Studies of the Toxicological Potential of Capsinoids V: Genotoxicity Studies of Dihydrocapsiate Bruce K. Bernard SRA International, Inc., Cambridge, Maryland, USA Eri Watanabe Terutaka Kodama Shoji Tsubuku Akira Otabe Toxicology and Pathology, Nonclinical Developmental Research Dept., Pharmaceutical Research Laboratories, Pharmaceutical Company, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan Madoka Nakajima Shoji Masumori Sawako Shimada Jin Tanaka Biosafety Research Center, Foods, Drugs and Pesticides, Iwata, Shizuoka, Japan Takeshi Masuyama Toxicology and Pathology, Nonclinical Developmental Research Dept., Pharmaceutical Research Laboratories, Pharmaceutical Company, Ajinomoto Co., Inc., Kawasaki, Kanagawa, Japan RUNNING TITLE: DIHYDROCAPSIATE: GENOTOXICITY CORRESPONDENCE TO: Bruce K. Bernard, Ph.D., President, SRA International, Inc., 5235 Ragged Point Road, Cambridge, MD 21613, USA, Telephone: 410-228-1400, Fax: 410-228-1450, E-mail:
[email protected]
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ABSTRACT A series of studies was performed to evaluate the safety of dihydrocapsiate (4-hydroxy-3methoxybenzyl 8-methylnonanoate, CAS No. 205687-03-2). This study evaluated the potential genotoxicity of this compound using a variety of in vitro and in vivo test systems including bacterial reverse mutation test, chromosomal aberration test, micronucleus test, gene mutation assay with transgenic rats and single cell gel (SCG) assay (comet assay). In vitro tests (bacterial reverse mutation test and chromosomal aberration test) produced positive results in the absence of metabolic activation, but negative results in the presence of metabolic activation. The in vivo gene mutation assay (with transgenic rats) produced negative results, as did the in vivo mouse micronucleus assay which failed to induce micronucleated polychromatic erythrocytes. While the rat SCG assay produced statistically-significant increases in the Olive tail moment and % tail DNA of the liver and intestine in the 2000 mg/kg group (compared with the negative control group), a number of factors caused the authors’ to question the validity of these findings. Taken together, these results suggest that dihydrocapsiate has a low or extremely low likelihood of inducing genotoxicity. Keywords: Capsiate, Capsinoids, Clastogenicity, Dihydrocapsiate, Genotoxicity, Mutagenicity
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INTRODUCTION Capsinoids are capsaicin analogues isolated from a fruit of CH-19 Sweet extract, the nonpungent cultivar of chili pepper (Yazawa et al. 2004). Kobata et al. (1998 and 1999) determined the structures of capsinoids and named a number of the family members including capsiate (4hydroxy-3-methoxybenzyl
(E)-8-methyl-6-nonenoate),
dihydrocapsiate
(4-hydroxy-3-
methoxybenzyl 8-methylnonanoate) and nordihydrocapsiate (4-hydroxy-3-methoxybenzyl 7methyloctanoate), respectively. Structurally, capsinoids are distinguished from capsaicin in that the former have an ester bond located between the vanillyl moiety and fatty acid chain; whereas capsaicin has an amide bond in that location. CH-19 Sweet Extract is an ingredient for oral use in a dietary supplement, which is recommended to be taken once a day at the dose containing 3 mg capsinoids. Until now, the increase in the daily intake of capsinoids proved difficult, because many of the common varieties of chili peppers available to consumers have a low ratio of capsinoids to capsaicinoids, resulting in a high consumption of a pungent fruit and leading to gastrointestinal discomfort before the desired amount of capsinoids could be consumed (Westerterp-Plantenga et al. 2006). However, CH-19 Sweet extract can be used to increase capsinoids supplementation, because it is derived from a variety of chili peppers containing none of the pungent capsaicinoids (Yazawa et al. 2004). Although capsinoids occur naturally in various cultivars of capsicum pepper, the amount of capsaicinoids is usually much larger than that of capsinoids. The exception to this is CH-19 Sweet (Yazawa et al. 2004). Kobata et al. (2002) reported enzymatic synthesis of capsinoids using vanillyl alcohol and fatty acid. Recently, Ajinomoto Co., Inc. has developed a process for
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synthesizing dihydrocapsiate using this method. Dihydrocapsiate could provide an alternative source of capsinoids for use as a food ingredient. Capsaicin and chili extracts have been shown to have genotoxic potential in the bacterial reverse mutation test and the micronucleus test in mice (Nagabhushan and Bhide 1985). However, the genotoxicity tests of CH-19 Sweet extract which contains capsinoids revealed neither mutagenicity nor in vitro or in vivo clastogenicity (Watanabe et al. 2008b). The purpose of this study was to evaluate directly the genotoxicity of dihydrocapsiate and it is one in a series of toxicity studies to assess the safety of capsinoids. Each genotoxicity test was performed at the Biosafety Research Center, Foods, Drugs and Pesticides (Shizuoka, Japan). These studies are referenced in the following guidelines or scientific literature: OECD Guidelines for the Testing of Chemicals Section 4 Test No.471, No.473 and No.474 (OECD guidelines, 1997a, 1997b and 1997c, respectively), the literature described by Tice et al. (2000), Hartman et al. (2003), Burlinson et al. (2007), and WHO guideline (2006). These tests were conducted in compliance with the Law Concerning the Protection and Control of Animals, Law No. 105, October 1, 1973, revised on December 22, 1999, partly revised Law No. 68 on June 22, 2005; Standards Relating to the Care and Management, etc. of Experimental Animals, Notification No. 6, March 27, 1980 of the Prime Minister’s Office, Japan, revised on May 28, 2002; and Guidelines for Animal Experimentation, the Japanese Association for Laboratory Animal Science, May 22, 1987 or the Guidelines for Animal Experimentation, Biosafety Research Center, Foods, Drugs and Pesticides, December 1, 2003. MATERIALS AND METHODS
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Test Substances The test substance, dihydrocapsiate (Figure 1), is a viscous, colorless to yellow liquid. Two lots of test substances were supplied by Ajinomoto Co., Inc. (Tokyo, Japan). One (Lot No. WKU05137ZBa) was used for the bacterial reverse mutation test, the in vitro chromosomal aberration test, and the in vivo micronucleus test and SCG assay. The other (Lot No. 060807) was used for gene mutation assay which employed transgenic rats. The test substance contained small amounts of 8-methylenonanoic acid which is the acyl residue of dihydrocapsiate. The purity of dihydrocapsiate in each lot test substance was 95.8% in WKU05137ZBa and 94.0% in 060807, respectively. The test substances were stored in a freezer throughout the study to protect them from heat and light. Medium chain triglyceride (MCT, Product Name Actor M-2), which was selected as the vehicle for the in vivo tests, was manufactured by Riken Vitamin Co., Ltd. (Tokyo, Japan). Dimethylsulfoxide (DMSO), which was selected as the vehicle for the in vitro tests, was manufactured by Merck Ltd. (Tokyo, Japan). Positive Control Agents The positive control substances used in the various assays (and their suppliers) were as follows: 2-(2-Furyl)-3-(5-nitro-2-furyl) acrylamide (AF-2, Oriental Yeast Co., Ltd., Tokyo, Japan), Sodium azide (NaN3, Oriental Yeast Co., Ltd.), 9-Aminoacridine hydrochloride (9-AA, Oriental Yeast Co., Ltd.), 2-Aminoanthracene (2-AA, Oriental Yeast Co., Ltd.), Mitomycin C (MMC, Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan), Cyclophosphamide (CP, Shionogi Co., Ltd., Osaka, Japan), 7,12-dimethylbenzo[a]anthracene (DMBA, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan) and Ethyl methanesulphonate (EMS, Sigma-Aldrich Japan K. K., Tokyo, Japan).
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Rat Liver Homogenate (S9) Mix S9 mix was purchased from Kikkoman Co., Ltd. (Chiba, Japan). S9 had been prepared from the supernatant fraction of a homogenate derived from the liver of 7-week-old male Sprague-Dawley rats pretreated with phenobarbital (PB) and 5, 6-benzoflavone (BF). S9 mix had been prepared by mixing S9, MgCl2, KCl, Glucose-6-phosphatase (G-6-P), NADPH, NADH and sodium phosphate buffer (pH 7.4). (Table 1) Experimental Procedures Reverse mutation assay (Ames test) This study is referenced in the OECD Guidelines for the Testing of Chemicals Section 4 Test No.471 (OECD guideline, 1997a), and was conducted according to the pre-incubation method of Ames et al. (1973) employing Salmonella typhimurium and Escherichia coli. S. typhimurium (TA100, TA98, TA1535 and TA1537) were obtained from Dr. Bruce N. Ames at the University of California and E. coli (WP2uvrA) was obtained from the National Institute of Health Sciences, Japan. Based on the results of dose finding studies for strain TA100 in the –S9 assay, 6 doses including the dose which would show a maximum specific activity, were selected. Similarly, for strain TA100 in the +S9 assay and for the other strains either with or without S9, 6 to 7 additional doses including the highest dose at which bacterial growth would be inhibited were selected. All plates were run in duplicates. (Table 2) Into each test tube, 100 µL of DMSO, test substance suspension in DMSO, or positive control substance solution was dispensed, followed by addition of either 500 µL of 0.1 mol/L sodium phosphate buffer (pH 7.4) in the -S9 assay or 500 µL of the S9 mix in the +S9 assay. After an addition of 100 µL of pre-cultured test strain suspension, the mixture was pre-incubated at 37°C, and shaken at 120 strokes/minute for 20 minutes. At the end of pre-incubation, 2 mL of
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top agar was added to mix the contents. Subsequently, the mixture was poured onto plates and spread. Each plate was incubated at 37°C for 48 hours. Precipitation on each plate and other changes were macroscopically examined at the start of exposure and when colonies were counted for each treatment. To determine whether the test substance had a growth inhibitory effect, growth of test strains (background lawn) on the plate was observed with a stereoscopic microscope. The number of revertant colonies was counted with a colony analyzer. With correction for the area size and count failure, the number of colonies was calculated. The colony analyzer could not be used because of precipitation of the test substance at doses of 4,000 µg/plate or greater in the -S9 assay; thus the number of colonies was counted manually. The results were judged positive if the number of revertant colonies was at least twice that of negative control colonies and the increase was dose-dependent. In the event of positive results, specific activity, as an index of the mutagenic intensity, was calculated using the following equation. Calculation was based on the rounded values for the mean number of colonies.
((No. of colonies per plate at the dose)-(No. of colonies of negative control per plate)) / Dose (mg/plate) In vitro chromosome aberration test This study is referenced in the OECD Guidelines for the Testing of Chemicals Section 4 Test No.473 (OECD guideline, 1997b). The study was conducted by short-term treatment with (+S9 assay) or without (-S9 assay) metabolic activation, and by continuous treatment (Ishidate et al. 1988) with a fibroblast cell line derived from the lungs of the Chinese hamster (CHL/IU) supplied by the National Institute of Health Sciences. Based on the results of the cell growth
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inhibition test, a dose at which the relative cell growth rate was less than 50% was set as the maximum dose for the chromosome aberration test. All plates were run in duplicates. For the short-term treatment, 5 mL of cell suspension containing 8 × 103 cells/mL (4 × 104 cells) was seeded onto each 60 mm plate and incubated for 3 days. Following the incubation period, 0.03 mL of the test substance suspension in DMSO was treated with 3 mL of the culture medium for the -S9 assay or 2.5 mL of the culture medium and 0.5 mL of the S9 mix for the +S9 assay. After a 6-hour exposure, the medium was removed from the plates and the cells were washed with Dulbecco’s phosphate-buffered saline (Sigma-Aldrich Japan K. K., Tokyo, Japan). Thereafter, 3 mL of fresh medium was added to the cells, and the cells were incubated for 18 additional hours. For the continuous treatment, 5 mL of cell suspension containing 8 × 103 cells/mL were seeded onto each 60 mm plate and incubated for 3 days. Following the incubation period, 0.03 mL of the test substance suspension in DMSO was treated with 3 mL of the culture medium. After a 24-hour exposure, the medium was removed from the plates and the cells were washed with Dulbecco’s phosphate-buffered saline (Sigma-Aldrich Japan K.K., Tokyo, Japan). Thereafter, 3 mL of fresh medium was added to the cells, and the cells were incubated for 2 additional hours. Chromosome sample slides were prepared by adding a colcemid solution (Invitrogen Japan K.K., Tokyo, Japan) to the treated cells to block mitosis at metaphase. Two hours later, the cells were detached from the plate by treatment with 0.25% trypsin (Invitrogen Japan K.K., Tokyo, Japan) and the cell suspension was transferred into a centrifuge tube with the culture medium. After centrifugation of the cell suspension, the supernatant was removed and 5 mL of 75 mmol/L potassium chloride solution warmed to 37°C was added to the cells. The cells were
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subjected to hypotonic treatment at 37°C for 16 minutes. After the solution was removed by centrifuging, the cells were fixed in a cold fixative solution (methanol: acetic acid = 3:1), washed twice with a fresh fixative solution, and re-suspended in a small volume of a fresh fixative solution. After the cell density was adjusted, two chromosome slides for chromosome aberration were prepared. The slides were allowed to dry completely and then stained for about 12 minutes with 1.2% Giemsa solution (Merck Ltd., Tokyo, Japan) diluted with 1/100 mol/L of sodium phosphate buffer solution (Buffer tablets pH 6.8: Merck Ltd., Tokyo, Japan). Finally, the slides were rinsed with water and dried. Three chromosome slides (one for cell density observation, two for chromosome observation) were prepared per plate. All slides were coded before scoring. One hundred metaphases per plate (i.e., 200 metaphases/dose) were examined microscopically (under approx. 600× magnification) and classified into the following groups depending on the morphologic change: gap (gap), chromatid break (ctb), chromosome break (csb), chromatid exchange (cte), chromosome exchange (cse), and others (oth). The cells with these morphological changes were counted as aberrant cells A gap was recorded when the chromatid or chromosome contained an unstained region, or a cutting such as a chromatid break was observed, and the width of the split (unstained) part was clearly narrower than the chromatid and not dislocated from the axis. The number of polyploid cells was counted by observing 200 metaphases per dose. If the incidence of aberrant cells was less than 5%, it was determined to be negative. If the incidence of aberrant cells was between 5% and 10% and this incidence was reproducible, it was determined to be inconclusive. If the incidence of aberrant cells was 10% or more and this incidence was reproducible or dose-dependent, it was determined to be positive. Cells with only gaps were excluded from the total aberrant cells. In addition, D20 value, which is the minimum dose (mg/ml) at which aberrations were found in
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20% of metaphases, and the translocation (TR) value were calculated from the incidence of aberrant cells (Ishidate et al., 1998). Micronucleus test This study is referenced in the OECD Guidelines for the Testing of Chemicals Section 4 Test No.474 (OECD guideline, 1997c). Thirty eight male BDF1 strain SPF mice (Japan SLC, Inc., Shizuoka, Japan) were purchased at 8 weeks of age. After 6 days of quarantine, healthy animals were randomly assigned to groups (6 animals/group) using a stratified body weight procedure. Unassigned animals were euthanized by CO2 inhalation. Individual body weights on the day of grouping were in the range of 24.2 to 29.0 g. Based on the results of the dose-finding study, 2000 mg/kg was selected as the high dose level, and a total of 3 dose levels including 1000 and 500 mg/kg were selected. Oral route was selected for administration of the vehicle and the test substance because this is the intended human exposure route. The test substance was dissolved in the vehicle and administered once daily for 2 consecutive days (at a 24-hour interval) according to the guidelines for genotoxicity tests by gavage using flexible stomach tubes to mice. The dose volume was 5 mL/kg body weight (BW). The positive control substance (MMC) was injected once intraperitoneally at a dose level of 0.5 mg/kg BW. (Table 3) Individual body weights were measured on the day of the first dosing, and just before preparation of bone marrow samples. Animals were observed for clinical signs at 1 and 24 hours after the first administration, at 1 hour after the final administration and just before preparation of bone marrow samples. Mice were euthanized by CO2 inhalation 24 hours after the final administration. One femur was removed and the bone marrow cells were flushed out with fetal bovine serum (Invitrogen Japan K.K., Tokyo, Japan inactivated at 56°C for 30 minutes) into a centrifuge tube.
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After excess serum was removed by centrifugation, three bone marrow smears per animal (1 slide for state of the smears and 2 slides for microscopic observation) were prepared. The smears were allowed to dry and were subsequently fixed in methanol. Two smears from each animal were stained with 3% Giemsa solution (Merck Ltd., Tokyo, Japan) for 30 minutes for microscopic evaluation. The smears were rinsed with 1/100 mol/L sodium phosphate buffer (Merck Ltd., Tokyo, Japan; Buffer tablets pH 6.8) and purified water and allowed to dry. The smears were then rinsed with 0.001% citric acid solution and purified water and allowed to dry again. Bone marrow smears were prepared from all animals. All smears were coded and only smears from 5 animals in each group were examined in a blind manner. The incidence (%) of micronucleated polychromatic erythrocytes (MNPCE) was calculated based on the observation of 2000 polychromatic erythrocytes (PCE) per animal. To investigate the effect on bone marrow cell proliferation, the ratio (%) of PCEs to 500 erythrocytes was calculated. The Conditional Binomial test was conducted to compare the incidences of MNPCE between the negative control group and each treatment group or positive control group (Kastenbaum and Bowman 1970). The ratio of PCE was analyzed statistically as follows. First, the data from the negative control group and all test substance treatment groups were tested using Bartlett’s test for homogeneity of variance (Sokal and Rohlf 1969). Since the variances were homogeneous, Dunnett’s multiple comparison test was conducted (Dunnett 1964). The data from the negative control group were compared with those from the positive control group by the F test for homogeneity of variance between each of the two groups (JIS 1965). Since the variances were homogeneous, Student’s t-test (JIS 1965) was conducted. The results were evaluated as positive when the incidences of MNPCE in the treatment groups were significantly different from those in the negative control group. For the final judgment, the biological relevance to the results (the historical background
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data for the testing facility, dose relationship, and the potential to induce chromosomal aberration in vitro) was taken into consideration in this study. Gene mutation assay in transgenic rats This study is referenced in the WHO guideline (2006). Twenty-five male rats (Big BlueTM transgenic rat, Stratagene Japan K.K., Tokyo, Japan) purchased at 7 weeks of age were randomly assigned to groups (6 animals/group) after 9 days of quarantine. Unassigned animals were euthanized by CO2 inhalation. Individual body weights on the day of grouping were in the range of 171 to 250 g. Based on the results of the 13-week toxicity study of the test substance (Watanabe et al. 2008c), the low-dose adverse effect level in males (1,000 mg/kg/day) was selected as the high dose in this study and half of that dose (i.e., 500 mg/kg/.day) was chosen as the low dose. The test substance was dissolved in the vehicle and administered to rats orally once daily for 28 consecutive days by gavage at a volume of 5 mL/kg BW. The positive control substance (DMBA) was administered once orally at a dose level of 100 mg/kg BW. Individual body weights were measured on Day 1, 5, 8, and 22, the day after the last dosing and the day of tissue sampling. Animals were observed for clinical signs at least once daily until tissue sampling. After a 3-day sampling time for gene mutation following the final treatment, the liver, kidney, and duodenum were isolated from the animals euthanized by inhalation of carbon dioxide. The target organs were selected based on findings in previous studies. The 13-week repeated administration study revealed findings suggesting effects on the liver (Watanabe et al., 2008c) and the kidney is the major excretion pathway (Bernard et al. 2008a). Oral administration would result in a relatively high concentration of the test material in the intestinal tract. The tissues from the positive control animals were collected at 14 days after dosing. The sampling
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time was selected according to “Transgenic Animal Mutagenicity Assays (United Nations WHO)” (Wahnshaffe et al. 2005). Since all animals survived, 5 out of 6 animals in a group were selected for gene mutation assay by the ascending order of animal ID number. Tissue samples from the liver, kidney, stomach and duodenum were removed from the unselected animals and stored. Four biopsied liver samples were prepared from the left lateral lobe. The left kidney was sliced in about 1-mm pieces (4 pieces in total). A 5-cm piece from the pyloric part of the stomach was cut out and duodenal contents were washed out with physiological saline solution. These samples were frozen in liquid nitrogen (LN2) and then stored in an ultra-low temperature freezer. As described in Kohara et al (2001), the tissue samples were treated as follows. Frozen tissue fragments were homogenized and centrifuged. The supernatant was discarded, and RNase (Nippon Gene Co., Ltd., Tokyo, Japan) was added to the remaining plates to make a suspension of cells containing nuclei. This suspension was incubated with proteinase K solution comprised of proteinase K (Wako Pure Chemical Industries, Osaka, Japan), sodium dodecyl sulfate (Wako Pure Chemical Industries, Tokyo, Japan) and EDTA (Nippon Gene Co., Ltd., Tokyo, Japan) adjusted to a pH of 7.5 with hydrochloric acid at 50°C until it became a clear solution. The solution was mixed with Phenol/Chloroform mixture (a mixture of Tris-hydroxymethylaminomethane-EDTA (TE) saturated phenol (Nippon Gene Co., Ltd., Tokyo, Japan) and chloroform at a ratio of 1:1) and centrifuged, and then the upper layer (water layer) was collected. This procedure was repeated twice. The water layer was mixed with chloroform/isoamyl alcohol (Wako Pure Chemical Industries, Osaka, Japan) at a ratio of 24:1 and centrifuged. The water layer was then mixed with ethanol and genomic DNA was extracted. After extraction, the DNA was dissolved in TE buffer and packaged into phage particles using Transpack packaging extract
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(Stratagene Japan K.K., Tokyo, Japan). For calculating mutant frequency, the packaged DNA sample was mixed with a suspension of E. coli hfl- (G1250) in a tube; the tube was incubated at room temperature for 20 to 30 minutes to allow phage to infect E. coli. Top agar was added to the tube, and thereafter the contents were poured over a LB (Lysogeny Broth) agar plate composed of Bacto tryptone (Becton, Dickinson and Company, NJ, USA), Bacto yeast extract (Becton, Dickinson and Compan, NJ, USAy), NaCl and Bacto agar (Becton, Dickinson and Company, NJ, USA). The agar plate was incubated at 24°C to 25°C for 44 to 48 hours. The number of plaques on the plate was identified as the number of mutant plaques. For calculating total number of plaques, a part of the suspension prepared for calculating mutant frequency was diluted, and thereafter the diluted solution was transferred to a tube and stirred. Top agar was added to the tube and thereafter the contents were poured over a LB agar plate. The agar plate was incubated at 37°C for 16 to 24 hours. The total number of plaques was calculated using the number of plaques on the plate. The mutant frequency in a specific organ was calculated by dividing the number of mutant plaques by the total number of plaques. Differences in the mutant frequency were analyzed for significance by the Conditional Binomial test (Kastenbaum and Bowman 1970). The results were evaluated as positive when the mutant frequency in the test substance-dose group was significantly different from that in the negative control group. Final judgment was made in consideration of biological relevance under the test conditions. Single Cell Gel (SCG) Assay in Rats- Comet Assays
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Twenty SD strain SPF male rats (Crl:CD(SD, Charles River Laboratories Japan, Inc., Kanagawa, Japan) were purchased at 7 weeks of age. After 8 days of quarantine, healthy animals were randomly assigned to groups (4 animals/group) using a stratified body weight procedure. Unassigned animals were euthanized by CO2 inhalation. Individual body weights on the day of grouping were in the range of 272 to 289 g. No severe toxic effects were observed after 13-week repeated administration of the test substance at 1,000 mg/kg (Kodama et al. 2008). In this study, where the administration period of this test was only 2 days, 2,000 mg/kg, the maximum feasible dose level, was selected as the high-dose level and 1,000 mg/kg as the low dose. The test substance was dissolved in the vehicle and administered to rats orally once daily for 2 consecutive days, 24-hours apart, using a disposable syringe fitted with a teflon intubation tube. The dosage volume was set at 5 mL/kg BW. The positive control substance (EMS) was injected once intraperitoneally at a dose level of 300 mg/kg BW. Individual body weights were measured on the day of the first dosing, and just before sampling of the tissues. Animals were observed for clinical signs at 1 and 24 hours after the first administration, at 1 hour after the final administration and just before sampling of the tissues. As described in Tice et al. (2000), this assay was conducted as follows. Rats were euthanized with by CO2 inhalation 3 hours after the final administration. The liver, kidney and duodenum (a 3-cm piece from the pyloric part of the stomach) were removed. The target organs were selected based on findings from previous studies. The 13-week repeated administration study revealed findings suggesting effects on the liver (Watanabe et al., 2008c) and the kidney is the major excretion pathway (Bernard et al. 2008a). Oral administration would result in a relatively high concentration of the test material in the intestinal tract. Portions of the liver (about 5-mm square from the left lateral lobe), kidney (about one third of the left
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kidney) were collected. About 5 mL of homogenizing buffer was added and the liver sections were homogenized using a Dounce homogenizer (Wheaton Science Products, NJ, USA.). Cell suspensions were chilled on ice for 5 minutes and 10 µL of supernatant was transferred to a microtube. The homogenate was centrifuged at 800 rpm for 5 minutes, supernatant was removed and the precipitate was suspended in homogenizing buffer, which was a mixture of Hanks balanced salt solution, EDTA-2Na and DMSO adjusted to a pH of 7.5 with NaOH. The cell suspension (10 µL) was transferred to a microtube. A superfrost glass slide was pre-coated with 0.6% agarose (Type I, Low EEO) gel dissolved in Dulbecco’s phosphate buffer. Seventy-five microliters of 0.6% low-melting agarose (Lonza Ltd., Basel, Switzerland) gel dissolved in Dulbecco’s phosphate buffer was added to the microtube described above and 75 µL of cell-agarose mixture was placed on the pre-coated slide and covered with a non-coated slide glass. After the agarose gelled, the non-coated slide glass was removed. Another layer of 0.6% low-melting agarose (75 µL) was added in the same manner. Each slide was placed in lysing solution, composed of NaCl, EDTA-2Na, Tris-hydroxymethyl aminomethane, NaOH, N-lauroylsacrosine, Triton-X and DMSO, and stored overnight under refrigerated and light-protected conditions. Three slides per organ were prepared. The slides were then placed in cold electrophoresis buffer (pH >13), composed of sodium hydroxide and EDTA-2Na, for 20 minutes. Electrophoresis for the slides was conducted at a constant voltage of 1 V/cm (36V) (initial current: 300 mA) for 15 minutes under the condition of pH >13. After electrophoresis, the slides were dehydrated with ethanol (≥99.6%). All slides were coded and examined by the masking method. Fifty microliters of ethidium bromide solution (20 µg/mL, Nippon Gene Co., Ltd., Tokyo, Japan) were dropped on the slides after electrophoresis and covered with a coverslip. The length of DNA migration and the
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proportion of DNA with altered migration were measured by fluorescence microscope on a CCD camera monitor with IG excitation and auxiliary absorbing filter (Olympus Co., Tokyo, Japan) and by image analysis using a Comet assay analyzer (Rainbow Star System, Toyobo Co., Ltd., Osaka, Japan). One hundred cells per organ (50 cells per slide), i.e., 400 cells per group (4 animals), were examined. The percentage of DNA in the tail (% tail DNA) and Olive tail moment (Olive et al, 1990) were used as parameters. Olive tail moment = (Tail.mean - Head.mean) × % tail DNA / 100 Tail.mean: The center of gravity of tail Head.mean: The center of gravity of head The Olive tail moment and % tail DNA were analyzed by Dunnett multiple comparison test (Dunnett 1964) between the negative control group and each dose group, and also by AspinWelch t-test (Welch 1938) between the negative control group and the positive control group. DNA-damaging by the test substance was determined based on the presence or absence of significant differences in the Olive tail moment and % tail DNA between the negative control group and dose group. However, final judgment was made in consideration of biological relevance under the test conditions and toxicity information of the test substance. RESULTS Bacterial reverse mutation assay (Ames test) Study results are shown in Table 4 and 5. In TA100 in the -S9 assay, treatment with the test substance produced a dose-dependent and twofold or more increase in the number of revertant colonies compared with the negative control group. The number of revertant colonies in TA98 in the -S9 assay also increased (1.77 fold) compared to the negative control group, but did
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not achieve the two-fold threshold. No increase in the number of revertant colonies was observed for the other test strains in the -S9 assay or in ANY strain in the +S9 assay. The test substance had an inhibitory effect on the growth of TA1537 at >250 µg/plate, on the other strains at >500 µg/plate in the -S9 assay, and all strains at 2500 µg/plate in the +S9 assay. The positive control substances markedly induced reversion in all test strains. Specific activity, as an index of the mutagenic intensity, was 1758 (colonies/mg/plate) In vitro chromosome aberration test The results are presented in Table 6. In the short-term treatment -S9 assay, the incidences of cells with structural chromosome aberration in the groups treated with the test substance were 1.0% at 70.0 µg/mL, 1.5% at 117 µg/mL, and 9.0% at 194 µg/mL; therefore only one dose (194 µg/mL) was evaluated as inconclusive. The incidences of polyploid cells were 0.5% at 70.0 µg/mL, 3.5% at 117 µg/mL, and 8.0% at 194 µg/mL; only one dose (194 µg/mL) was evaluated as inconclusive. Moreover, dose-dependent decreases in the relative cell growth rate were noted, and the relative cell growth rate was 38.6% at 194 µg/mL, which was the highest dose in the evaluation groups for chromosome aberration. In the cells treated with the positive control substance (MMC), a large number of chromosome structural aberrations were observed, and the incidence was 49.0%. In the short-term treatment +S9 assay, the incidences of cells with structural chromosome aberration in the groups treated with the test substance were 2.5% at 540 µg/mL, 2.5% at 900 µg/mL, and 3.5% at 1500 µg/mL, which was less than 5% in each dose. The incidences of polyploid cells were 2.5% at 540 µg/mL, 4.0% at 900 µg/mL, and 2.5% at 1500 µg/mL, which was less than 5% in each dose. Moreover, dose-dependent decreases in the relative cell growth rate were noted, and the relative cell growth rate was 49.7% at 1500 µg/mL, which was the highest dose in the evaluation groups for chromosome
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aberration. In the cells treated with the positive control substance (CP), the incidence of chromosome structural aberrations was 38.0%. In the continuous treatment 24-hour assay, the incidences of cells with structural chromosome aberration in the groups treated with the test substance were 1.0% at 70.0 µg/mL, 0.5% at 117 µg/mL, and 11.0% at 194 µg/mL; as such, only one dose (194 µg/mL) was evaluated as positive. The incidences of polyploid cells were 0.0% at 70.0 µg/mL, 1.5% at 117 µg/mL, and 1.5% at 194 µg/mL, which was less than 5% in each dose. Moreover, dosedependent decreases in the relative cell growth rate were noted, and the relative cell growth rate was 37.1% at 194 µg/mL. This was the highest dose in the evaluation groups for chromosome aberration. The D20 value (mg/mL) and TR value (per mg/mL), were 0.197 and 56.1, respectively. In the cells treated with the positive control substance (MMC), the incidence of structural aberrations was 44.5%. Micronucleus test Each group showed slight decreases in body weights at preparation of bone marrow sample, but there were no markedly differences between the negative control group and the test article-treated groups. No clinical signs were observed throughout the study (data not shown). The results of the micronucleus test are presented in Table 7. In the negative control group, there were 2 to 4 micronucleated cells (MNPCE) in 2000 polychromatic erythrocytes per animal and the incidence of MNPCE was 0.14%. The ratio of polychromatic erythrocytes to the total number of analyzed erythrocytes (ratio of PCE) was 57.1%. The incidences of MNPCE after the administration of the test substance were 0.14% in the 500 mg/kg group, 0.19% in the 1000 mg/kg group, and 0.21% in the
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2000 mg/kg group. No statistically-significant increase was noted in any of the treatment groups compared with the negative control group. These values were almost same as or smaller than the historical data in the testing facility (0.22 %). The ratios of PCE, an index of the effect of the test substance on the bone marrow cells were 58.9%, 59.2%, and 61.6% in the 500, 1000, and 2000 mg/kg groups, respectively. No statistically-significant difference was noted in any of the treatment groups compared with the negative control group. On the other hand, the incidence of MNPCE in the positive control group markedly increased to 0.99% (11 to 25 MNPCE in 2000 PCE), and this increase was statisticallysignificant (p≤0.025) compared with the negative group. The ratio of PCE was 57.8%. Gene mutation assay in transgenic rats Neither apparent suppression of body weight gain nor clinical sign was observed in any animal in the test article treated groups or positive control group. The results of the gene mutation assay are presented in Table 8. For the liver, in the negative control group, 101 mutant plaques out of 2,828,700 total plaques appeared and the mutant frequency was 35.7 × 10-6. The mean among the individuals was 36.7 × 10-6. The mutant frequencies in the test substance treatment groups were 24.5 × 10-6 (mutant plaques/total plaques: 52/2,122,200) for 500 mg/kg and 28.1 × 10–6 (mutant plaques/total plaques: 57/2,026,800) for 1000 mg/kg, and were comparable to the negative control group. The means among the individuals in the 500 and 1000 mg/kg groups were 24.0 × 10-6 and 28.2 × 10-6, respectively. In the positive control group, the mutant frequency was 118.3 × 10-6 (mutant plaques/total plaques: 249/2,104,200) and a statistically-significant (p
