29
Brain (g)	2.18	±	0.06	2.12	±	0.07	2.12	±	0.09	2.16	±	0.10		1.99	±	0.11	1.97	±	0.09	1.95	±	0.05	1.97	±	0.09
Pituitary (mg)	12.8	±	1.0	12.0	±	1.3	12.0	±	1.7	12.0	±	2.0		15.5	±	1.5	15.2	±	1.8	15.4	±	1.7	13.9	±	1.9
Thyroid (mg)	24.8	±	4.0	24.4	±	3.1	27.4	±	2.9	27.8	±	4.4		17.5	±	2.2	17.5	±	2.2	17.3	±	2.8	18.9	±	1.9
Salivary gland (mg)	828	±	76	783	±	81	800	±	106	775	±	101		493	±	45	503	±	49	484	±	38	520	±	62
Thymus (mg)	292	±	43	278	±	73	308	±	69	255	±	57		254	±	59	275	±	68	262	±	77	258	±	40
Heart (g)	1.46	±	0.08	1.39	±	0.14	1.43	±	0.11	1.42	±	0.14		0.95	±	0.06	0.94	±	0.06	0.93	±	0.09	0.95	±	0.06
Lung (g)	1.52	±	0.14	1.49	±	0.08	1.43	±	0.13	1.50	±	0.08		1.14	±	0.08	1.12	±	0.09	1.08	±	0.09	1.09	±	0.10
Liver (g)	12.75	±	1.07	11.96	±	1.41	11.69	±	1.19	12.92	±	1.23		6.90	±	0.39	7.02	±	0.56	7.01	±	0.84	7.20	±	0.89
Spleen (g)	0.81	±	0.10	0.72	±	0.08	0.74	±	0.09	0.73	±	0.09		0.50	±	0.06	0.47	±	0.06	0.49	±	0.08	0.52	±	0.16
Kidney (g)	3.18	±	0.20	2.96	±	0.34	2.94	±	0.28	3.19	±	0.27		1.88	±	0.13	1.84	±	0.11	1.85	±	0.20	1.84	±	0.16
Adrenal (mg)	56	±	9	55	±	11	54	±	6	54	±	7		63	±	8	62	±	6	63	±	8	65	±	9
Testis (g)	3.36	±	0.19	3.38	±	0.23	3.44	±	0.24	3.49	±	0.21		-			-			-			-
Prostate (g)	1.21	±	0.20	1.13	±	0.15	1.17	±	0.21	1.19	±	0.17		-			-			-			-
Seminal vesicle (g)	1.38	±	0.11	1.20	±	0.31	1.35	±	0.23	1.30	±	0.11		-			-			-			-
Ovary (mg)	-			-			-			-				83.9	±	12.9	78.4	±	8.0	71.6	±	19.4	80.1	±	18.1
Uterus (mg)	-			-			-			-				590	±	150	563	±	126	521	±	78	497	±	102
Relative organ weights
Final body weight (g)	516	±	34	488	±	49	481	±	43	500	±	48		297	±	20	293	±	25	280	±	30	275	±	29
Brain (g/100 g)	0.42	±	0.03	0.44	±	0.04	0.44	±	0.03	0.43	±	0.04		0.68	±	0.07	0.68	±	0.04	0.71	±	0.07	0.72	±	0.10
Pituitary (mg/100 g)	2.5	±	0.2	2.5	±	0.3	2.5	±	0.4	2.4	±	0.4		5.2	±	0.4	5.2	±	0.7	5.5	±	0.6	5.1	±	0.6
Thyroid (mg/100 g)	4.8	±	0.7	5.0	±	0.6	5.7	±	0.7*	5.6	±	0.8*		5.9	±	0.7	6.0	±	0.9	6.2	±	0.6	6.9	±	1.0*
Salivary gland (mg/100 g)	161	±	19	161	±	15	166	±	13	155	±	17		167	±	18	173	±	17	174	±	17	190	±	19*
Thymus (mg/100 g)	57	±	9	57	±	13	64	±	16	51	±	8		85	±	17	94	±	25	94	±	24	94	±	16
Heart (g/100 g)	0.29	±	0.02	0.29	±	0.03	0.30	±	0.03	0.29	±	0.02		0.32	±	0.01	0.32	±	0.02	0.34	±	0.02	0.35	±	0.03*
Lung (g/100 g)	0.30	±	0.02	0.31	±	0.02	0.30	±	0.02	0.30	±	0.03		0.38	±	0.03	0.39	±	0.03	0.39	±	0.02	0.40	±	0.02
Liver (g/100 g)	2.47	±	0.15	2.45	±	0.12	2.43	±	0.09	2.59	±	0.14		2.33	±	0.08	2.40	±	0.12	2.51	±	0.17**	2.61	±	0.10**
Spleen (g/100 g)	0.16	±	0.02	0.15	±	0.02	0.15	±	0.01	0.15	±	0.02		0.17	±	0.02	0.16	±	0.02	0.18	±	0.03	0.19	±	0.04
Kidney (g/100 g)	0.62	±	0.04	0.61	±	0.05	0.61	±	0.05	0.64	±	0.07		0.64	±	0.04	0.63	±	0.03	0.66	±	0.03	0.67	±	0.05
Adrenal (mg/100 g)	11	±	2	11	±	2	11	±	1	11	±	2		21	±	2	21	±	3	23	±	3	24	±	4
Testis (g/100 g)	0.65	±	0.05	0.70	±	0.07	0.72	±	0.07	0.70	±	0.07		-			-			-			-
Prostate (g/100 g)	0.24	±	0.04	0.23	±	0.03	0.24	±	0.04	0.24	±	0.04		-			-			-			-
Seminal vesicle (g/100 g)	0.27	±	0.03	0.25	±	0.06	0.28	±	0.03	0.26	±	0.03		-			-			-			-
Ovary (mg/100 g)	-			-			-			-				28.3	±	4.1	26.9	±	3.2	25.7	±	6.3	29.2	±	5.6
Uterus (mg/100 g)	-			-			-			-				198	±	44	196	±	58	188	±	33	182	±	37

Male and female SD rats were used (n=10/group). Data are expressed as mean ± S.D. MHE, matured hop extract.

* P < 0.05, ** P < 0.01 using Dunnet's test (two-side) and compared to the control.

Based on the results of the subchronic safety studies, the no-observed-adverse-effect-level (NOAEL) of MHE is considered to be over 3,484 mg/kg body weight/day for male and 4,022 mg/kg body weight/day for female rats under the study conditions.

In conclusion, although the structural chromosomal aberration was positive in the high dose group, the Ames and in vivo micronucleus tests suggests MHE has no potential for mutagenicity and genotoxicity under the in vivo conditions. The acute toxicity level of MHE is higher than 2,000 mg/kg body weight. NOAEL of MHE in the subchronic oral safety was considered to be over 3,484 and 4,022 mg/kg body weight/day for male and female rats, respectively. These results indicate that there is no safety concern for MHE in the present preclinical study.

ACKNOWLEDGMENTS

We appreciate the valuable experimental data from BoZo Research Center Inc. We thank our group members of Kirin Co., Ltd. for their valuable discussions.

Conflict of interest

SS, YMK, CT, YT and MK are employees of Kirin Co., Ltd., the study sponsor. There are no other conflicts of interest to be declared.

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