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Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats
Mutation Research 780-781 (2015) 76–80
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Mutation Research/Genetic Toxicology and Environmental Mutagenesis journal homepage: www.elsevier.com/locate/gentox Community address: www.elsevier.com/locate/mutres
Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats Shoji Matsumura a , Naohiro Ikeda a,∗ , Shuichi Hamada b , Wakako Ohyama c , Yumi Wako b , Kazufumi Kawasako b , Toshio Kasamatsu a , Naohiro Nishiyama a a
Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan c Yakult Honsha Co. Ltd., 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan b
a r t i c l e
i n f o
Article history: Received 4 September 2014 Accepted 5 September 2014 Available online 16 September 2014 Keywords: Liver micronucleus assay Gastrointestinal tract micronucleus assay CI Solvent Yellow 14 Sudan I Repeated dose toxicity study
a b s t r a c t The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action. © 2014 Published by Elsevier B.V.
1. Introduction This study was conducted to evaluate the efficacy of repeateddose liver and gastrointestinal tract micronucleus (MN) assays using young adult rats. We examined CI Solvent Yellow 14 (Sudan I) as part of a collaborative study by the Mammalian Mutagenicity Study (MMS) Group, a subgroup of the Japanese Environmental Mutagen Society (JEMS). Sudan I is a water-insoluble mono-azo dye consisting of aniline and 1-amino-2-hydroxynaphthalene (Fig. 1). This dye has been widely used to color hydrocarbon solvents, oils, fats, waxes, shoes and floor polishes [1]. In the 1940s, the dye was used to color margarine, but at present, it is not used in foods, drugs, or cosmetics. In a National Toxicology Program (NTP) bioassay,
∗ Corresponding author at: Safety Science Research, Research and Development, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan. Tel.: +81 285 68 7447; fax: +81 285 68 7452. E-mail address: [email protected] (N. Ikeda). http://dx.doi.org/10.1016/j.mrgentox.2014.09.002 1383-5718/© 2014 Published by Elsevier B.V.
Sudan I was reported to be carcinogenic in rats, causing neoplastic nodules in the liver. In contrast, Sudan I was not carcinogenic in mice [1]. A number of studies have been performed on Sudan I to investigate its genotoxicity in vitro and in vivo; these examinations include mutation, chromosomal aberration and DNA repair end point assays. In vitro studies have produced both positive and negative results in the Salmonella reverse mutation assay with and without metabolic activation [1–3]. Negative results were observed for Sudan I in a CHO chromosomal aberration study [4]. Positive results were reported for in vivo studies using the rat bone marrow micronucleus (BMMN) assay [5–7]. However, negative results were observed in rat liver unscheduled DNA synthesis (UDS) assays [5,8]. Although several contradictory results have been reported, based on the clear evidence of MN induction in rat BMMN assays, Sudan I is considered to be genotoxic in rats. Furthermore, given the carcinogenic potential of Sudan I to rat liver in the NTP bioassay, it is suspected that Sudan I will be genotoxic in the rat liver. Nonetheless, given that Sudan I induced DNA damage in the mouse colon in vivo [9], it is suspected that genotoxicity will also be observed in
S. Matsumura et al. / Mutation Research 780-781 (2015) 76–80
OH N N
Fig. 1. Sudan I. CI Solvent Yellow 14 (CI 12005, Sudan I). CAS No. 842-07-9. CAS Name 1-phenylazo-2-naphthalenol.
the gastrointestinal tract. For the present study, we performed MN assays in the liver, bone marrow and gastrointestinal tract using Sudan I to assess the efficacy of these assays. 2. Materials and methods 2.1. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Yokohama, Japan) and used at 6 weeks of age. The animals were housed in an air-conditioned room with a 12 h light/dark cycle and free access to food and drinking water. Prior to the start of these studies, the experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee. 2.2. Chemicals CI Solvent Yellow 14 (Sudan I, CAS No. 842-07-9, >95% purity) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sudan I was suspended in 0.5% (w/v) Methyl Cellulose Solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) each day before dosing. For the liver MN assay, collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) was dissolved (100 units/mL) in Hanks’ balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA). Acridine orange and 4 ,6-diamidino-2-phenylindole dihydrochloride were purchased from Invitrogen (Oregon, USA). 2.3. Doses levels and treatments The maximum dose for the repeated-dose studies was defined as the dose that induced clinical signs without causing lethality, i.e., the 14-day maximum tolerated dose. This maximum dose was determined according to the published literature [1], as well as from the results of dose range tests conducted in our laboratory. Three dose levels were set at 150, 300, and 600 mg/kg/day. Animals that received the vehicle alone served as the negative control group. Five animals were used in each repeated-dose group. 2.4. MN assays Twenty-four hours after the 14-day administration, the rats were euthanized under anesthesia. The MN assays using the liver, glandular stomach, colon and bone marrow were performed according to the protocol described in the summary report of this collaborative study [10].
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2.4.3. Gastrointestinal tract MN assay The gastrointestinal tract MN assays were performed according to the methods reported by Hamada et al. [10]. Briefly, the stomach was dissected and the forestomach was removed, leaving the gastric cardia. After the contents of the stomach were rinsed out with HBSS, a glass rod was inserted into the gastric cardia, and the glandular stomach was everted on a ball at the end of the glass rod. The everted tissue was incubated in a tube containing a solution composed of 1 mM ethylenediamine-tetraacetic acid disodium salt (EDTA) and 2 mM dithiothreitol in HBSS at 35 ◦ C for 1 h. After incubation, the tissue was vortexed for several minutes to isolate the epithelial cells. The colons were dissected, rinsed with HBSS, and everted on a glass rod. These colons were incubated in a solution of 1 mM EDTA in HBSS at 35 ◦ C for 30 min. The tissue was vortexed every 15 min to isolate the epithelial cells. The collected cells were strained through nylon mesh to remove cell clumps, and fixed in 10% neutral-buffered formalin. Immediately before observation, the cells were stained with AO and DAPI. The stained cells were analyzed under a fluorescence microscope with U excitation. Two thousand intact cells were scored per animal to determine the frequency of micronucleated gastrointestinal tract cells (MNGTCs).
2.5. Histopathological examinations After sacrifice, the liver tissue remaining from the left lateral lobe, after the isolation of the HEPs, was fixed with 10% phosphate-buffered formalin, paraffinembedded, and then stained with hematoxylin and eosin (H.E.) according to the standard method. Histopathological examination was performed using a light microscope.
2.6. Statistical analyses Statistical analyses were also performed according to the summary report of this collaborative study [10]. Differences in the incidences of MNHEPs and MNGTCs between the test and the vehicle control groups were analyzed using the conditional binomial test reported by Kastenbaum and Bowman [11] at the upper-tailed significance levels of 5% and 1%. The exception in our study is that a Chi-squared test was performed to analyze differences in the incidences of micronucleated immature erythrocytes (MNIMEs) between the test groups and the vehicle-treated control group. The data were also statistically analyzed by a multiple comparison test according to the requirements of the organizing committee. Specifically, the homogeneity of the variance was examined using Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied. In cases of heterogeneous variance, the Kruskal–Wallis test was applied. When a statistically significant difference was demonstrated between groups, the difference was assessed by Dunnett’s test or a Dunnett-type multiple comparison test.
3. Results 3.1. Dose setting
2.4.1. Liver MN assay The hepatocyte preparations were obtained according to methods reported by Hamada et al. [10]. Briefly, the livers were excised, and a portion of the left lateral lobe was sliced into several pieces. These strips were rinsed with Hanks’ balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA) and treated with HBSS containing 100 units/mL of collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) in a flask shaken for 1 h at 37 ◦ C. The resulting material was repeatedly pipetted to break apart cell clumps and then forced through a cell strainer. The obtained cell suspension was then fixed with 10% neutral buffered formalin. Immediately before observation, the cell suspension was stained with acridine orange (AO) and 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI), and specimens were prepared. The specimens were microscopically observed with U-excitation (Ultraviolet ray excitation), and the number of micronucleated hepatocytes (MNHEPs) per 2000 parenchymal hepatocytes (HEPs), including mono-, bi-, and multi-nucleated cells, was counted for each animal. MNHEPs were identified using the criteria required by the organizing committee. The number of mitotic phase cells among the 2000 HEPs was also counted to determine the mitotic index (MI). 2.4.2. Bone Marrow MN assay After the removal of the livers as described in the liver MN assay, the femurs were removed from the same animals. BM cells were collected from the femurs according to methods reported by Hamada et al. [10]. Immediately prior to microscopic observation, smear preparations were made with each sample and then stained with an AO solution and covered with a cover slip. The specimens were observed under a fluorescent microscope with B-excitation (Blue light excitation), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 immature erythrocytes (IMEs) was counted for each animal.
We performed a dose range finding study, using as a reference the results of a 14-day oral repeated-dose NTP bioassay [1]. The animals were dosed once daily for 7 days with 75, 150, 300, and 600 mg/kg/day of Sudan I and were monitored for clinical signs and body weight changes. A decrease in body weight was observed in the 600 mg/kg/day group compared with the vehicle control group, but no lethality or decrease in motor activity was observed. Based on these observations, we determined the highest dose as 600 mg/kg/day and set the lower doses with the common ratio of two (300 and 150 mg/kg/day).
3.2. MN assay 3.2.1. Liver MN assay The results of the liver MN assay are shown in Table 1. The mean MI values in the vehicle-treated control groups were 0.05%. The MI values for the groups treated with the test material were similar to those of the vehicle-treated control groups. The MNHEPs frequency in the vehicle-treated group was 0.01%. There was no statistically significant increase in the incidences of MNHEPs with the administration of Sudan I.
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Table 1 Results of the liver MN assay with Sudan I. Sudan I (mg/kg/day)
No. of animals
0
5
150
5
300
5
600
5
MNHEPs (%) individual data (mean ± SD)
MI (%) individual data (mean ± SD)
0.00, 0.00, 0.05, 0.00, 0.00 (0.01 ± 0.02) 0.05, 0.00, 0.05, 0.05, 0.00 (0.03 ± 0.03) 0.00, 0.00, 0.05, 0.00, 0.10 (0.03 ± 0.04) 0.00, 0.00, 0.10, 0.10, 0.10 (0.06 ± 0.05)
0.00, 0.00, 0.05, 0.05, 0.15 (0.05 ± 0.06) 0.00, 0.00, 0.00, 0.10, 0.10 (0.04 ± 0.05) 0.00, 0.00, 0.00, 0.10, 0.15 (0.05 ± 0.07) 0.00, 0.00, 0.10, 0.05, 0.10 (0.05 ± 0.05)
Table 2 Results of the gastrointestinal tract MN assay with Sudan I. Sudan I (mg/kg/day)
No. of animals
0
5
150
5
300
5
600
5
3.2.2. Gastrointestinal tract MN assay The results of the gastrointestinal tract MN assays are shown in Table 2. We examined the oblong epithelial cells isolated from the glandular stomach and colon. In both MN assays, there were no statistically significant increases in the incidences of micronucleated epithelial cells.
3.2.3. Bone marrow MN assay The results of the BMMN assays are shown in Table 3. The MNIME frequencies significantly increased in a dose-dependent manner after the repeated administration of Sudan I. Statistical significance was noted at doses of 150 mg/kg/day or more.
3.3. Histopathology After the 14-day administration of Sudan I, livers were isolated from all of the animals, and the organ weights were measured. Histopathological examination was conducted using the residual liver tissue of the left lateral lobe that was not used for the isolation of HEPs. A dose-dependent increase in the relative liver weight was observed (Table 4). Single cell necrosis was noted at 600 mg/kg/day and hypertrophy of the hepatocytes was observed at 300 and 600 mg/kg/day in a dose-dependent manner. These results indicate that there was sufficient exposure of Sudan I to the rat livers to cause liver injury.
Table 3 Results of the BMMN assay with Sudan I. Sudan I (mg/kg/day)
No. of animals
0
5
150
5
300
5
600
5
*
p < 0.01, Chi-squared test.
MNIMEs (%) individual data (mean ± SD) 0.60, 0.65, 0.75, 0.25, 0.50 (0.55 ± 0.19) 0.75, 1.25, 1.75, 1.10, 1.25 (1.22 ± 0.35* ) 0.65, 1.30, 2.00, 3.25, 1.65 (1.77 ± 0.96* ) 1.80, 2.40, 3.25, 1.75, 2.70 (2.38 ± 0.63* )
Glandular stomach MN cells (%) individual data (mean ± SD)
Colon MN cells (%) individual data (mean ± SD)
0.15, 0.10, 0.05, 0.10, 0.05
0.25, 0.15, 0.05, 0.00, 0.05
(0.09 ± 0.04) 0.15, 0.20, 0.05, 0.00, 0.10 (0.10 ± 0.08) 0.20, 0.10, 0.10, 0.00, 0.05 (0.09 ± 0.08) 0.10, 0.20, 0.05, 0.05, 0.15 (0.11 ± 0.07)
(0.10 ± 0.10) 0.15, 0.05, 0.20, 0.10, 0.05 (0.11 ± 0.07) 0.00, 0.15, 0.15, 0.15, 0.10 (0.11 ± 0.07) 0.05, 0.10, 0.10, 0.15, 0.00 (0.08 ± 0.06)
4. Discussion Repeated-dose rat liver MN assays have been conducted with several hepatocarcinogens to date and are indicated as effective assays for predicting liver carcinogenicity [12,13]. In the present study, we performed repeated-dose liver, gastrointestinal tract, and BMMN assays using Sudan I, which has been reported to be carcinogenic to rat liver with the NTP bioassay [1]. Some of the reported in vitro and in vivo studies have suggested a genotoxic potential for Sudan I in the rat [1–7]. The clear evidence of MN induction during in vivo BMMN assays [5–7], together with the results of the NTP bioassay, suggested that Sudan I may be a genotoxic carcinogen in rats. However, under the conditions of the present study, no significant increase in the MNHEPs was noted, suggesting that the liver carcinogenicity by Sudan I may not be due to genotoxicity. Nevertheless, a statistically significant and dosedependent increase in MNIMEs was observed in the BMMN assay; this effect has been observed in previous studies [5–7]. One of the explanations for these contradictory results is that the amount of the test material was insufficient to form hepatic MN in the rats. However, this explanation is unlikely given that sufficient exposure of the test material to the liver was indicated by the histopathological examination (e.g., single cell necrosis of the hepatocyte, Table 4). An alternate explanation is that the micronucleated cells were induced by the administration of Sudan I but were eliminated by processes like apoptosis during the test period. The number of initiated cells in the liver formed by the administration of genotoxic materials is known to decrease after the treatment, and apoptosis is believed to contribute to the initiated cell elimination [14]. However, this explanation appears unlikely because previous studies with other chemicals have reported that MNHEPs appear to accumulate during the test period [12,13]. Immuno-staining analysis of the liver or time-course monitoring of the MN frequency may answer this question. An explanation supported by our results is that Sudan I is activated by peroxidase in the blood cells and that the resulting metabolites are genotoxic to bone marrow cells. Several reports have shown that Sudan I can be oxidized by peroxidase in vitro, and this reaction generates metabolites that can form guanosine DNA adducts [15,16]. Sudan I DNA adducts derived from peroxidase metabolites were also identified in vivo in the urinary bladders
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Table 4 Results of the histopathological examination. Sudan I (mg/kg/day)
0
150
300
600
No. of animals Organ weight (% to body weight) Hypertrophy, hepatocyte, perilobular − + ++ +++ ++++ Single cell necrosis, hepatocyte, diffuse − + ++ +++ ++++
5 3.1 ± 0.2
5 4.4 ± 0.3*
5 5.4 ± 0.8*
5 6.4 ± 0.7*
5 – – – –
5 – – – –
1 1 3 – –
– – 2 3 –
5 – – – –
5 – – – –
5 – – – –
2 3 – – –
Grade: −: none; +: minimal; ++: mild; +++: moderate; ++++: severe. * p < 0.01, Dunnett’s test.
of rats, which have a relatively high level of tissue peroxidases [17,18]. In contrast to the results in the urinary bladder, no increase in hepatic Sudan I DNA adducts was observed in this report [17]. Under this hypothesis, it is perhaps reasonable that a genotoxic effect was not observed in the liver, while a clear positive result was observed with the BMMN assay. Another explanation for a lack of liver MN induction might be possible under the hypothesis that Sudan I and its metabolites were readily eliminated from the liver by phase II detoxification enzymes. It has been reported that the azo-dye and its main biotransformation product, an aromatic amine, are conjugated to glucuronate or glutathione [19,20]. In our study, hepatic enzyme induction was suggested from the observation of the liver histopathology [21]. In either case, previous rat liver UDS assays demonstrated that Sudan I was not mutagenic to the liver [5,8]. In these reports, the test material also induced an increase in MN in a BMMN assay. Additionally, Sudan I was reported to induce hepatocellular altered foci in a medium term liver carcinogenicity study in which the test material was administered as a promoting agent following initiation by diethylnitrosamine [22]. Therefore, the mode of action of the liver carcinogenicity of Sudan I is believed in part to be a non-genotoxic mechanism. These reports in the literature agree with our results. In the gastrointestinal tract MN assays, no treatment-related increase in micronucleated cells was observed. Sudan I has been reported to induce DNA strand breaks in the colon tissue of mice [9]. However, the dose was higher than the dose used in the present studies (i.e., 1000 mg/kg). Azo compounds may be less likely to induce DNA strand breaks in rat colon than in mice due to differences in their gut micro floral metabolism; Shimada et al. demonstrated that several azo-based colorants did not induce DNA damage to rat colon or stomach, but did induce DNA damage in mice [23]. It is therefore reasonable that Sudan I did not exhibit genotoxicity to the epithelial cells of the gastrointestinal tract under the tested conditions. In conclusion, Sudan I was genotoxic in bone marrow but not in the liver or the gastrointestinal tract. Based on our results and other relevant information, the repeated-dose rat liver and gastrointestinal tract MN assays are useful methods that enable the assessment of genotoxic potential in multiple organs. These methods also contribute to a reduction in the number of animals studied because they can be easily integrated into a repeated-dose general toxicity study.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments A part of this work was supported by the Health and Labour Sciences Research Grant H24-Chemistry-Designation-008 (Ministry of Health, Labour and Welfare). We would like to give special thanks to Dr. Osamu Morita for proofreading and for his many valuable suggestions for the improvement of the manuscript. References [1] Carcinogenesis Bioassay of C.I. Solvent Yellow 14 (CAS No. 842-07-9) in F344/N Rats and B6C3F1 Mice (Feed Study), Natl. Toxicol. Program Tech. Rep. Ser. 226 (1982) 1–164. [2] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals, Environ. Mol. Mutagen. 11 (Suppl. 12) (1988) 1–157. [3] P. Møller, H. Wallin, Genotoxic hazards of azo pigments and other colorants related to 1-phenylazo-2-hydroxynaphthalene, Mutat. Res. 462 (2000) 13–30. [4] J.L. Ivett, B.M. Brown, C. Rodgers, B.E. Anderson, M.A. Resnick, E. Zeiger, Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals, Environ. Mol. Mutagen. 14 (1989) 165–187. [5] B.M. Elliott, K. Griffiths, J.M. Mackay, J.D. Wade, CI solvent yellow 14 shows activity in the bone marrow micronucleus assay in both the rat and mouse, Mutagenesis 12 (1997) 255–258. [6] C. Westmoreland, D.G. Gatehouse, The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observations on species and tissue specificity), Carcinogenesis 12 (1991) 1403–1407. [7] A. Wakata, Y. Miyamae, S. Sato, T. Suzuki, T. Morita, N. Asano, T. Awogi, K. Kondo, M. Hayashi, Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group, Environ. Mol. Mutagen. 32 (1998) 84–100. [8] J.C. Mirsalis, C.K. Tyson, K.L. Steinmetz, E.K. Loh, C.M. Hamilton, J.P. Bakke, J.W. Spalding, Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds, Environ. Mol. Mutagen. 14 (1989) 155–164. [9] S. Tsuda, N. Matsusaka, H. Madarame, S. Ueno, N. Susa, K. Ishida, N. Kawamura, K. Sekihashi, Y.F. Sasaki, The comet assay in eight mouse organs: results with 24 azo compounds, Mutat. Res. 465 (2000) 11–26. [10] S. Hamada, W. Ohyama, R. Takashima, K. Shimada, K. Matsumoto, S. Kawakami, F. Uno, H. Sui, Y. Shimada, T. Imamura, S. Matsumura, H. Sanada, K. Inoue, S. Muto, I. Ogawa, A. Hayashi, T. Takayanagi, Y. Ogiwara, A. Maeda, E. Okada, Y. Terashima, H. Takasawa, K. Narumi, Y. Wako, K. Kawasako, M. Sano, N. Ohashi, T. Morita, H. Kojima, M. Honma, M. Hayashi, Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: Summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 780–781 (2015) 2–17. [11] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutat. Res. 9 (1970) 527–549. [12] K. Narumi, K. Ashizawa, R. Takashima, H. Takasawa, S. Katayama, Y. Tsuzuki, H. Tatemoto, T. Morita, M. Hayashi, S. Hamada, Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene, Mutat. Res. 747 (2012) 234–239.
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Repeated-dose liver and gastrointestinal tract micronucleus assays with CI Solvent Yellow 14 (Sudan I) using young adult rats Shoji Matsumura a , Naohiro Ikeda a,∗ , Shuichi Hamada b , Wakako Ohyama c , Yumi Wako b , Kazufumi Kawasako b , Toshio Kasamatsu a , Naohiro Nishiyama a a
Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan c Yakult Honsha Co. Ltd., 1796 Yaho, Kunitachi-shi, Tokyo 186-8650, Japan b
a r t i c l e
i n f o
Article history: Received 4 September 2014 Accepted 5 September 2014 Available online 16 September 2014 Keywords: Liver micronucleus assay Gastrointestinal tract micronucleus assay CI Solvent Yellow 14 Sudan I Repeated dose toxicity study
a b s t r a c t The in vivo genotoxicity of CI Solvent Yellow 14 (Sudan I) was examined using repeated-dose liver and gastrointestinal tract micronucleus (MN) assays in young adult rats. Sudan I is a mono-azo dye based on aniline and 1-amino-2-hydroxynaphthalene. This dye was demonstrated as a rat liver carcinogen in a National Toxicology Program (NTP) bioassay, and genotoxicity was noted in a rat bone marrow micronucleus (BMMN) assay. In the present study, Sudan I was administered orally to rats for 14-days, and the MN frequency in the liver, stomach, colon, and bone marrow were analyzed. The frequency of micronucleated hepatocytes (MNHEPs) was not significantly increased by the administration of the Sudan I. Gastrointestinal tract MNs were also not induced. However, in the BMMN assay, a significant increase in micronucleated immature erythrocytes (MNIMEs) was observed in a dose-dependent manner. While Sudan I has been reported to lack hepatic genotoxicity, it has also exhibited tumor-promoting activities. These results are consistent with the lack of induction of MN in the hepatocytes. The lack of MN induction in cells of the gastrointestinal tract was also logical because azo-compounds are reported to be unlikely to induce DNA damage in the rat gut. The repeated-dose rat liver and gastrointestinal tract MN assays have the potential to be used in the evaluation of the genotoxicity of a chemical in each organ in accordance with its mode of action. © 2014 Published by Elsevier B.V.
1. Introduction This study was conducted to evaluate the efficacy of repeateddose liver and gastrointestinal tract micronucleus (MN) assays using young adult rats. We examined CI Solvent Yellow 14 (Sudan I) as part of a collaborative study by the Mammalian Mutagenicity Study (MMS) Group, a subgroup of the Japanese Environmental Mutagen Society (JEMS). Sudan I is a water-insoluble mono-azo dye consisting of aniline and 1-amino-2-hydroxynaphthalene (Fig. 1). This dye has been widely used to color hydrocarbon solvents, oils, fats, waxes, shoes and floor polishes [1]. In the 1940s, the dye was used to color margarine, but at present, it is not used in foods, drugs, or cosmetics. In a National Toxicology Program (NTP) bioassay,
∗ Corresponding author at: Safety Science Research, Research and Development, Kao Corporation, 2606 Akabane, Ichikai-machi, Haga-gun, Tochigi 321-3497, Japan. Tel.: +81 285 68 7447; fax: +81 285 68 7452. E-mail address: [email protected] (N. Ikeda). http://dx.doi.org/10.1016/j.mrgentox.2014.09.002 1383-5718/© 2014 Published by Elsevier B.V.
Sudan I was reported to be carcinogenic in rats, causing neoplastic nodules in the liver. In contrast, Sudan I was not carcinogenic in mice [1]. A number of studies have been performed on Sudan I to investigate its genotoxicity in vitro and in vivo; these examinations include mutation, chromosomal aberration and DNA repair end point assays. In vitro studies have produced both positive and negative results in the Salmonella reverse mutation assay with and without metabolic activation [1–3]. Negative results were observed for Sudan I in a CHO chromosomal aberration study [4]. Positive results were reported for in vivo studies using the rat bone marrow micronucleus (BMMN) assay [5–7]. However, negative results were observed in rat liver unscheduled DNA synthesis (UDS) assays [5,8]. Although several contradictory results have been reported, based on the clear evidence of MN induction in rat BMMN assays, Sudan I is considered to be genotoxic in rats. Furthermore, given the carcinogenic potential of Sudan I to rat liver in the NTP bioassay, it is suspected that Sudan I will be genotoxic in the rat liver. Nonetheless, given that Sudan I induced DNA damage in the mouse colon in vivo [9], it is suspected that genotoxicity will also be observed in
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OH N N
Fig. 1. Sudan I. CI Solvent Yellow 14 (CI 12005, Sudan I). CAS No. 842-07-9. CAS Name 1-phenylazo-2-naphthalenol.
the gastrointestinal tract. For the present study, we performed MN assays in the liver, bone marrow and gastrointestinal tract using Sudan I to assess the efficacy of these assays. 2. Materials and methods 2.1. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. (Yokohama, Japan) and used at 6 weeks of age. The animals were housed in an air-conditioned room with a 12 h light/dark cycle and free access to food and drinking water. Prior to the start of these studies, the experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee. 2.2. Chemicals CI Solvent Yellow 14 (Sudan I, CAS No. 842-07-9, >95% purity) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sudan I was suspended in 0.5% (w/v) Methyl Cellulose Solution (Wako Pure Chemical Industries, Ltd., Osaka, Japan) each day before dosing. For the liver MN assay, collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) was dissolved (100 units/mL) in Hanks’ balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA). Acridine orange and 4 ,6-diamidino-2-phenylindole dihydrochloride were purchased from Invitrogen (Oregon, USA). 2.3. Doses levels and treatments The maximum dose for the repeated-dose studies was defined as the dose that induced clinical signs without causing lethality, i.e., the 14-day maximum tolerated dose. This maximum dose was determined according to the published literature [1], as well as from the results of dose range tests conducted in our laboratory. Three dose levels were set at 150, 300, and 600 mg/kg/day. Animals that received the vehicle alone served as the negative control group. Five animals were used in each repeated-dose group. 2.4. MN assays Twenty-four hours after the 14-day administration, the rats were euthanized under anesthesia. The MN assays using the liver, glandular stomach, colon and bone marrow were performed according to the protocol described in the summary report of this collaborative study [10].
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2.4.3. Gastrointestinal tract MN assay The gastrointestinal tract MN assays were performed according to the methods reported by Hamada et al. [10]. Briefly, the stomach was dissected and the forestomach was removed, leaving the gastric cardia. After the contents of the stomach were rinsed out with HBSS, a glass rod was inserted into the gastric cardia, and the glandular stomach was everted on a ball at the end of the glass rod. The everted tissue was incubated in a tube containing a solution composed of 1 mM ethylenediamine-tetraacetic acid disodium salt (EDTA) and 2 mM dithiothreitol in HBSS at 35 ◦ C for 1 h. After incubation, the tissue was vortexed for several minutes to isolate the epithelial cells. The colons were dissected, rinsed with HBSS, and everted on a glass rod. These colons were incubated in a solution of 1 mM EDTA in HBSS at 35 ◦ C for 30 min. The tissue was vortexed every 15 min to isolate the epithelial cells. The collected cells were strained through nylon mesh to remove cell clumps, and fixed in 10% neutral-buffered formalin. Immediately before observation, the cells were stained with AO and DAPI. The stained cells were analyzed under a fluorescence microscope with U excitation. Two thousand intact cells were scored per animal to determine the frequency of micronucleated gastrointestinal tract cells (MNGTCs).
2.5. Histopathological examinations After sacrifice, the liver tissue remaining from the left lateral lobe, after the isolation of the HEPs, was fixed with 10% phosphate-buffered formalin, paraffinembedded, and then stained with hematoxylin and eosin (H.E.) according to the standard method. Histopathological examination was performed using a light microscope.
2.6. Statistical analyses Statistical analyses were also performed according to the summary report of this collaborative study [10]. Differences in the incidences of MNHEPs and MNGTCs between the test and the vehicle control groups were analyzed using the conditional binomial test reported by Kastenbaum and Bowman [11] at the upper-tailed significance levels of 5% and 1%. The exception in our study is that a Chi-squared test was performed to analyze differences in the incidences of micronucleated immature erythrocytes (MNIMEs) between the test groups and the vehicle-treated control group. The data were also statistically analyzed by a multiple comparison test according to the requirements of the organizing committee. Specifically, the homogeneity of the variance was examined using Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied. In cases of heterogeneous variance, the Kruskal–Wallis test was applied. When a statistically significant difference was demonstrated between groups, the difference was assessed by Dunnett’s test or a Dunnett-type multiple comparison test.
3. Results 3.1. Dose setting
2.4.1. Liver MN assay The hepatocyte preparations were obtained according to methods reported by Hamada et al. [10]. Briefly, the livers were excised, and a portion of the left lateral lobe was sliced into several pieces. These strips were rinsed with Hanks’ balanced salt solution (HBSS; GIBCO-Invitrogen, Carlsbad, CA, USA) and treated with HBSS containing 100 units/mL of collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) in a flask shaken for 1 h at 37 ◦ C. The resulting material was repeatedly pipetted to break apart cell clumps and then forced through a cell strainer. The obtained cell suspension was then fixed with 10% neutral buffered formalin. Immediately before observation, the cell suspension was stained with acridine orange (AO) and 4 ,6-diamidino-2-phenylindole dihydrochloride (DAPI), and specimens were prepared. The specimens were microscopically observed with U-excitation (Ultraviolet ray excitation), and the number of micronucleated hepatocytes (MNHEPs) per 2000 parenchymal hepatocytes (HEPs), including mono-, bi-, and multi-nucleated cells, was counted for each animal. MNHEPs were identified using the criteria required by the organizing committee. The number of mitotic phase cells among the 2000 HEPs was also counted to determine the mitotic index (MI). 2.4.2. Bone Marrow MN assay After the removal of the livers as described in the liver MN assay, the femurs were removed from the same animals. BM cells were collected from the femurs according to methods reported by Hamada et al. [10]. Immediately prior to microscopic observation, smear preparations were made with each sample and then stained with an AO solution and covered with a cover slip. The specimens were observed under a fluorescent microscope with B-excitation (Blue light excitation), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 immature erythrocytes (IMEs) was counted for each animal.
We performed a dose range finding study, using as a reference the results of a 14-day oral repeated-dose NTP bioassay [1]. The animals were dosed once daily for 7 days with 75, 150, 300, and 600 mg/kg/day of Sudan I and were monitored for clinical signs and body weight changes. A decrease in body weight was observed in the 600 mg/kg/day group compared with the vehicle control group, but no lethality or decrease in motor activity was observed. Based on these observations, we determined the highest dose as 600 mg/kg/day and set the lower doses with the common ratio of two (300 and 150 mg/kg/day).
3.2. MN assay 3.2.1. Liver MN assay The results of the liver MN assay are shown in Table 1. The mean MI values in the vehicle-treated control groups were 0.05%. The MI values for the groups treated with the test material were similar to those of the vehicle-treated control groups. The MNHEPs frequency in the vehicle-treated group was 0.01%. There was no statistically significant increase in the incidences of MNHEPs with the administration of Sudan I.
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Table 1 Results of the liver MN assay with Sudan I. Sudan I (mg/kg/day)
No. of animals
0
5
150
5
300
5
600
5
MNHEPs (%) individual data (mean ± SD)
MI (%) individual data (mean ± SD)
0.00, 0.00, 0.05, 0.00, 0.00 (0.01 ± 0.02) 0.05, 0.00, 0.05, 0.05, 0.00 (0.03 ± 0.03) 0.00, 0.00, 0.05, 0.00, 0.10 (0.03 ± 0.04) 0.00, 0.00, 0.10, 0.10, 0.10 (0.06 ± 0.05)
0.00, 0.00, 0.05, 0.05, 0.15 (0.05 ± 0.06) 0.00, 0.00, 0.00, 0.10, 0.10 (0.04 ± 0.05) 0.00, 0.00, 0.00, 0.10, 0.15 (0.05 ± 0.07) 0.00, 0.00, 0.10, 0.05, 0.10 (0.05 ± 0.05)
Table 2 Results of the gastrointestinal tract MN assay with Sudan I. Sudan I (mg/kg/day)
No. of animals
0
5
150
5
300
5
600
5
3.2.2. Gastrointestinal tract MN assay The results of the gastrointestinal tract MN assays are shown in Table 2. We examined the oblong epithelial cells isolated from the glandular stomach and colon. In both MN assays, there were no statistically significant increases in the incidences of micronucleated epithelial cells.
3.2.3. Bone marrow MN assay The results of the BMMN assays are shown in Table 3. The MNIME frequencies significantly increased in a dose-dependent manner after the repeated administration of Sudan I. Statistical significance was noted at doses of 150 mg/kg/day or more.
3.3. Histopathology After the 14-day administration of Sudan I, livers were isolated from all of the animals, and the organ weights were measured. Histopathological examination was conducted using the residual liver tissue of the left lateral lobe that was not used for the isolation of HEPs. A dose-dependent increase in the relative liver weight was observed (Table 4). Single cell necrosis was noted at 600 mg/kg/day and hypertrophy of the hepatocytes was observed at 300 and 600 mg/kg/day in a dose-dependent manner. These results indicate that there was sufficient exposure of Sudan I to the rat livers to cause liver injury.
Table 3 Results of the BMMN assay with Sudan I. Sudan I (mg/kg/day)
No. of animals
0
5
150
5
300
5
600
5
*
p < 0.01, Chi-squared test.
MNIMEs (%) individual data (mean ± SD) 0.60, 0.65, 0.75, 0.25, 0.50 (0.55 ± 0.19) 0.75, 1.25, 1.75, 1.10, 1.25 (1.22 ± 0.35* ) 0.65, 1.30, 2.00, 3.25, 1.65 (1.77 ± 0.96* ) 1.80, 2.40, 3.25, 1.75, 2.70 (2.38 ± 0.63* )
Glandular stomach MN cells (%) individual data (mean ± SD)
Colon MN cells (%) individual data (mean ± SD)
0.15, 0.10, 0.05, 0.10, 0.05
0.25, 0.15, 0.05, 0.00, 0.05
(0.09 ± 0.04) 0.15, 0.20, 0.05, 0.00, 0.10 (0.10 ± 0.08) 0.20, 0.10, 0.10, 0.00, 0.05 (0.09 ± 0.08) 0.10, 0.20, 0.05, 0.05, 0.15 (0.11 ± 0.07)
(0.10 ± 0.10) 0.15, 0.05, 0.20, 0.10, 0.05 (0.11 ± 0.07) 0.00, 0.15, 0.15, 0.15, 0.10 (0.11 ± 0.07) 0.05, 0.10, 0.10, 0.15, 0.00 (0.08 ± 0.06)
4. Discussion Repeated-dose rat liver MN assays have been conducted with several hepatocarcinogens to date and are indicated as effective assays for predicting liver carcinogenicity [12,13]. In the present study, we performed repeated-dose liver, gastrointestinal tract, and BMMN assays using Sudan I, which has been reported to be carcinogenic to rat liver with the NTP bioassay [1]. Some of the reported in vitro and in vivo studies have suggested a genotoxic potential for Sudan I in the rat [1–7]. The clear evidence of MN induction during in vivo BMMN assays [5–7], together with the results of the NTP bioassay, suggested that Sudan I may be a genotoxic carcinogen in rats. However, under the conditions of the present study, no significant increase in the MNHEPs was noted, suggesting that the liver carcinogenicity by Sudan I may not be due to genotoxicity. Nevertheless, a statistically significant and dosedependent increase in MNIMEs was observed in the BMMN assay; this effect has been observed in previous studies [5–7]. One of the explanations for these contradictory results is that the amount of the test material was insufficient to form hepatic MN in the rats. However, this explanation is unlikely given that sufficient exposure of the test material to the liver was indicated by the histopathological examination (e.g., single cell necrosis of the hepatocyte, Table 4). An alternate explanation is that the micronucleated cells were induced by the administration of Sudan I but were eliminated by processes like apoptosis during the test period. The number of initiated cells in the liver formed by the administration of genotoxic materials is known to decrease after the treatment, and apoptosis is believed to contribute to the initiated cell elimination [14]. However, this explanation appears unlikely because previous studies with other chemicals have reported that MNHEPs appear to accumulate during the test period [12,13]. Immuno-staining analysis of the liver or time-course monitoring of the MN frequency may answer this question. An explanation supported by our results is that Sudan I is activated by peroxidase in the blood cells and that the resulting metabolites are genotoxic to bone marrow cells. Several reports have shown that Sudan I can be oxidized by peroxidase in vitro, and this reaction generates metabolites that can form guanosine DNA adducts [15,16]. Sudan I DNA adducts derived from peroxidase metabolites were also identified in vivo in the urinary bladders
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Table 4 Results of the histopathological examination. Sudan I (mg/kg/day)
0
150
300
600
No. of animals Organ weight (% to body weight) Hypertrophy, hepatocyte, perilobular − + ++ +++ ++++ Single cell necrosis, hepatocyte, diffuse − + ++ +++ ++++
5 3.1 ± 0.2
5 4.4 ± 0.3*
5 5.4 ± 0.8*
5 6.4 ± 0.7*
5 – – – –
5 – – – –
1 1 3 – –
– – 2 3 –
5 – – – –
5 – – – –
5 – – – –
2 3 – – –
Grade: −: none; +: minimal; ++: mild; +++: moderate; ++++: severe. * p < 0.01, Dunnett’s test.
of rats, which have a relatively high level of tissue peroxidases [17,18]. In contrast to the results in the urinary bladder, no increase in hepatic Sudan I DNA adducts was observed in this report [17]. Under this hypothesis, it is perhaps reasonable that a genotoxic effect was not observed in the liver, while a clear positive result was observed with the BMMN assay. Another explanation for a lack of liver MN induction might be possible under the hypothesis that Sudan I and its metabolites were readily eliminated from the liver by phase II detoxification enzymes. It has been reported that the azo-dye and its main biotransformation product, an aromatic amine, are conjugated to glucuronate or glutathione [19,20]. In our study, hepatic enzyme induction was suggested from the observation of the liver histopathology [21]. In either case, previous rat liver UDS assays demonstrated that Sudan I was not mutagenic to the liver [5,8]. In these reports, the test material also induced an increase in MN in a BMMN assay. Additionally, Sudan I was reported to induce hepatocellular altered foci in a medium term liver carcinogenicity study in which the test material was administered as a promoting agent following initiation by diethylnitrosamine [22]. Therefore, the mode of action of the liver carcinogenicity of Sudan I is believed in part to be a non-genotoxic mechanism. These reports in the literature agree with our results. In the gastrointestinal tract MN assays, no treatment-related increase in micronucleated cells was observed. Sudan I has been reported to induce DNA strand breaks in the colon tissue of mice [9]. However, the dose was higher than the dose used in the present studies (i.e., 1000 mg/kg). Azo compounds may be less likely to induce DNA strand breaks in rat colon than in mice due to differences in their gut micro floral metabolism; Shimada et al. demonstrated that several azo-based colorants did not induce DNA damage to rat colon or stomach, but did induce DNA damage in mice [23]. It is therefore reasonable that Sudan I did not exhibit genotoxicity to the epithelial cells of the gastrointestinal tract under the tested conditions. In conclusion, Sudan I was genotoxic in bone marrow but not in the liver or the gastrointestinal tract. Based on our results and other relevant information, the repeated-dose rat liver and gastrointestinal tract MN assays are useful methods that enable the assessment of genotoxic potential in multiple organs. These methods also contribute to a reduction in the number of animals studied because they can be easily integrated into a repeated-dose general toxicity study.
Conflict of interest The authors declare that there are no conflicts of interest.
Acknowledgments A part of this work was supported by the Health and Labour Sciences Research Grant H24-Chemistry-Designation-008 (Ministry of Health, Labour and Welfare). We would like to give special thanks to Dr. Osamu Morita for proofreading and for his many valuable suggestions for the improvement of the manuscript. References [1] Carcinogenesis Bioassay of C.I. Solvent Yellow 14 (CAS No. 842-07-9) in F344/N Rats and B6C3F1 Mice (Feed Study), Natl. Toxicol. Program Tech. Rep. Ser. 226 (1982) 1–164. [2] E. Zeiger, B. Anderson, S. Haworth, T. Lawlor, K. Mortelmans, Salmonella mutagenicity tests: IV. Results from the testing of 300 chemicals, Environ. Mol. Mutagen. 11 (Suppl. 12) (1988) 1–157. [3] P. Møller, H. Wallin, Genotoxic hazards of azo pigments and other colorants related to 1-phenylazo-2-hydroxynaphthalene, Mutat. Res. 462 (2000) 13–30. [4] J.L. Ivett, B.M. Brown, C. Rodgers, B.E. Anderson, M.A. Resnick, E. Zeiger, Chromosomal aberrations and sister chromatid exchange tests in Chinese hamster ovary cells in vitro. IV. Results with 15 chemicals, Environ. Mol. Mutagen. 14 (1989) 165–187. [5] B.M. Elliott, K. Griffiths, J.M. Mackay, J.D. Wade, CI solvent yellow 14 shows activity in the bone marrow micronucleus assay in both the rat and mouse, Mutagenesis 12 (1997) 255–258. [6] C. Westmoreland, D.G. Gatehouse, The differential clastogenicity of Solvent Yellow 14 and FD & C Yellow No. 6 in vivo in the rodent micronucleus test (observations on species and tissue specificity), Carcinogenesis 12 (1991) 1403–1407. [7] A. Wakata, Y. Miyamae, S. Sato, T. Suzuki, T. Morita, N. Asano, T. Awogi, K. Kondo, M. Hayashi, Evaluation of the rat micronucleus test with bone marrow and peripheral blood: summary of the 9th collaborative study by CSGMT/JEMS. MMS. Collaborative Study Group for the Micronucleus Test. Environmental Mutagen Society of Japan. Mammalian Mutagenicity Study Group, Environ. Mol. Mutagen. 32 (1998) 84–100. [8] J.C. Mirsalis, C.K. Tyson, K.L. Steinmetz, E.K. Loh, C.M. Hamilton, J.P. Bakke, J.W. Spalding, Measurement of unscheduled DNA synthesis and S-phase synthesis in rodent hepatocytes following in vivo treatment: testing of 24 compounds, Environ. Mol. Mutagen. 14 (1989) 155–164. [9] S. Tsuda, N. Matsusaka, H. Madarame, S. Ueno, N. Susa, K. Ishida, N. Kawamura, K. Sekihashi, Y.F. Sasaki, The comet assay in eight mouse organs: results with 24 azo compounds, Mutat. Res. 465 (2000) 11–26. [10] S. Hamada, W. Ohyama, R. Takashima, K. Shimada, K. Matsumoto, S. Kawakami, F. Uno, H. Sui, Y. Shimada, T. Imamura, S. Matsumura, H. Sanada, K. Inoue, S. Muto, I. Ogawa, A. Hayashi, T. Takayanagi, Y. Ogiwara, A. Maeda, E. Okada, Y. Terashima, H. Takasawa, K. Narumi, Y. Wako, K. Kawasako, M. Sano, N. Ohashi, T. Morita, H. Kojima, M. Honma, M. Hayashi, Evaluation of the repeated-dose liver and gastrointestinal tract micronucleus assays with 22 chemicals using young adult rats: Summary of the collaborative study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/The Japanese Environmental Mutagen Society (JEMS) Mammalian Mutagenicity Study Group (MMS), Mutat. Res. 780–781 (2015) 2–17. [11] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutat. Res. 9 (1970) 527–549. [12] K. Narumi, K. Ashizawa, R. Takashima, H. Takasawa, S. Katayama, Y. Tsuzuki, H. Tatemoto, T. Morita, M. Hayashi, S. Hamada, Development of a repeated-dose liver micronucleus assay using adult rats: an investigation of diethylnitrosamine and 2,4-diaminotoluene, Mutat. Res. 747 (2012) 234–239.
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