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The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats
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The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats Kenji Inoue a,∗ , Akimu Ochi a , Akira Koda a , Yumi Wako b , Kazufumi Kawasako b , Takaaki Doi a a b
Maruho Co., Ltd., 93 Awata-cho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan
a r t i c l e
i n f o
Article history: Received 9 April 2014 Accepted 10 April 2014 Available online xxx Keywords: Liver micronucleus assay Methapyrilene hydrochloride Rat Repeated-dose toxicity study Hepatocarcinogen Integration
a b s t r a c t The repeated dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect genotoxic hepatocarcinogens that can be integrated into a general toxicity study. The assay methods were thoroughly validated by 19 Japanese facilities. Methapyrilene hydrochloride (MP), known to be a non-genotoxic hepatocarcinogen, was examined in the present study. MP was dosed orally at 10, 30 and 100 mg/kg/day to 6-week-old male Crl:CD (SD) rats daily for 14 days. Treatment with MP resulted in an increase in micronucleated hepatocytes (MNHEPs) with a dosage of only 100 mg/kg/day. At this dose level, cytotoxicity followed by regenerative cell growth was noted in the liver. These findings suggest that MP may induce clastogenic effects indirectly on the liver or hepatotoxicity of MP followed by regeneration may cause increase in spontaneous incidence of MNHEPs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A collaborative study by the Mammalian Mutagenicity Study (MMS) Group, which is a subgroup of the Japanese Environmental Mutagen Society (JEMS), was conducted to evaluate the suitability of the repeated dose liver and gastrointestinal tract micronucleus assays using young adult rats. This study, using methapyrilene hydrochloride (MP), was performed as part of a collaborative study at Maruho Co., Ltd. MP is an antihistaminic compound that was once used as a popular over-the-counter sleep-aid component and was also used in cold and allergy medications. However, MP was found to induce hepatocellular carcinomas and cholangiocarcinomas in rats [1], and it was subsequently withdrawn from the market. MP gave positive [2] and negative [3] results under metabolism-activated conditions for the in vitro mouse lymphoma assay (MLA), but MP gave negative results for the Ames assay [4], in vivo Comet assay [5], UDS assay [6] and DNA adduct assay [7–9]. These data are consistent with the hypothesis that MP is carcinogenic in rats via non-genotoxic mechanisms [6,10].
∗ Corresponding author at: Kyoto R&D Center, Toxicological Research, Maruho Co., Ltd., 93 Awata-cho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan. Tel.: +81 75 325 3255; fax: +81 75 325 3222. E-mail address: inoue [email protected] (K. Inoue).
In this study, MP was used as a non-genotoxic hepatocarcinogen to examine the selectivity of the liver MN assay. To assess the possibility that the liver MN assay could be incorporated into a general toxicity study, 6-week-old rats, which are usually used in these types of studies, were used. At the same time, we attempted to assess whether the dose levels in the 4-week, repeated-dose toxicity study would be compatible with the dosage levels required for an acceptable liver MN assay. 2. Materials and methods 2.1. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. and were aged 6 weeks with weights between 180 and 200 g at the beginning of the experiments. The animals were housed one per cage in an air-conditioned room with a 12 h light/dark cycle and free access to food and drinking water. The animal experiments were approved by the Institutional Animal Care and Use Committee prior to conducting the experiments.
2.2. Chemicals Methapyrilene hydrochloride (MP; CAS No. 135-23-9, 99.6% purity) was purchased from Sigma–Aldrich Co., LLC (Tokyo, Japan). MP was dissolved in water (Otsuka Pharmaceutical Factory, Inc., Japan) at concentrations of 1, 3 and 10 mg/mL before use. A positive control, diethylnitrosamine (DEN; CAS No. 55-18-5, 99.9% purity), was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). DEN was dissolved in water at a concentration of 1.25 mg/mL before use.
http://dx.doi.org/10.1016/j.mrgentox.2014.04.004 1383-5718/© 2014 Elsevier B.V. All rights reserved.
Please cite this article in press as: K. Inoue, et al., The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.04.004
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2 2.3. Doses levels and treatment
2.6. Histopathological examination
The maximum dose for repeated dose studies was defined as the dose inducing clinical signs without causing lethality. The high dose for MP (100 mg/kg/day) was determined according to published literature information [5]. Three dose levels were set, and the mid- and low-dose levels were each approximately 1/3 of the next higher dose. DEN given at 12.5 mg/kg/day served as a positive control for the liver MN assay. Animals were divided into groups of 5 and received the test chemicals or the vehicle alone by oral gavage (10 mL/kg of body weight volume) once a day for 14 consecutive days. Clinical signs and mortality were observed every day, and the body weights were measured twice a week. At 24 h following the last administration, fasted rats were euthanised under isoflurane anaesthesia. Blood samples were used for blood chemistry examinations. Livers were subjected to the liver MN assay and histopathological examination after weighting. Femurs were used for the bone marrow MN (BM MN) assay.
Upon euthanasia, the residual liver tissue of the left lateral lobe after the isolation of HEPs was fixed with 10% phosphate buffered formalin, embedded in paraffin, and then stained with haematoxylin and eosin (H.E.) according to the standard method. The histopathological examination was performed under a light microscope.
2.4. MN assays Both hepatocytes (HEPs) and bone marrow immature erythrocytes (IMEs) were analysed.
2.4.1. Liver MN assay The HEPs were prepared by the method of Narumi et al. [11], except for the slice method, the concentrations of collagenase and acridine orange and the conditions for centrifugation. The livers were excised, and an approximately 1 g portion of the left lateral lobe was sliced into several strips, each approximately 1-mm in width, using a razor blade. These strips were rinsed with Hanks’ balanced salt solution (HBSS; Sigma–Aldrich Co., LCC, Tokyo, Japan) and treated with HBSS containing 100 U/mL of collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) in a screw-capped flask with shaking (approximately 50 rpm) at 37 ◦ C for 1 h. During this time, they were shaken at the maximum speed (approximately 180 rpm) for 1 min after 30 min have passed. The resulting material was repeatedly pipetted vigorously to break apart cell clumps, and then was forced through a cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ, USA). The resulting cell suspension was then rinsed with 10% neutral buffered formalin and centrifuged at 50 × g for 5 min. Finally, the HEP pellet was resuspended in 10% neutral buffered formalin and kept at room temperature until further analysis was performed. Immediately before observation, 10 L of the HEP suspension was mixed with an equal volume of an acridine orange (AO; 250 g/mL) and 4 ,6-diamidino2-phenylindole dihydrochloride (DAPI; 10 g/mL) staining solution, dropped onto a glass slide, and covered with a coverslip (24 mm × 32 mm). The specimens were observed under a fluorescent microscope at 400× magnification with U-excitation (ultraviolet rays excitation, wave length: 330–385 nm), and the number of micronucleated HEPs (MNHEPs) per 2000 HEPs, including mono-, bi-, and multi-nucleate cells, was counted for each animal. MNHEPs were defined as HEPs with round or distinct micronuclei stained with the same colour as the main nuclei, and with diameters of 1/4 or less than those of the main nuclei. MNHEPs were confirmed by focusing up and down while taking into account the thickness of the HEPs. The number of metaphase (M-phase) cells among the 2000 HEPs was also counted to determine the mitotic index (MI). M-phase cells were identified as cells in a division stage from prophase to telophase which had identifiable chromosomes, or a poorly defined nuclear envelope, or where the appearance of the single nucleus looked like two unevenly shaped nuclei but fluoresced strongly.
2.4.2. BM 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 a method reported by Hayashi et al. [12], and the resultant BM cell suspensions were stored at room temperature until used for microscopic observation. Immediately prior to microscopic observation, 20 L of each BM sample were placed onto an AO-coated slide and covered with a 24 mm × 32 mm cover slip. The specimens were observed under a fluorescent microscope at 800× magnification with B-excitation (blue light excitation, wave length: 460–495 nm), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 erythrocytes was counted for each animal. As a parameter of hematopoietic function in BM, the proportion of immature erythrocytes (IMEs) among 500 erythrocytes was also calculated for each animal.
2.5. Blood chemistry examination Blood chemistry was examined using blood samples collected from the posterior vena cava of all animals in each group. Blood chemistry parameters included total protein (TP), albumin (Alb), albumin/globulin ratio (A/G), total bilirubin (T-Bil), direct bilirubin (D-Bil), gamma glutamyl transpeptidase (␥-GTP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (T-Cho), triglycerides (TG), glucose (Glu), blood urea nitrogen (UN), creatinine, phospholipids (PL), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K) and chlorine (Cl).
2.7. Statistical analysis Differences in the incidences of MNHEPs and MNIMEs between the test and the vehicle control groups were analysed using the conditional binomial test, reported by Kastenbaum and Bowman [13], at an upper-tailed significance level of 5%. Additional quantitative data were analysed for their statistical significance by the multiple comparison test for the MP and the vehicle control groups at a significance level of 5%. Namely, the homogeneity of variance was examined by Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied; however, when a heterogeneous variance was observed, the Kruskal–Wallis test was applied. When a statistical significance was demonstrated between the groups, the difference was assessed by Dunnett’s test or the Dunnett-type multiple comparison test. In addition, the homogeneity of variance was examined by the F-test between the positive and vehicle control groups at a significance level of 5%. When a homogeneous variance was demonstrated, Student’s t-test was applied, whereas the Aspin–Welch t-test was applied when a heterogeneous variance was observed.
3. Results 3.1. Liver MN assay The results of the liver MN assay are shown in Table 1. The MNHEP frequency significantly increased at 100 mg/kg/day of MP treatment. The mean value in the 100 mg/kg/day group was 0.23%, and the mean value in the vehicle control group was 0.03%. The mean MI value in the HEPs tended to be higher in the MP treated groups when compared with the vehicle control group, although there was no statistically significant difference between the MP treated groups and the vehicle control group, and this seemed comparable to the magnitude of MI values among the three MP treated groups. In the positive control group, the MNHEP frequency and the MI significantly increased. 3.2. BM MN assay The results of the BM MN assay are given in Table 1. The MNIME frequencies and the IME ratios in the MP treated groups were similar to those in the vehicle control group. There was no statistically significant difference in the MNIME frequency among the samples in the positive control group. The IME ratio significantly decreased at 10 mg/kg/day, but it was considered incidental because there was no dose-dependency. 3.3. Clinical signs, body weight and liver weight The results pertaining to the clinical signs, body weight and liver weight are shown in Table 2. Decreased body weight gain was noted at 100 mg/kg/day. The relative liver weight significantly increased at 100 mg/kg/day. In regards to any clinical signs, no MP-related abnormalities were noted in any of the animals during the administration period. In the positive control group, decreased body weight gain and a low absolute and relative liver weight were noted. 3.4. Blood chemistry examination The results of the blood chemistry examination are shown in Table 3. Na was significantly low in the 30 and 100 mg/kg/day groups and K was significantly high in the same groups. UN was significantly high or tended to be high in the 30 and 100 mg/kg/day groups. There were significantly high T-Bil, D-Bil, ␥-GTP, AST, ALT and ALP levels and significantly low TP and Alb
Please cite this article in press as: K. Inoue, et al., The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.04.004
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Table 1 Results of the liver and bone marrow micronucleus assay. Test substance
Dose (mg/kg)
Water
*
BM MN assay
MNHEP (%) individual data (mean ± SD)
MI (%) individual data (mean ± SD)
MNIME (%) individual data (mean ± SD)
IME ratio (%) individual data (mean ± SD)
5
0.05, 0.00, 0.10, 0.00, 0.00 (0.03 ± 0.04)
0.05, 0.00, 0.00, 0.00, 0.05 (0.02 ± 0.03)
0.10, 0.05, 0.15, 0.20, 0.10 (0.12 ± 0.06)
80.6, 80.4, 86.8, 87.6, 79.4 (83.0 ± 3.9)
10
5
30
5
100
5
0.05, 0.00, 0.10, 0.05, 0.05 (0.05 ± 0.04) 0.15, 0.00, 0.05, 0.05, 0.00 (0.05 ± 0.06) 0.15, 0.15, 0.30, 0.40, 0.15 (0.23 ± 0.12@ )
0.15, 0.00, 0.00, 0.05, 0.10 (0.06 ± 0.07) 0.15, 0.05, 0.10, 0.10, 0.05 (0.09 ± 0.04) 0.00, 0.20, 0.05, 0.10, 0.05 (0.08 ± 0.08)
0.20, 0.05, 0.15, 0.15, 0.25 (0.16 ± 0.07) 0.15, 0.10, 0.15, 0.15, 0.10 (0.13 ± 0.03) 0.15, 0.20, 0.15, 0.25, 0.15 (0.18 ± 0.04)
68.2, 76.6, 78.2, 78.8, 71.6 (74.7 ± 4.6* ) 80.0, 85.2, 85.0, 88.4, 87.0 (85.1 ± 3.2) 86.4, 82.2, 80.8, 86.6, 82.2 (83.6 ± 2.7)
1.40, 1.60, 1.15, 1.70, 1.45 (1.46 ± 0.21@ )
0.10, 0.10, 0.25, 0.15, 0.15 (0.15 ± 0.06$ )
0.25, 0.15, 0.20, 0.20, 0.10 (0.18 ± 0.06)
85.0, 83.0, 79.4, 82.8, 84.6 (83.0 ± 2.2)
12.5
DEN
$
Liver MN assay
0
MP
@
No. of animals
5
p < 0.05 by Kastenbaum and Bowman method (significantly different from vehicle control). p < 0.05 by Student’s t-test (significantly different from vehicle control). p < 0.05 by parametric Dunnett test (significantly different from vehicle control).
Table 2 Results of the body weight and liver weight. Test substance
Water MP
DEN
Dose (mg/kg)
No. of animals
Body weight (g) Day 1
Day 4
Day 8
Day 11
Day 14
Day 15
0
5
188.6 ± 3.6
217.6 ± 4.8
254.7 ± 8.1
281.5 ± 8.9
306.7 ± 10.9
275.0 ± 9.1
10 30 100
5 5 5
189.3 ± 5.0 187.6 ± 4.1 187.3 ± 4.6
219.3 ± 8.1 217.2 ± 5.2 210.3 ± 6.3
255.3 ± 11.4 253.6 ± 9.0 241.9 ± 9.0
280.5 ± 14.4 284.6 ± 12.4 264.6 ± 8.6
308.2 ± 15.7 313.4 ± 17.2 278.7 ± 11.2*
277.0 ± 13.6 278.6 ± 15.8 246.3 ± 9.8*
5
188.5 ± 3.7
208.4 ± 4.8$
233.2 ± 6.5$
251.7 ± 10.8$
270.8 ± 12.3$
245.4 ± 11.2$
12.5
Test substance
Dose (mg/kg)
Water MP
DEN
No. of animals
Absolute liver weight (g)
Relative liver weight (g/100 g B.W.)
0
5
9.3836 ± 0.6459
3.4158 ± 0.2597
10 30 100
5 5 5
9.1123 ± 0.6815 9.8009 ± 0.7222 10.5697 ± 1.4235
3.2879 ± 0.1320 3.5175 ± 0.1421 4.2817 ± 0.4318*
12.5
6.9591 ± 0.3958$
5
2.8378 ± 0.1674$
Data indicated mean ± SD. * p < 0.05 by parametric Dunnett test (significantly different from vehicle control). $ p < 0.05 by Student’s t-test (significantly different from vehicle control).
Table 3 Results of the blood chemistry examination. Test substance Water MP
DEN Test substance Water MP
DEN
Dose (mg/kg)
D-Bil (mg/dL)
␥-GT (U/L)
0.04 ± 0.01
0.00 ± 0.01
1±0
66 ± 6
0.04 ± 0.01 0.04 ± 0.00 0.14 ± 0.07#
0.01 ± 0.01 0.01 ± 0.00 0.05 ± 0.04#
1±0 1±0 4 ± 1*
91 ± 10# 79 ± 13 227 ± 97#
0.08 ± 0.02$
0.02 ± 0.01$
1±1
109 ± 8$
No. of animals
TP (g/dL)
Alb (g/dL)
T-Bil (mg/dL)
0
5
4.8 ± 0.2
2.4 ± 0.1
10 30 100
5 5 5
4.7 ± 0.3 4.6 ± 0.3 4.2 ± 0.3*
2.4 ± 0.2 2.3 ± 0.2 2.1 ± 0.1*
5
4.3 ± 0.2$
2.2 ± 0.1
12.5 Dose (mg/kg)
No. of animals
ALT (U/L)
0
5
27 ± 3
10 30 100
5 5 5
34 ± 6 27 ± 2 41 ± 8*
5
47 ± 6$
1030 ± 47$
12.5
ALP (U/L)
AST (U/L)
UN (mg/dL)
Na (mEq/L)
K (mEq/L)
765 ± 136
15.5 ± 1.1
144.1 ± 0.6
4.11 ± 0.16
843 ± 114 724 ± 78 957 ± 95*
15.9 ± 3.0 19.7 ± 2.5 21.7 ± 4.7*
143.6 ± 0.4 143.0 ± 0.7* 141.6 ± 0.8*
4.26 ± 0.32 4.53 ± 0.19* 4.84 ± 0.28*
15.4 ± 2.6
142.5 ± 0.9$
4.73 ± 0.12$
Data indicated mean ± SD. * p < 0.05 by parametric Dunnett test (significantly different from vehicle control). # p < 0.05 by Steel test (significantly different from vehicle control). $ p < 0.05 by Student’s t-test (significantly different from vehicle control).
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in the 100 mg/kg/day group. The AST significantly increased at 10 mg/kg/day, but it was considered incidental because there was no dose-dependency. 3.5. Histopathological examination The results of the histopathological examination are shown in Table 4. Focal single cell necrosis of hepatocytes in the perilobular region was observed in the 10 mg/kg/day and higher groups, and the hypertrophy of hepatocytes in the perilobular region was observed in the 30 and 100 mg/kg/day groups. These findings in the 100 mg/kg/day group were more severe than those in the lower dose groups. In the 100 mg/kg/day group, infiltration of inflammatory cells, proliferation of oval cells and prominent nucleolus of the hepatocytes in the perilobular region were observed. 4. Discussion Treatment with MP resulted in a statistically significant increase in MNHEP at only 100 mg/kg/day, and MP was negative in the BM MN assay. MP is known to be carcinogenic in the rat liver [1]. MP gave positive [2] and negative [3] results under metabolismactivated conditions for the in vitro MLA, but it gave negative results in the Ames assay [4], in vivo Comet assay [5], UDS assay [6] and DNA adduct assay [7–9]. Therefore, MP is considered a non-genotoxic hepatocarcinogen, and it is unlikely that the genotoxic activity of MP increased the levels of MNHEPs. MP is known to induce hepatotoxicity with bile stasis [14,15], and this characteristic is consistent with the cytotoxic findings in the perilobular region of the liver, and thus an increase in D-Bil levels was observed in this study. In the 100 mg/kg/day group, there were increases in the blood chemistry parameters related to hepatotoxicity and the liver weight, and a higher grade of severity in HEP single cell necrosis and oval cell proliferation were found in the histopathology, indicating that necrosis followed by regenerative cell growth in the liver might increase the levels of MNHEPs. This hypothesis may be supported by a report that subchronic administration of MP to rats clearly increased cell proliferation in HEPs [16]. In contrast to the clear changes observed in the blood chemistry and the histopathology at 100 mg/kg/day, the magnitude of the MI values seemed comparable between the three doses groups, suggesting that MI values may not be a good indicator for cell proliferation assessment in the liver, which would be an important factor to consider when interpreting the toxicological significance of increased MNHEP levels. There is
also a possibility that bile acid may cause the increase in MNHEP frequency because chenodeoxycholic acid, one component of bile acid, is reported to induce DNA oxidative damage [17]. These findings suggest that MP may induce clastogenic effects indirectly on the liver. As other possibility, MP-induced liver regeneration may cause errors of cell division, and that whole chromosomes may have been lost into the MN. However, further investigation would be necessary to clarify the mechanism that leads to increased MNHEPs during MP treatment. The ICH guidelines recommend the integration of an in vivo genotoxicity endpoint into routine, general toxicity studies, with the aim of reducing the number of experimental animals used [18]. Thus, based on the study results from 2 weeks, we assessed whether the dose levels in the 4-week, repeated-dose toxicity study would be compatible with the dosage levels required for an acceptable liver MN assay. In the liver, focal single cell necrosis of the hepatocytes in the perilobular region was observed in the 10 mg/kg/day and higher groups, and hypertrophy of the hepatocytes in the perilobular region was observed in the 30 mg/kg/day and higher groups. These findings in the 100 mg/kg/day group were more severe than the results found in the lower dose groups. In the 100 mg/kg/day group, there were high levels of T-Bil, D-Bil, ␥-GTP, AST and ALT, increased liver weight, infiltration of the inflammatory cells, proliferation of oval cells and prominent hepatocyte nucleoli in the perilobular region. The present results suggest a hepatotoxicity related to bile stasis, which was similar to previous reports [14,15]. Based on the present results, the no-observed-adverse effect level (NOAEL) in the 4-week, repeated dose toxicity study of MP would be less than 10 mg/kg/day. Therefore, in the 4-week, repeated dose toxicity study, 100 mg/kg/day could be estimated as a high dose group, and a common ratio of more than 4 would be preferable for determining the NOAEL. However, the increase of MNHEP for multiple doses would be desirable to clarify the relationship between the increase of MNHEP and the dosage level. To satisfy this requirement, less than 2 as a common ratio would be preferable when the high dose level is set at 100 mg/kg/day. Therefore, the integration of the liver MN assay into a 4-week, repeated dose toxicity study is considered possible, if the high dose is set appropriately. However, a judgement is difficult, when the weak positive reaction is noted in the high dose group only. In that case, an additional short-term study with higher exposure is desirable. In the present study, DEN was used as a positive control for the liver MN assay to confirm the reproducibility of the assay.
Table 4 Results of the histopathological examination. Organ and finding
Digestive system Liver Hypertrophy, hepatocyte, perilobular Single cell necrosis, hepatocyte, perilobular, focal Cell infiltration, inflammatory, perilobular Proliferation, oval cell, perilobular Prominent, nucleolus, hepatocyte, perilobular Hypertrophy, hepatocyte, centrilobular Single cell necrosis, hepatocyte, centrilobular Cell infiltration, inflammatory, centrilobular Brown pigment deposition, Kupper’s cell, centrilobular Necrosis, hepatocyte, focal
Test substance
Water
MP
Dose (mg/kg)
0
10
Number of animals
5
Grade sign
−
+
++
−
+
++
−
+
++
−
+
++
−
+
++
5 5 5 5 5 5 5 5 5 5
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
5 2 5 5 5 5 5 5 5 4
0 3 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0
0 0 5 5 5 5 5 5 5 5
5 5 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 1 1 0 5 5 5 5 5
3 0 3 4 5 0 0 0 0 0
2 5 1 0 0 0 0 0 0 0
5 5 5 5 5 0 1 0 2 5
0 0 0 0 0 5 2 5 3 0
0 0 0 0 0 0 2 0 0 0
DEN 30
5
100
5
12.5
5
5
Values indicated the number of animals with the finding. −, none, +, minimal; ++, mild.
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Thus, a positive result was noted for DEN only in the liver MN assay. This finding is consistent with the assay results from a previous collaborative study, which was conducted to investigate the reproducibility of the assay results [19]. Therefore, the present experiment was considered to be successfully conducted, and the liver MN assay results of MP were deemed acceptable. However, a negative result was noted in the BM MN assay. Therefore, the slides from the other studies with a positive control that showed an increase in BM-MN is needed to justify the observation procedure, when both the BM MN assay and the liver MN assay are integrated into the general GLP toxicity study. In conclusion, the RDLMN assay has the potential to detect hepatocarcinogens. However, the positive results were presumed to be caused by non-genotoxic mechanisms because MP is considered a non-genotoxic hepatocarcinogen. An investigation using other test chemicals that induce hepatotoxicity with bile stasis, without genotoxicity, will provide useful information for elucidating the toxicological significance of the positive results. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements Support for this study was provided by the Mammalian Mutagenicity Study (MMS) Group in the Japanese Environmental Mutagen Society (JEMS) as well as by a Health and Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare (MHLW), Japan. References [1] M. Habs, P. Shubik, G. Eisenbrand, Carcinogenicity of methapyrilene hydrochloride, mepyramine hydrochloride, thenyldiamine hydrochloride, and pyribenzamine hydrochloride in Sprague-Dawley rats, J. Cancer Res. Clin. Oncol. 111 (1) (1986) 71–74. [2] N.T. Turner, J.L. Woolley, J.C. Hozier, J.R. Sawyer, D. Clive, Methapyrilene is a genotoxic carcinogen. Studies on methapyrilene and pyrilamine in the L5178Y tk+/− mouse lymphoma assay, Mutat. Res. 189 (3) (1987) 285–297. [3] D.B. Mcgregor, A.G. Brown, S. Howgate, D. Mcbride, C. Riach, W.J. Caspary, Responses of the L5178Y mouse lymphoma cell forward mutation assay V: 27 coded chemicals, Environ. Mol. Mutagen 17 (3) (1991) 196– 219.
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[4] T.J. Oberly, J.C. Scheuring, K.A. Richardson, F.C. Richardson, M.L. Garriott, The evaluation of methapyrilene for bacterial mutation with metabolic activation by aroclor-induced, methapyrilene-induced and noninduced rat-liver S-9, Mutat. Res. 299 (2) (1993) 77–84. [5] A. Rothfuss, M. O’Donovan, M. De Boeck, D. Brault, A. Czich, L. Custer, et al., Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies, Mutat. Res. 702 (1) (2010) 40–69. [6] K.L. Steinmetz, Examination of genotoxicity, toxicity and morphologic alterations in hepatocytes following in vivo or in vitro exposure to methapyrilene, Carcinogenesis 9 (6) (1988) 959–963. [7] D.A. Casciano, G. Talaska, D. Clive, The potent hepatocarcinogen methapyrilene induces mutations in L5178Y mouse lymphoma cells in the apparent absence of DNA adduct formation, Mutat. Res. 263 (2) (1991) 127–132. [8] W. Lijinsky, K. Yamashita, Lack of binding of methapyrilene and similar antihistamines to rat liver DNA examined by 32 P postlabeling, Cancer Res. 48 (22) (1988) 6475–6477. [9] D.A. Casciano, J.G. Shaddock, G. Talaska, The potent hepatocarcinogen methapyrilene does not form DNA adducts in livers of Fischer 344 rats, Mutat. Res. 208 (3/4) (1988) 129–135. [10] J.C. Mirsalis, Genotoxicity, toxicity, and carcinogenicity of the antihistamine methapyrilene, Mutat. Res. 185 (3) (1987) 309–317. [11] 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. [12] M. Hayashi, T. Morita, Y. Kodama, T. Sofuni, M. Ishidate Jr., The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutat. Res. 245 (4) (1990) 245–249. [13] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutat. Res. 9 (1970) 527–549. [14] G.S. Ratra, S. Cottrell, C.J. Powell, Effects of induction and inhibition of cytochromes P450 on the hepatotoxicity of methapyrilene, Toxicol. Sci. 46 (1) (1998) 185–196. [15] H.K. Hamadeh, Methapyrilene toxicity: anchorage of pathologic observations to gene expression alterations, Toxicol. Pathol. 30 (4) (2002) 470–482. [16] NTP Technical Report on the Hepatotoxicity Studies of the Liver Carcinogen No. 46 (2000). [17] P. Rosignoli, R. Fabiani, A. De Bartolomeo, R. Fuccelli, M.A. Pelli, G. Morozzi, Genotoxic effect of bile acids on human normal and tumour colon cells and protection by dietary antioxidants and butyrate, Eur. J. Nutr. 47 (6) (2008) 301–309. [18] ICH, Harmonized Tripartite Guideline, Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, S2(R2), Step 4 Version, 2011. [19] 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), 2014 (in press).
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The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats Kenji Inoue a,∗ , Akimu Ochi a , Akira Koda a , Yumi Wako b , Kazufumi Kawasako b , Takaaki Doi a a b
Maruho Co., Ltd., 93 Awata-cho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan LSI Medience Corporation, 14-1 Sunayama, Kamisu-shi, Ibaraki 314-0255, Japan
a r t i c l e
i n f o
Article history: Received 9 April 2014 Accepted 10 April 2014 Available online xxx Keywords: Liver micronucleus assay Methapyrilene hydrochloride Rat Repeated-dose toxicity study Hepatocarcinogen Integration
a b s t r a c t The repeated dose liver micronucleus (RDLMN) assay using young adult rats has the potential to detect genotoxic hepatocarcinogens that can be integrated into a general toxicity study. The assay methods were thoroughly validated by 19 Japanese facilities. Methapyrilene hydrochloride (MP), known to be a non-genotoxic hepatocarcinogen, was examined in the present study. MP was dosed orally at 10, 30 and 100 mg/kg/day to 6-week-old male Crl:CD (SD) rats daily for 14 days. Treatment with MP resulted in an increase in micronucleated hepatocytes (MNHEPs) with a dosage of only 100 mg/kg/day. At this dose level, cytotoxicity followed by regenerative cell growth was noted in the liver. These findings suggest that MP may induce clastogenic effects indirectly on the liver or hepatotoxicity of MP followed by regeneration may cause increase in spontaneous incidence of MNHEPs. © 2014 Elsevier B.V. All rights reserved.
1. Introduction A collaborative study by the Mammalian Mutagenicity Study (MMS) Group, which is a subgroup of the Japanese Environmental Mutagen Society (JEMS), was conducted to evaluate the suitability of the repeated dose liver and gastrointestinal tract micronucleus assays using young adult rats. This study, using methapyrilene hydrochloride (MP), was performed as part of a collaborative study at Maruho Co., Ltd. MP is an antihistaminic compound that was once used as a popular over-the-counter sleep-aid component and was also used in cold and allergy medications. However, MP was found to induce hepatocellular carcinomas and cholangiocarcinomas in rats [1], and it was subsequently withdrawn from the market. MP gave positive [2] and negative [3] results under metabolism-activated conditions for the in vitro mouse lymphoma assay (MLA), but MP gave negative results for the Ames assay [4], in vivo Comet assay [5], UDS assay [6] and DNA adduct assay [7–9]. These data are consistent with the hypothesis that MP is carcinogenic in rats via non-genotoxic mechanisms [6,10].
∗ Corresponding author at: Kyoto R&D Center, Toxicological Research, Maruho Co., Ltd., 93 Awata-cho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan. Tel.: +81 75 325 3255; fax: +81 75 325 3222. E-mail address: inoue [email protected] (K. Inoue).
In this study, MP was used as a non-genotoxic hepatocarcinogen to examine the selectivity of the liver MN assay. To assess the possibility that the liver MN assay could be incorporated into a general toxicity study, 6-week-old rats, which are usually used in these types of studies, were used. At the same time, we attempted to assess whether the dose levels in the 4-week, repeated-dose toxicity study would be compatible with the dosage levels required for an acceptable liver MN assay. 2. Materials and methods 2.1. Animals Male Crl:CD (SD) rats were purchased from Charles River Japan Inc. and were aged 6 weeks with weights between 180 and 200 g at the beginning of the experiments. The animals were housed one per cage in an air-conditioned room with a 12 h light/dark cycle and free access to food and drinking water. The animal experiments were approved by the Institutional Animal Care and Use Committee prior to conducting the experiments.
2.2. Chemicals Methapyrilene hydrochloride (MP; CAS No. 135-23-9, 99.6% purity) was purchased from Sigma–Aldrich Co., LLC (Tokyo, Japan). MP was dissolved in water (Otsuka Pharmaceutical Factory, Inc., Japan) at concentrations of 1, 3 and 10 mg/mL before use. A positive control, diethylnitrosamine (DEN; CAS No. 55-18-5, 99.9% purity), was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). DEN was dissolved in water at a concentration of 1.25 mg/mL before use.
http://dx.doi.org/10.1016/j.mrgentox.2014.04.004 1383-5718/© 2014 Elsevier B.V. All rights reserved.
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2 2.3. Doses levels and treatment
2.6. Histopathological examination
The maximum dose for repeated dose studies was defined as the dose inducing clinical signs without causing lethality. The high dose for MP (100 mg/kg/day) was determined according to published literature information [5]. Three dose levels were set, and the mid- and low-dose levels were each approximately 1/3 of the next higher dose. DEN given at 12.5 mg/kg/day served as a positive control for the liver MN assay. Animals were divided into groups of 5 and received the test chemicals or the vehicle alone by oral gavage (10 mL/kg of body weight volume) once a day for 14 consecutive days. Clinical signs and mortality were observed every day, and the body weights were measured twice a week. At 24 h following the last administration, fasted rats were euthanised under isoflurane anaesthesia. Blood samples were used for blood chemistry examinations. Livers were subjected to the liver MN assay and histopathological examination after weighting. Femurs were used for the bone marrow MN (BM MN) assay.
Upon euthanasia, the residual liver tissue of the left lateral lobe after the isolation of HEPs was fixed with 10% phosphate buffered formalin, embedded in paraffin, and then stained with haematoxylin and eosin (H.E.) according to the standard method. The histopathological examination was performed under a light microscope.
2.4. MN assays Both hepatocytes (HEPs) and bone marrow immature erythrocytes (IMEs) were analysed.
2.4.1. Liver MN assay The HEPs were prepared by the method of Narumi et al. [11], except for the slice method, the concentrations of collagenase and acridine orange and the conditions for centrifugation. The livers were excised, and an approximately 1 g portion of the left lateral lobe was sliced into several strips, each approximately 1-mm in width, using a razor blade. These strips were rinsed with Hanks’ balanced salt solution (HBSS; Sigma–Aldrich Co., LCC, Tokyo, Japan) and treated with HBSS containing 100 U/mL of collagenase (Collagenase Yakult-S, Yakult Pharmaceutical Industry, Co., Ltd., Tokyo, Japan) in a screw-capped flask with shaking (approximately 50 rpm) at 37 ◦ C for 1 h. During this time, they were shaken at the maximum speed (approximately 180 rpm) for 1 min after 30 min have passed. The resulting material was repeatedly pipetted vigorously to break apart cell clumps, and then was forced through a cell strainer (Becton Dickinson and Co., Franklin Lakes, NJ, USA). The resulting cell suspension was then rinsed with 10% neutral buffered formalin and centrifuged at 50 × g for 5 min. Finally, the HEP pellet was resuspended in 10% neutral buffered formalin and kept at room temperature until further analysis was performed. Immediately before observation, 10 L of the HEP suspension was mixed with an equal volume of an acridine orange (AO; 250 g/mL) and 4 ,6-diamidino2-phenylindole dihydrochloride (DAPI; 10 g/mL) staining solution, dropped onto a glass slide, and covered with a coverslip (24 mm × 32 mm). The specimens were observed under a fluorescent microscope at 400× magnification with U-excitation (ultraviolet rays excitation, wave length: 330–385 nm), and the number of micronucleated HEPs (MNHEPs) per 2000 HEPs, including mono-, bi-, and multi-nucleate cells, was counted for each animal. MNHEPs were defined as HEPs with round or distinct micronuclei stained with the same colour as the main nuclei, and with diameters of 1/4 or less than those of the main nuclei. MNHEPs were confirmed by focusing up and down while taking into account the thickness of the HEPs. The number of metaphase (M-phase) cells among the 2000 HEPs was also counted to determine the mitotic index (MI). M-phase cells were identified as cells in a division stage from prophase to telophase which had identifiable chromosomes, or a poorly defined nuclear envelope, or where the appearance of the single nucleus looked like two unevenly shaped nuclei but fluoresced strongly.
2.4.2. BM 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 a method reported by Hayashi et al. [12], and the resultant BM cell suspensions were stored at room temperature until used for microscopic observation. Immediately prior to microscopic observation, 20 L of each BM sample were placed onto an AO-coated slide and covered with a 24 mm × 32 mm cover slip. The specimens were observed under a fluorescent microscope at 800× magnification with B-excitation (blue light excitation, wave length: 460–495 nm), and the number of micronucleated immature erythrocytes (MNIMEs) per 2000 erythrocytes was counted for each animal. As a parameter of hematopoietic function in BM, the proportion of immature erythrocytes (IMEs) among 500 erythrocytes was also calculated for each animal.
2.5. Blood chemistry examination Blood chemistry was examined using blood samples collected from the posterior vena cava of all animals in each group. Blood chemistry parameters included total protein (TP), albumin (Alb), albumin/globulin ratio (A/G), total bilirubin (T-Bil), direct bilirubin (D-Bil), gamma glutamyl transpeptidase (␥-GTP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total cholesterol (T-Cho), triglycerides (TG), glucose (Glu), blood urea nitrogen (UN), creatinine, phospholipids (PL), inorganic phosphorus (IP), calcium (Ca), sodium (Na), potassium (K) and chlorine (Cl).
2.7. Statistical analysis Differences in the incidences of MNHEPs and MNIMEs between the test and the vehicle control groups were analysed using the conditional binomial test, reported by Kastenbaum and Bowman [13], at an upper-tailed significance level of 5%. Additional quantitative data were analysed for their statistical significance by the multiple comparison test for the MP and the vehicle control groups at a significance level of 5%. Namely, the homogeneity of variance was examined by Bartlett’s test. When a homogeneous variance was demonstrated, one-way analysis of variance was applied; however, when a heterogeneous variance was observed, the Kruskal–Wallis test was applied. When a statistical significance was demonstrated between the groups, the difference was assessed by Dunnett’s test or the Dunnett-type multiple comparison test. In addition, the homogeneity of variance was examined by the F-test between the positive and vehicle control groups at a significance level of 5%. When a homogeneous variance was demonstrated, Student’s t-test was applied, whereas the Aspin–Welch t-test was applied when a heterogeneous variance was observed.
3. Results 3.1. Liver MN assay The results of the liver MN assay are shown in Table 1. The MNHEP frequency significantly increased at 100 mg/kg/day of MP treatment. The mean value in the 100 mg/kg/day group was 0.23%, and the mean value in the vehicle control group was 0.03%. The mean MI value in the HEPs tended to be higher in the MP treated groups when compared with the vehicle control group, although there was no statistically significant difference between the MP treated groups and the vehicle control group, and this seemed comparable to the magnitude of MI values among the three MP treated groups. In the positive control group, the MNHEP frequency and the MI significantly increased. 3.2. BM MN assay The results of the BM MN assay are given in Table 1. The MNIME frequencies and the IME ratios in the MP treated groups were similar to those in the vehicle control group. There was no statistically significant difference in the MNIME frequency among the samples in the positive control group. The IME ratio significantly decreased at 10 mg/kg/day, but it was considered incidental because there was no dose-dependency. 3.3. Clinical signs, body weight and liver weight The results pertaining to the clinical signs, body weight and liver weight are shown in Table 2. Decreased body weight gain was noted at 100 mg/kg/day. The relative liver weight significantly increased at 100 mg/kg/day. In regards to any clinical signs, no MP-related abnormalities were noted in any of the animals during the administration period. In the positive control group, decreased body weight gain and a low absolute and relative liver weight were noted. 3.4. Blood chemistry examination The results of the blood chemistry examination are shown in Table 3. Na was significantly low in the 30 and 100 mg/kg/day groups and K was significantly high in the same groups. UN was significantly high or tended to be high in the 30 and 100 mg/kg/day groups. There were significantly high T-Bil, D-Bil, ␥-GTP, AST, ALT and ALP levels and significantly low TP and Alb
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Table 1 Results of the liver and bone marrow micronucleus assay. Test substance
Dose (mg/kg)
Water
*
BM MN assay
MNHEP (%) individual data (mean ± SD)
MI (%) individual data (mean ± SD)
MNIME (%) individual data (mean ± SD)
IME ratio (%) individual data (mean ± SD)
5
0.05, 0.00, 0.10, 0.00, 0.00 (0.03 ± 0.04)
0.05, 0.00, 0.00, 0.00, 0.05 (0.02 ± 0.03)
0.10, 0.05, 0.15, 0.20, 0.10 (0.12 ± 0.06)
80.6, 80.4, 86.8, 87.6, 79.4 (83.0 ± 3.9)
10
5
30
5
100
5
0.05, 0.00, 0.10, 0.05, 0.05 (0.05 ± 0.04) 0.15, 0.00, 0.05, 0.05, 0.00 (0.05 ± 0.06) 0.15, 0.15, 0.30, 0.40, 0.15 (0.23 ± 0.12@ )
0.15, 0.00, 0.00, 0.05, 0.10 (0.06 ± 0.07) 0.15, 0.05, 0.10, 0.10, 0.05 (0.09 ± 0.04) 0.00, 0.20, 0.05, 0.10, 0.05 (0.08 ± 0.08)
0.20, 0.05, 0.15, 0.15, 0.25 (0.16 ± 0.07) 0.15, 0.10, 0.15, 0.15, 0.10 (0.13 ± 0.03) 0.15, 0.20, 0.15, 0.25, 0.15 (0.18 ± 0.04)
68.2, 76.6, 78.2, 78.8, 71.6 (74.7 ± 4.6* ) 80.0, 85.2, 85.0, 88.4, 87.0 (85.1 ± 3.2) 86.4, 82.2, 80.8, 86.6, 82.2 (83.6 ± 2.7)
1.40, 1.60, 1.15, 1.70, 1.45 (1.46 ± 0.21@ )
0.10, 0.10, 0.25, 0.15, 0.15 (0.15 ± 0.06$ )
0.25, 0.15, 0.20, 0.20, 0.10 (0.18 ± 0.06)
85.0, 83.0, 79.4, 82.8, 84.6 (83.0 ± 2.2)
12.5
DEN
$
Liver MN assay
0
MP
@
No. of animals
5
p < 0.05 by Kastenbaum and Bowman method (significantly different from vehicle control). p < 0.05 by Student’s t-test (significantly different from vehicle control). p < 0.05 by parametric Dunnett test (significantly different from vehicle control).
Table 2 Results of the body weight and liver weight. Test substance
Water MP
DEN
Dose (mg/kg)
No. of animals
Body weight (g) Day 1
Day 4
Day 8
Day 11
Day 14
Day 15
0
5
188.6 ± 3.6
217.6 ± 4.8
254.7 ± 8.1
281.5 ± 8.9
306.7 ± 10.9
275.0 ± 9.1
10 30 100
5 5 5
189.3 ± 5.0 187.6 ± 4.1 187.3 ± 4.6
219.3 ± 8.1 217.2 ± 5.2 210.3 ± 6.3
255.3 ± 11.4 253.6 ± 9.0 241.9 ± 9.0
280.5 ± 14.4 284.6 ± 12.4 264.6 ± 8.6
308.2 ± 15.7 313.4 ± 17.2 278.7 ± 11.2*
277.0 ± 13.6 278.6 ± 15.8 246.3 ± 9.8*
5
188.5 ± 3.7
208.4 ± 4.8$
233.2 ± 6.5$
251.7 ± 10.8$
270.8 ± 12.3$
245.4 ± 11.2$
12.5
Test substance
Dose (mg/kg)
Water MP
DEN
No. of animals
Absolute liver weight (g)
Relative liver weight (g/100 g B.W.)
0
5
9.3836 ± 0.6459
3.4158 ± 0.2597
10 30 100
5 5 5
9.1123 ± 0.6815 9.8009 ± 0.7222 10.5697 ± 1.4235
3.2879 ± 0.1320 3.5175 ± 0.1421 4.2817 ± 0.4318*
12.5
6.9591 ± 0.3958$
5
2.8378 ± 0.1674$
Data indicated mean ± SD. * p < 0.05 by parametric Dunnett test (significantly different from vehicle control). $ p < 0.05 by Student’s t-test (significantly different from vehicle control).
Table 3 Results of the blood chemistry examination. Test substance Water MP
DEN Test substance Water MP
DEN
Dose (mg/kg)
D-Bil (mg/dL)
␥-GT (U/L)
0.04 ± 0.01
0.00 ± 0.01
1±0
66 ± 6
0.04 ± 0.01 0.04 ± 0.00 0.14 ± 0.07#
0.01 ± 0.01 0.01 ± 0.00 0.05 ± 0.04#
1±0 1±0 4 ± 1*
91 ± 10# 79 ± 13 227 ± 97#
0.08 ± 0.02$
0.02 ± 0.01$
1±1
109 ± 8$
No. of animals
TP (g/dL)
Alb (g/dL)
T-Bil (mg/dL)
0
5
4.8 ± 0.2
2.4 ± 0.1
10 30 100
5 5 5
4.7 ± 0.3 4.6 ± 0.3 4.2 ± 0.3*
2.4 ± 0.2 2.3 ± 0.2 2.1 ± 0.1*
5
4.3 ± 0.2$
2.2 ± 0.1
12.5 Dose (mg/kg)
No. of animals
ALT (U/L)
0
5
27 ± 3
10 30 100
5 5 5
34 ± 6 27 ± 2 41 ± 8*
5
47 ± 6$
1030 ± 47$
12.5
ALP (U/L)
AST (U/L)
UN (mg/dL)
Na (mEq/L)
K (mEq/L)
765 ± 136
15.5 ± 1.1
144.1 ± 0.6
4.11 ± 0.16
843 ± 114 724 ± 78 957 ± 95*
15.9 ± 3.0 19.7 ± 2.5 21.7 ± 4.7*
143.6 ± 0.4 143.0 ± 0.7* 141.6 ± 0.8*
4.26 ± 0.32 4.53 ± 0.19* 4.84 ± 0.28*
15.4 ± 2.6
142.5 ± 0.9$
4.73 ± 0.12$
Data indicated mean ± SD. * p < 0.05 by parametric Dunnett test (significantly different from vehicle control). # p < 0.05 by Steel test (significantly different from vehicle control). $ p < 0.05 by Student’s t-test (significantly different from vehicle control).
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4
in the 100 mg/kg/day group. The AST significantly increased at 10 mg/kg/day, but it was considered incidental because there was no dose-dependency. 3.5. Histopathological examination The results of the histopathological examination are shown in Table 4. Focal single cell necrosis of hepatocytes in the perilobular region was observed in the 10 mg/kg/day and higher groups, and the hypertrophy of hepatocytes in the perilobular region was observed in the 30 and 100 mg/kg/day groups. These findings in the 100 mg/kg/day group were more severe than those in the lower dose groups. In the 100 mg/kg/day group, infiltration of inflammatory cells, proliferation of oval cells and prominent nucleolus of the hepatocytes in the perilobular region were observed. 4. Discussion Treatment with MP resulted in a statistically significant increase in MNHEP at only 100 mg/kg/day, and MP was negative in the BM MN assay. MP is known to be carcinogenic in the rat liver [1]. MP gave positive [2] and negative [3] results under metabolismactivated conditions for the in vitro MLA, but it gave negative results in the Ames assay [4], in vivo Comet assay [5], UDS assay [6] and DNA adduct assay [7–9]. Therefore, MP is considered a non-genotoxic hepatocarcinogen, and it is unlikely that the genotoxic activity of MP increased the levels of MNHEPs. MP is known to induce hepatotoxicity with bile stasis [14,15], and this characteristic is consistent with the cytotoxic findings in the perilobular region of the liver, and thus an increase in D-Bil levels was observed in this study. In the 100 mg/kg/day group, there were increases in the blood chemistry parameters related to hepatotoxicity and the liver weight, and a higher grade of severity in HEP single cell necrosis and oval cell proliferation were found in the histopathology, indicating that necrosis followed by regenerative cell growth in the liver might increase the levels of MNHEPs. This hypothesis may be supported by a report that subchronic administration of MP to rats clearly increased cell proliferation in HEPs [16]. In contrast to the clear changes observed in the blood chemistry and the histopathology at 100 mg/kg/day, the magnitude of the MI values seemed comparable between the three doses groups, suggesting that MI values may not be a good indicator for cell proliferation assessment in the liver, which would be an important factor to consider when interpreting the toxicological significance of increased MNHEP levels. There is
also a possibility that bile acid may cause the increase in MNHEP frequency because chenodeoxycholic acid, one component of bile acid, is reported to induce DNA oxidative damage [17]. These findings suggest that MP may induce clastogenic effects indirectly on the liver. As other possibility, MP-induced liver regeneration may cause errors of cell division, and that whole chromosomes may have been lost into the MN. However, further investigation would be necessary to clarify the mechanism that leads to increased MNHEPs during MP treatment. The ICH guidelines recommend the integration of an in vivo genotoxicity endpoint into routine, general toxicity studies, with the aim of reducing the number of experimental animals used [18]. Thus, based on the study results from 2 weeks, we assessed whether the dose levels in the 4-week, repeated-dose toxicity study would be compatible with the dosage levels required for an acceptable liver MN assay. In the liver, focal single cell necrosis of the hepatocytes in the perilobular region was observed in the 10 mg/kg/day and higher groups, and hypertrophy of the hepatocytes in the perilobular region was observed in the 30 mg/kg/day and higher groups. These findings in the 100 mg/kg/day group were more severe than the results found in the lower dose groups. In the 100 mg/kg/day group, there were high levels of T-Bil, D-Bil, ␥-GTP, AST and ALT, increased liver weight, infiltration of the inflammatory cells, proliferation of oval cells and prominent hepatocyte nucleoli in the perilobular region. The present results suggest a hepatotoxicity related to bile stasis, which was similar to previous reports [14,15]. Based on the present results, the no-observed-adverse effect level (NOAEL) in the 4-week, repeated dose toxicity study of MP would be less than 10 mg/kg/day. Therefore, in the 4-week, repeated dose toxicity study, 100 mg/kg/day could be estimated as a high dose group, and a common ratio of more than 4 would be preferable for determining the NOAEL. However, the increase of MNHEP for multiple doses would be desirable to clarify the relationship between the increase of MNHEP and the dosage level. To satisfy this requirement, less than 2 as a common ratio would be preferable when the high dose level is set at 100 mg/kg/day. Therefore, the integration of the liver MN assay into a 4-week, repeated dose toxicity study is considered possible, if the high dose is set appropriately. However, a judgement is difficult, when the weak positive reaction is noted in the high dose group only. In that case, an additional short-term study with higher exposure is desirable. In the present study, DEN was used as a positive control for the liver MN assay to confirm the reproducibility of the assay.
Table 4 Results of the histopathological examination. Organ and finding
Digestive system Liver Hypertrophy, hepatocyte, perilobular Single cell necrosis, hepatocyte, perilobular, focal Cell infiltration, inflammatory, perilobular Proliferation, oval cell, perilobular Prominent, nucleolus, hepatocyte, perilobular Hypertrophy, hepatocyte, centrilobular Single cell necrosis, hepatocyte, centrilobular Cell infiltration, inflammatory, centrilobular Brown pigment deposition, Kupper’s cell, centrilobular Necrosis, hepatocyte, focal
Test substance
Water
MP
Dose (mg/kg)
0
10
Number of animals
5
Grade sign
−
+
++
−
+
++
−
+
++
−
+
++
−
+
++
5 5 5 5 5 5 5 5 5 5
0 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
5 2 5 5 5 5 5 5 5 4
0 3 0 0 0 0 0 0 0 1
0 0 0 0 0 0 0 0 0 0
0 0 5 5 5 5 5 5 5 5
5 5 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0
0 0 1 1 0 5 5 5 5 5
3 0 3 4 5 0 0 0 0 0
2 5 1 0 0 0 0 0 0 0
5 5 5 5 5 0 1 0 2 5
0 0 0 0 0 5 2 5 3 0
0 0 0 0 0 0 2 0 0 0
DEN 30
5
100
5
12.5
5
5
Values indicated the number of animals with the finding. −, none, +, minimal; ++, mild.
Please cite this article in press as: K. Inoue, et al., The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.04.004
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Thus, a positive result was noted for DEN only in the liver MN assay. This finding is consistent with the assay results from a previous collaborative study, which was conducted to investigate the reproducibility of the assay results [19]. Therefore, the present experiment was considered to be successfully conducted, and the liver MN assay results of MP were deemed acceptable. However, a negative result was noted in the BM MN assay. Therefore, the slides from the other studies with a positive control that showed an increase in BM-MN is needed to justify the observation procedure, when both the BM MN assay and the liver MN assay are integrated into the general GLP toxicity study. In conclusion, the RDLMN assay has the potential to detect hepatocarcinogens. However, the positive results were presumed to be caused by non-genotoxic mechanisms because MP is considered a non-genotoxic hepatocarcinogen. An investigation using other test chemicals that induce hepatotoxicity with bile stasis, without genotoxicity, will provide useful information for elucidating the toxicological significance of the positive results. Conflicts of interest The authors declare that there are no conflicts of interest. Acknowledgements Support for this study was provided by the Mammalian Mutagenicity Study (MMS) Group in the Japanese Environmental Mutagen Society (JEMS) as well as by a Health and Labour Sciences Research Grant from the Ministry of Health, Labour and Welfare (MHLW), Japan. References [1] M. Habs, P. Shubik, G. Eisenbrand, Carcinogenicity of methapyrilene hydrochloride, mepyramine hydrochloride, thenyldiamine hydrochloride, and pyribenzamine hydrochloride in Sprague-Dawley rats, J. Cancer Res. Clin. Oncol. 111 (1) (1986) 71–74. [2] N.T. Turner, J.L. Woolley, J.C. Hozier, J.R. Sawyer, D. Clive, Methapyrilene is a genotoxic carcinogen. Studies on methapyrilene and pyrilamine in the L5178Y tk+/− mouse lymphoma assay, Mutat. Res. 189 (3) (1987) 285–297. [3] D.B. Mcgregor, A.G. Brown, S. Howgate, D. Mcbride, C. Riach, W.J. Caspary, Responses of the L5178Y mouse lymphoma cell forward mutation assay V: 27 coded chemicals, Environ. Mol. Mutagen 17 (3) (1991) 196– 219.
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[4] T.J. Oberly, J.C. Scheuring, K.A. Richardson, F.C. Richardson, M.L. Garriott, The evaluation of methapyrilene for bacterial mutation with metabolic activation by aroclor-induced, methapyrilene-induced and noninduced rat-liver S-9, Mutat. Res. 299 (2) (1993) 77–84. [5] A. Rothfuss, M. O’Donovan, M. De Boeck, D. Brault, A. Czich, L. Custer, et al., Collaborative study on fifteen compounds in the rat-liver Comet assay integrated into 2- and 4-week repeat-dose studies, Mutat. Res. 702 (1) (2010) 40–69. [6] K.L. Steinmetz, Examination of genotoxicity, toxicity and morphologic alterations in hepatocytes following in vivo or in vitro exposure to methapyrilene, Carcinogenesis 9 (6) (1988) 959–963. [7] D.A. Casciano, G. Talaska, D. Clive, The potent hepatocarcinogen methapyrilene induces mutations in L5178Y mouse lymphoma cells in the apparent absence of DNA adduct formation, Mutat. Res. 263 (2) (1991) 127–132. [8] W. Lijinsky, K. Yamashita, Lack of binding of methapyrilene and similar antihistamines to rat liver DNA examined by 32 P postlabeling, Cancer Res. 48 (22) (1988) 6475–6477. [9] D.A. Casciano, J.G. Shaddock, G. Talaska, The potent hepatocarcinogen methapyrilene does not form DNA adducts in livers of Fischer 344 rats, Mutat. Res. 208 (3/4) (1988) 129–135. [10] J.C. Mirsalis, Genotoxicity, toxicity, and carcinogenicity of the antihistamine methapyrilene, Mutat. Res. 185 (3) (1987) 309–317. [11] 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. [12] M. Hayashi, T. Morita, Y. Kodama, T. Sofuni, M. Ishidate Jr., The micronucleus assay with mouse peripheral blood reticulocytes using acridine orange-coated slides, Mutat. Res. 245 (4) (1990) 245–249. [13] M.A. Kastenbaum, K.O. Bowman, Tables for determining the statistical significance of mutation frequencies, Mutat. Res. 9 (1970) 527–549. [14] G.S. Ratra, S. Cottrell, C.J. Powell, Effects of induction and inhibition of cytochromes P450 on the hepatotoxicity of methapyrilene, Toxicol. Sci. 46 (1) (1998) 185–196. [15] H.K. Hamadeh, Methapyrilene toxicity: anchorage of pathologic observations to gene expression alterations, Toxicol. Pathol. 30 (4) (2002) 470–482. [16] NTP Technical Report on the Hepatotoxicity Studies of the Liver Carcinogen No. 46 (2000). [17] P. Rosignoli, R. Fabiani, A. De Bartolomeo, R. Fuccelli, M.A. Pelli, G. Morozzi, Genotoxic effect of bile acids on human normal and tumour colon cells and protection by dietary antioxidants and butyrate, Eur. J. Nutr. 47 (6) (2008) 301–309. [18] ICH, Harmonized Tripartite Guideline, Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, S2(R2), Step 4 Version, 2011. [19] 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), 2014 (in press).
Please cite this article in press as: K. Inoue, et al., The 14-day repeated dose liver micronucleus test with methapyrilene hydrochloride using young adult rats, Mutat. Res.: Genet. Toxicol. Environ. Mutagen. (2014), http://dx.doi.org/10.1016/j.mrgentox.2014.04.004