Abstract
Ubiquinol (the reduced form of coenzyme Q10) is the two-electron reduction product of ubiquinone (the oxidized form of coenzyme Q10), and has been shown to be an integral part of living cells, where it functions as an antioxidant in both mitochondria and lipid membranes. To provide information to enable a Generally Regarded as Safe (GRAS) evaluation for the use of ubiquinol in selected foods, a series of Organisation of Economic Cooperation and Development (OECD) and good laboratory practice (GLP) toxicological studies was conducted to evaluate the mutagenic and genotoxic potential of Kaneka QH brand of ubiquinol. Ubiquinol did not induce reverse mutations in Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2uvrA at concentrations up to 5000 μg/plate, in either the absence and presence of exogenous metabolic activation by rat liver S9. Likewise, ubiquinol did not induce chromosome aberrations in Chinese hamster lung fibroblast (CHL/IU) cells in short-term (6-h) tests with or without rat liver S9 at concentrations up to 5000 μg/ml or in a continuous (24-h) treatment test at concentrations up to 1201 μg/ml. Finally, no mortalities, no abnormal clinical signs, and no significant increase in chromosome damage were observed in an in vivo micronucleus test when administered orally at doses up to 2000 mg/kg/day. Thus, ubiquinol was evaluated as negative in the bacterial reverse mutation, chromosomal aberration, and rat bone marrow micronucleus tests under the conditions of these assays.
The term “coenzyme Q” (CoQ) refers to a class of homologous benzoquinones that have been identified in all plants and animals, as well as in a majority of microorganisms (Budavari et al. 1996; Nohl, Gille, and Staniek 1998). Benzoquinone homologs consist of a redox active quinoid moiety, and a hydrophobic side chain comprising 6 to 10 isoprenoid units, depending on the species (Ibrahim et al. 2000; Matthews et al. 1998; Lenaz 2001). In humans and most mammals, including dogs, the predominant form of CoQ is coenzyme Q10 (CoQ10), also referred to as ubiquinone, which consists of 10 isoprenoid units in the side chain (Ramasarma 1985). In rats and mice, the primary form is coenzyme Q9, which contains nine isoprenoid units. However, low levels of CoQ10 have also been reported in rats and mice (Battino et al. 1992).
CoQ10 is located in the hydrophobic interior of nearly every cellular membrane, and to varying degrees in all tissues (Nohl, Gille, and Staniek 1998; Lass and Sohal 1999).Since its discovery in 1957 by Crane and colleagues (Bertelli and Ronca 1990), CoQ10 has been extensively studied for its key role in mitochondrial energy production, where it acts as both an electron carrier and proton translocator during cellular respiration and adenosine triphosphate (ATP) production (Nohl, Gille, and Staniek 1998; Hughes et al. 2002; Nohl et al. 2001).
Ubiquinol (the reduced form of coenzyme Q10) is the two-electron reduction product of ubiquinone (the oxidized form of coenzyme Q10) (Frei, Kim, and Ames 1990; Schoepp 1997; Pepping 1999). The conversion between ubiquinol and ubiquinone is shown in Figure 1.
Like ubiquinone, ubiquinol has been shown to be an integral part of virtually all living cells, where it has been shown to be a potent lipophilic antioxidant for the protection of lipids in both mitochondria and lipid membranes in a number of biological and model systems (Frei, Kim, and Ames 1990; Forsmark-Andree et al. 1997; Noack, Kube, and Augustin 1994; Ernster and Forsmark-Andree 1993).
Ubiquinol is the most common form of CoQ10 in vivo. The reduced form represents more than 80% of the total ubiquinol + ubiquinone pool in human plasma, intestine, and liver (Edlund 1988; Okamoto et al. 1989; Åberg et al. 1992). In the plasma of healthy adults, ubiquinol accounts for approximately 95% of the total concentration of CoQ10, whereas ubiquinone accounts for only 5% (Yamashita and Yamamoto 1997); in human urine, ubiquinol accounts for approximately 59% of the total CoQ10 concentration (Okamoto et al. 1989). High levels of reduction (70% to 100%) were also observed in human tissues, including the liver, pancreas, and intestine (Åberg et al. 1992). Only in the brain and lung, most of the CoQ10 (approximately 80%) was in the oxidized state. In contrast, the degree of ubiquinone reduction in all rat tissues was less than in corresponding human tissues.
Furthermore, Hosoe et al. (2007) reported that after 28 days intake of ubiquinol as a dietary supplement by healthy volunteers, daily dose of 90, 150, and 300 mg attained plasma ubiquinol concentrations of 2.84, 3.84, and 7.28 μg/ml, respectively, whereas the base line plasma ubiquinol levels prior to each intake ranged from 0.57 to 0.66 μg/ml.
Ubiquinol is the only known lipid-soluble antioxidant that animals can synthesize de novo, and for which there exist mechanisms that can regenerate it from its oxidized product formed as a result of its antioxidant activity (Ernster and Dallner 1995). Although ubiquinol becomes oxidized as a result of its antioxidant function, a substantial amount is maintained in its reduced state in the plasma membrane and endomembranes (Takahashi et al. 1993), as well as in lipoproteins (Stocker and Frei 1991).
It has been suggested that reduction of ubiquinone to ubiquinol by the liver plays an important role in maintaining its level in plasma (Stocker and Suarna 1993). Various electron transfer systems recycle ubiquinol from its oxidized form (Crane, Sun, and Sun 1993). In the plasma membrane, reduction of ubiquinone is achieved through the involvement of several CoQ reductases (e.g., DT-diaphorase and NADPH-CoQ reductase) that may be either integral membrane proteins or cytosolic enzymes (Arroyo et al. 2000). It was also reported that natural ubiquinones are readily reduced after dietary uptake (Stocker and Suarna 1993). Although it is generally accepted that oxidized coenzyme Q10 is the final product of its biosynthetic path-way, some authors have proposed that the de novo synthesis of the hydroquinone also contributes, at least in part, to the high levels of ubiquinol observed in vivo (Stocker and Suarna 1993; Schultz et al. 1996).
In addition to this de novo synthesis and regeneration, ubiquinol and ubiquinone are present in the diet in meats, fish, fruits, and vegetables (Kubo et al. 2007). Because CoQ10 has essential roles in mitochondrial energy production and its reduced form of ubiquinol is a potent lipophilic antioxidant protecting cells from oxidative stress or free radical damage, and evidence for deficiency of CoQ10 based on genetic failure, age, disease, or drug that inhibit biosynthesis of CoQ10 has been found, ubiquinone has been widely used as a dietary supplement by health-conscious individuals and those who with ailments including various cardiac disorders (Crane 2001; Overvad et al. 1999; Greenberg and Frishman 1990; Hendler and Rorvik, 2001; Tran et al. 2001; Jones et al. 2002). In addition to these important biological roles of CoQ10, ubiquinol has higher bioavailability than ubiquinone (Mae et al. 2001). Therefore ubiquinol (Kaneka QH) was initially developed as a dietary supplement superior to ubiquinone. Kaneka QH has now been proposed for addition to foods in order to increase option for ubiquinol intake. Accordingly, as part of the process for attaining “Generally Recognized as Safe” (GRAS) status for Kaneka QH, a standard battery of genotoxicity studies, including rat bone marrow micronucleus, chromosomal aberration, and bacterial reverse mutation tests, was conducted to evaluate its in vivo and in vitro mutagenic and genotoxic potential (FDA 2000). Results regarding ubiquinol, the reduced form of CoQ10, are reported here for the first time although the mutagenic potential of ubiquinone, the oxidized form of CoQ10, has been reported (Kitano et al. 2006).
MATERIALS AND METHODS
Study Organization
Studies were conducted at the Biosafety Research Center, Foods, Drugs and Pesticides (58202, Arahama Shioshinden Fukude-Cho Iwata-Gun, Shizuoka 437-1213 Japan) in accordance with “Guidelines for Genotoxicity Tests on Drugs” (Pharmaceutical and Medical Safety Bureau, Japanese Ministry of Health and Welfare [now Japanese Ministry of Health, Labor and Welfare], Notification Iyakushin No. 1604, November 1, 1999), and under “GLP Standard Ordinance for Nonclinical Laboratory Studies on Safety of Drugs” (Japanese Ministry of Health and Welfare, Ordinance No. 21, March 26, 1997). The Japanese guidelines were in accordance with the Organisation for Economic Cooperation and Development (OECD) guidelines (471, 473, 474) with several minor exceptions. Specifically, in the Ames assay, triplicate plating and statistical analysis for demonstration of repeatability and dose relationships are required by the OECD guideline but not by the Japanese Ministry of Health and Welfare (JMHW). Likewise, in the chromosomal aberration test, confirmation of chromosome count, criteria for establishing the highest dose, and the requirement for a 48-h treatment in cases of negative results in the OECD guidelines are not mandated by the JMHW guidelines. Finally, the use of both sexes is needed in the OECD guidelines on micronucleus test; herein, only males were tested per the JMHW requirement.
Test Article and Chemicals
Ubiquinol (CAS no. 992-78-9) was manufactured by Kaneka Corporation (Kaneka QH; lot no. QH-P01, purity 98.71%). The bacterial mutation test was performed with ubiquinol dissolved in acetone (Kanto Chemical Industries Co., Ltd.; lot no. 307F1754). The S9 fraction (lot no. RAA-469 produced in August 2002, Kikkoman Corporation, protein content 26.86 mg/ml) was prepared from the livers of 7-week-old male Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone. Positive-control substances were as follows: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2; lot no. PAE1151), sodium azide (NaN3; lot no. KSE1776), and 2-aminoanthracene (2-AA; lot no. KSE1917) obtained from Wako Pure Chemical Industries, Ltd., and 9-aminoacridine hydrochloride (lot no. 03024JR) obtained from Aldrich Chemical Co., Inc.
The chromosome aberration test was performed with ubiquinol suspended in 0.5% (w/v) sodium carboxymethyl cellulose solution (CMC; Wako Pure Chemical Industries, Ltd.; lot no. WTH1105). The reason for this is because acetone was not suitable in the chromosome aberration test. The S9 fraction (lot no. RAA-468, produced in August 2002 by Kikkoman Corporation, protein content 27.90 mg/ml) was prepared as previously described. Mitomycin C (MMC; Kyowa Hakko Kogyo Co., Ltd.; lot no. 339AJH) dissolved in sterile water for injection (The Japanese Pharmacopoeia; Otsuka Pharmaceutical Factory, Inc.; lot no. K1D78) was diluted in physiological saline (The Japanese Pharmacopoeia; Otsuka Pharmaceutical Factory, Inc.; lot no. K1D74). It was subdivided to 1 ml and stored frozen. This positive control was used at doses of 0.1 and 0.05 μg/ml for the 6- and 24-hour treatments (–S9), respectively. Cyclophosphamide (CP; Shionogi & Co., Ltd.; lot no. 1013) dissolved in sterile water for injection (lot no. K1D78) was diluted with physiological saline (lot no. K1D74). It was subdivided to 1 ml and stored frozen. This positive control was used in the presence of S9 at a dose of 12.5 μg/ml.
The rat bone marrow micronucleus test was performed with ubiquinol dissolved in corn oil (Nakarai Tesque, Inc.; lot no. V2M8974). MMC was selected as the positive control, because it is frequently used in micronucleus assays and proposed in the guideline. MMC (2 mg potency with 48 mg NaCl in vial; Kyowa Hakko Kogyo Co., Ltd.; lot no. 378ABC) was dissolved in sterile water for injection (Otsuka Pharmaceutical Factory, Inc.; lot no. K1K76).
Bacterial Reverse Mutation Test
Ubiquinol was tested in Salmonella typhimurium strains TA100, TA1535, TA98, and TA1537 and Escherichia coli strain WP2uvrA in the absence and presence of exogenous metabolic activation by rat liver S9, according to previously published methods (Ames, Lee, and Durston 1973; Ames et al. 1973;Ames, McCann, and Yamasaki 1975; Yahagi 1975; Ministry of Labor Industrial Safety and Health Department 1991; Ishidate 1991) and the recommendations of “Guidelines for Genotoxicity Tests on Drugs.” The Salmonella typhimurium strains were received from Dr. Bruce N. Ames (University of California) on September 9, 1983, and the Escherichia coli strain was provided by the National Institute of Health Sciences (Japan) on March 16, 1983. The composition of S9 mix used in this test was as follows: S9 0.1 ml, MgCl2 8 μmol, KCl 33 μmol, glucose-6-phosphate (G-6-P) 5 μmol, NADPH 4 μmol, NADH 4 μmol, and sodium phosphate buffer 100 μmol in 1 ml of S9 mix.
Based on the guideline and the results of a concentration range–finding test (data not shown), the highest concentration in the mutagenicity test was established at 5000 μg/plate. The positive-control substances were AF2, 9-AA, 2-AA, and NaN3; a vehicle control (acetone) was also included. Tests were performed by the preincubation method.
After the 48-h incubation period, the number of revertants was counted using an automated colony analyzer (System Science Co.; CA-11). Plates with severe precipitation of the test substance were counted manually. Any changes, including precipitation of the test article on the plates or growth inhibition of tester strains induced by the test article, were evaluated macroscopically. Two plates were used for each concentration of the test substance and positive controls whereas three plates were used for the negative control according to the recommendation of Ministry of Labor Industrial Safety and Health Department (1991). The mean number of revertants was calculated for each group. The results were judged to be positive when the mean number of revertants in each concentration increased to twofold or more than the negative control and reproducibility and concentration-dependent relationship were observed. The reproducibility was confirmed by the two results of concentration range–finding test (pilot study) and bacterial reversion assay (main study).
Chromosomal Aberration Test in CHL/IU Cells
Ubiquinol was evaluated for its potential to produce chromosome aberrations in Chinese hamster lung fibroblast (CHL/IU) cells in vitro in the absence and presence of exogenous metabolic activation by rat liver S9 following 6-h treatments and in the absence of S9 following 24-h treatments according to the results of checking cell doubling time and previously published methods (Ishidate and Odashima 1977; Ishidate 1979, 1987; Evans 1976; Matsuoka, Hayashi, and Ishidate 1979; Report of the Ad Hoc Committee of the Environmental Mutagen Society and the Institute for Medical Research 1972). The cell subline passaged 27 times, under storage since it was supplied by the National Institute of Health Sciences on November 15, 1984, was cultured in 10% Eagle’s minimum essential medium (MEM; ASAHI Techno Glass Co., Ltd.; lot no. 809012) supplemented with heat inactivated calf serum (Invitrogen; lot no. 353445) of 10% final concentration in a CO2 incubator (5% CO2) at 37°C. Five milliliters of the cell suspension containing 8 × 103 cells/ml was seeded onto cell culture plate (60 mm; Sumitomo Bakelite Co., Ltd) and incubated for 3 days. Following incubation, 300 μl of the vehicle, test substance suspension, or positive-control substance solution was added; 500 μl of S9 mix was also added to the +S9 assay. The composition of S9 mix was as follows: S9 0.3 ml, MgCl2 5 μmol, KCl 33 μmol, G-6-P 5 μmol, NADP 4 μmol, HEPES buffer 4 μmol, and distilled water 0.1 ml in 1 ml of S9 mix. In the short-term treatment assays, the culture medium was removed after 6 h and the cells were washed with Dulbecco’s phosphate-buffered saline (Sigma Chemical; lot no. 62K2338). Three milliliters of fresh culture medium was added and the cells were incubated for another 18 h; in the continuous treatment assay, cells were incubated for 24 h after the addition of the vehicle, test substance suspension, or positive-control substance solution.
A growth inhibition test using the crystal violet method was performed to determine the concentration range of the test article for the chromosomal aberration test (data not shown). On this basis, the highest concentration for the chromosomal aberration test, which was expected to cause 50% or more cell growth inhibition, was established at 5000 μg/ml for the 6-h treatment and 1201 μg/ml for the 24-h treatment. In addition, six or seven lower concentrations were included (short-term treatment groups: 412, 588, 840, 1201, 1715, 2450, and 3500 μg/ml; continuous treatment group: 141, 202, 288, 412, 588, and 840 μg/ml). Two plates per concentration were prepared for the vehicle control (CMC), test article treatment, and positive-control groups. Three chromosomal slides per plate were prepared following incubation with the vehicle control, test substance, or positive-control substance as follows: Colcemid solution (Invitrogen; lot no. 1125546) was added to the treated cells at a final concentration of 0.2 μg/ml 2 h before slide preparation to inhibit mitosis at metaphase. The culture medium was transferred into a centrifuge tube. The cells were stripped from the plate using 0.25% trypsin (Invitrogen; lot no. 1129248) and the cell suspension was added to the culture medium in the centrifuge tube. Following centrifugation at 1000 rpm for 5 min, the culture medium was removed and the cells were subjected to hypotonic treatment in 5 ml of 75 mmol/L KCl for 16 min at 37°C. The hypotonic solution was removed by centrifugation and the cells were fixed in a cold (4°C) fixative solution (3:1 v/v methanol/acetic acid), washed three times, and resuspended in a small volume of fresh fixative solution. One or two drops of the suspension were placed on each clean, defatted slide. The slides were allowed to dry and stained for 12 min with 1.2% Giemsa solution (Merck KGaA; lot no. 040428561) diluted with 1/100 mol/L sodium phosphate buffer solution (pH 6.8; Merck KGaA; lot no. TP551574147) and then rinsed with water and dried.
Cell growth inhibition was determined at the preparation of chromosomal slides with an ATP spectrophotometer (Lumitester C-100LU; Kikkoman Corp.) for negative-control, treatment, and positive-control groups. Cell suspension after hypotonic treatment (50 μl) was transferred into a small test tube containing 2 ml of 1% Tween 80 solution and mixed. This mixture was left still for about 20 min and 100 μl of luminescent reagent of reagent kit for ATP determination (Lucifer 250; Kikkoman Corp.) was transferred into test tube for determination. The rate to the relative light unites (RLU) in the negative-control group (cell survival rate) was calculated for each dose and determined as cell growth inhibition.
Microscopic examination was performed on the all observable doses for observation of chromosome aberrations: 412 to 3500 μg/mL for the short-term treatment –S9 assay, 412 to 3500 μg/ml for the short-term treatment +S9 assay, and 141 to 1201 μg/ml for the continuous treatment (24-h) assay. After all the slides had been coded, they were examined under code. One hundred metaphases per plate (200 cells/dose) were examined microscopically (×600). The type of structural aberrations was classified into six groups: gap (gap), chromatid break (ctb), chromosome break (csb), chromatid exchange (cte), chromosome exchange (cse), and others (oth). A gap was recorded when the chromatid or chromosome with an unstained region existed, or a cutting like a chromatid break was observed, and the width of the unstained part was clearly narrower than the chromatid and not dislocated from the axis. For numerical aberrations, the number of polyploid cells was counted by observing 200 cells per dose.
Clastogenic potential was assessed with the incidence of the total aberrant cells excluding cells with only gap. If the incidence of structural or numerical aberrations was less than 5%, it was judged to be negative (–). If the incidence of structural or numerical aberration in each treatment group between was 5% and 10% and reproducibility was observed, it was judged to be inconclusive (±). If the incidence of structural or numerical aberration in each treatment group was 10% or more and reproducibility was observed, it was judged to be positive (+). These judgments were based on the methods of Ishidate (1987) and in-house background data. Statistical evaluation was not performed.
Rat Bone Marrow Micronucleus Test
The in vivo micronucleus was conducted in accordance with previously published methods (Hayashi, Sofuni, and Ishidate 1984; Schmid 1975, 1976; Salamone and Heddle 1980). Male, 7-week-old Crj:CD(SD)IGS[SPF] rats were obtained from Charles River Japan, Inc., and housed in a temperature- and humidity-controlled room (temperature range 23.0°C to 24.3°C, relative humidity 34% to 55%). The room air was changed 18 times per hour and a 12-h fluorescent, light/dark cycle was maintained. Paired animals were housed in Zyfone animal cages with bedding (ALPHA-dri; Shepherd Specialty Papers, Inc.; lot no. 10102) setting on the Micro-Isolator System rack (Lab Products, Inc.) with an automatic watering system. The animals were given a commercially available pellet diet (MF; Oriental Yeast Co., Ltd.; lot no. 021206) ad libitum. Animals were given tap water ad libitum via an automatic water-service nozzle. Animals were examined visually for the presence or absence of disease once a day for 6 days and were acclimated to the testing facility. None of the animals showed any sign of disease in general conditions or abnormality in body weight gain during this period. Animals were stratified by body weight on the day of the first dose and were grouped by a random sampling method (6 animals/group). Two animals each were housed in each cage, with a label showing the experiment number, the animal ID number, etc., after grouping, and each animal was individually identified by pen tail mark after grouping. At the time of the dosing, rats were 8 weeks old and their body weight ranged from 285 to 307 g.
In the preliminary acute toxicity test (data not shown), no deaths or abnormalities attributable to the test substance were observed in the 2500 or 5000 mg/kg group in the single oral administration study in rats. Furthermore, no sex differences were observed in the study. Therefore, 2000 mg/kg/day, the upper-limit dose in the guidelines, was set as the high and 1000 and 500 mg/kg/day as the lower doses using only male rats.
Groups of six male rats received two oral doses, separated by a 24-h interval, of ubiquinol at 500, 1000, or 2000 mg/kg/day (dose volume: 2 ml/100 g body weight). The solvent (corn oil) was used as the negative control and was likewise administered orally. The positive-control substance, MMC, was dissolved in 5 ml of water for injection and this solution was injected intravenously at a dosage level of 2 mg/kg (dose volume: 0.5 ml/100 g body weight) on a single occasion, approximately 24 h prior to euthanasia. Six animals were treated in all groups to ensure that data would be available for five animals in each group. Because no deaths were observed, the bone marrow samples were prepared from five animals in each group in order of the animal ID number. Surplus animals whose bone marrow samples were not prepared were euthanized by CO2 inhalation.
Animals were weighed and observed for general condition at dosing, 24 h after the first dose, and prior to the preparation of bone marrow samples. Rats were sacrificed by CO2 gas approximately 24 h after the last dose. One femur was removed from each rat and cells were flushed out with calf serum (Invitrogen; lot no. 364570; inactivated at 56°C for 30 min) into a centrifuge tube. Excess serum was removed by centrifugation. The bone marrow samples were prepared according to the method of Kawabata et al. (2007). The remainder of the cells was resuspended in Dulbecco’s phosphate-buffered saline (PBS; Sigma-Aldrich; lot no. 92K2322) to wash cells. This cell suspension was centrifuged and the supernatant removed; this washing procedure was repeated twice. The cells were then fixed in a 10% neutral-buffered formalin solution (for tissue fixative use: Wako Pure Chemical Industries; lot no. TCF8362). This cell suspension was centrifuged, and the supernatant removed. After this procedure had been repeated twice, the cell suspension was maintained at room temperature and the fixed cell suspension was dropped on a cover slip and put on a slide coated with acridine orange for observation.
Two thousand polychromatic erythrocytes (PEs) per rat were analyzed using a fluorescent microscope (×800) equipped with a blue extraction filter (BP470-490) and a barrier filter (BA515IF). To investigate the influence of the test substance on bone marrow cell proliferation, the number of PEs out of a total of 500 erythrocytes was then counted. The proportion of PEs to total erythrocytes was calculated as a percent as follows:
The incidence of micronucleated polychromatic erythrocytes (MNPEs) in relation to the total number of PEs was calculated as percent as follows:
The conditional binomial test (Kastenbaum and Bowman 1970) was performed to compare the incidence of MNPEs in the negative-control group to that in each dose group and the positive-control group. The conditional binomial test was conducted at the significance level of .05. The proportion of PEs was analyzed statistically using Dunnett’s t test (significance level of .05) (Dunnett 1964).
RESULTS
Bacterial Reverse Mutation Test
The mutagenicity test was conducted at five concentrations (313 to 5000 μg/plate), with a twofold dose interval in strains TA100, TA98, TA1537, and WP2uvrA. Because of the tendency to increase the numbers of revertant colonies at 20.5 or 128 μg/plate in strain TA1535 in the dose-finding study (results not shown), nine concentrations (19.5 to 5000 μg/plate with a twofold dose interval) were used for this strain.
As shown in Table 1, the number of revertants in main study did not increase to more than twice that of the negative control in any strain used, with or without S9 mix. It was judged that the tendency to increase the number of revertant colonies in the TA1535 strain observed in the concentration range–finding study was incidental because no reproducibility was observed in main study. Ubiquinol did not inhibit growth of any bacterial strain at any concentration up to the limit dose of 5000 μg/plate concentration with or without S9 mix. No microbial contamination was detected in the test solution or S9 mix in any of the tests. The mutagenicity of the positive-control substances confirmed the sensitivity of the test and the activity of the S9 mix and the numbers of revertants in positive and negative controls were within the range (mean ± 3 SD) of historical control values (data not shown).
Chromosomal Aberration Test in CHL/IU Cells
Marked cell growth inhibition was observed at higher doses in all treatments. The 50% cell growth inhibition dose was 2929 μg/ml for the 6-h treatment without S9, 2415 μg/ml for the 6-h treatment with S9, and 551 μg/ml for the 24-h treatment.
In all three treatment scenarios, the incidences of cells with chromosome structural aberrations and numerical aberrations in the groups treated with ubiquinol were similar to those in the negative-control group (Tables 2 to 4). In cultures treated with positive controls, high incidences of chromosomal aberrations were observed, confirming the sensitivity of the test and the activity of the S9 mix. The incidence of cells with chromosome aberrations in both negative- and positive-control groups was within the range of our historical data (data not shown), which supported the validity of this study.
Rat Bone Marrow Micronucleus Test
In the micronucleus test, no deaths occurred and no clinical signs were observed in any animal in any group, including positive- and negative-control groups. As shown in Table 5, 1 to 5 MNPEs in 2000 PEs per animal were noted in the negative-control group, with a group mean incidence of 0.12%. The proportion of PEs to the total number of erythrocytes was 67.7%, which is within the in-house historical data (64.1% ± 8.5%). The value is also similar to the reference data (61.0% ± 3.0%, 57.9% ± 5.5%) (Kawabata et al. 2007). The incidence of MNPEs after the administration of ubiquinol was 0.19% in the 500 mg/kg/day group, 0.16% in the 1000 mg/kg/day group, and 0.13% in the 2000 mg/kg/group. No significant increase was noted in any of the treatment groups compared to the negative-control group. The proportion of PEs to the total number of erythrocytes, a measure of the rate of bone marrow cell (erythroblast) division was 66.2%, 65.6%, and 59.0% in the 500, 1000, and 2000 mg/kg/day groups, respectively. A slight but not statistically significant decrease of 8% in the ratio of PEs was obtained for highest dose group. The ratio of PEs to the total number of erythrocytes of 59% seen at the highest dose level was still within the in-house historical control data (64.1% ± 8.5%). The incidence of MN-PEs in the positive-control group was markedly increased to 4.90% (70 to 142 micronuclei in 2000 PEs), and a statistically significant increase (p ≤ .05) was observed as compared with the negative-control group. Furthermore, the proportion of PEs was decreased to 54.8% (p ≤ .05), indicative of slight bone marrow toxicity. The incidence of micronuclei and proportion of PEs to the total numbers of erythrocytes in both the negative- and positive-control groups were within the range of our historical data (data not shown), which supported the validity of this study.
DISCUSSION
The test article, ubiquinol, was evaluated as negative in the bacterial reverse mutation, chromosomal aberration, and rat bone marrow micronucleus tests under the conditions of these assays.
The results of these assays demonstrate that ubiquinol (1) does not induce reverse mutations in Salmonella typhimurium and Escherichia coli at concentrations as high as 5000 μg/plate; (2) does not induce chromosomal aberration in cultured CHL/IU cells exposed to concentrations up to the limit of toxicity; and (3) is devoid of chromosome or mitotic apparatus-damaging activity in rat bone marrow when administered orally to rats at doses up to the standard limit of 2000 mg/kg/day.
In the chromosomal aberration test, slight increase in percentage of polyploidy cells in all treatment was observed. Because the values were within in-house background data using CMC as a vehicle (data not shown), they were judged to be negative according to the criteria of Ishidate (1987). However, the increase in percentage of polyploidy cells might be due to the physical effects of the deposition test article. A report shows that needle crystal contacts cells and inhibits cellular division physically (Kawaguchi et al. 1997). In addition, a number of compounds including reducing agents are known to induce polyploidy at high doses in vitro; the relevance of this effect to the in vivo situation is not known. We have confirmed that the plasma concentration of ubiquinol (about 4 μg/ml) increased by the first oral administration of 1200 mg/kg body weight in 13-week toxicity study on rats (Kitano et al. submitted). This data indicate that the exposure of ubiquinol to bone marrow cells was sufficient to confirm the presence or absence of mutagenicity in rat bone marrow micronucleus test.
The results reported here, along with those of subchronic rat and dog studies performed with ubiquinol (Kitano et al. submitted) and consideration of its known important physiologic and nutritive activities, provide evidence of the safety of ubiquinol.
Ubiquinol is a lipid-soluble compound and insoluble in water, saline, DMSO, and ethanol. Because ubiquinol is soluble in acetone at approximately 100 mg/ml, acetone was used as a solvent in the Ames assay. Ames et al. have confirmed that 12 solvents including acetone were satisfactory under the conditions for the Salmonella mutagenicity test (Maron, Katzenellenbogen, and Ames 1981). Japanese guidelines allow all precipitate at all test concentration in the Ames assay because Japanese guideline recommends use of higher concentrations unless those concentrations inhibit cell growth. In the chromosome aberration system, precipitation of the test article is considered acceptable because the cells are capable of phagocytosis. There is some evidence that dose-related genotoxic activity of certain insoluble compounds can be detected in both bacterial and mammalian cell geonotoxicity assays. Laboratories in Japan have experience in testing precipitates and have identified examples of substances that are clearly genotoxic only in the precipitating range of concentrations. A discussion of these factors is given in the report of the in vitro subgroup of the International Workshop on Standardisation of Genotoxicity Procedures, Melbourne, 1993 (Kirkland 1994; ICH Guidance 1995).
There is a close biological relationship between ubiquinol and ubiquinone. For example, ubiquinone is formed in vivo as results of the antioxidant activity of ubiquinol (Ernster and Dallner 1995). Natural ubiquinone is readily reduced to ubiquinol after dietary intake (Stocker and Suarna 1993). As a result of this relationship, reports of the safety of ubiquinone are relevant to the safety of ubiquinol. Nonclinical toxicity studies demonstrating that ubiquinone had no adverse acute, subacute, chronic, or reproductive developmental effects (Chiba et al. 1972a, 1972b; Notake et al. 1972; Williams et al. 1999) or mutagenic activity in the in vitro Ames and chromosome aberration assays, and in an in vivo bone marrow micronucleus test (Kitano et al. 2006) support the safety of ubiquinol demonstrated in the tests reported herein.
Footnotes
Figure and Tables
Conflict of Interest Statement. The authors except Fukutaro Mizuhashi are presently employed by Kaneka Corporation, who manufactures the ubiquinol product (Kaneka QHTM) used in the study and is the sponsor and sole source of funding for the present study.