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Effect of route of administration in the micronucleus test with potassium bromate
Mutation Research, 223 (1989) 399-402
399
Elsevier MUTGEN 01441
Effect of route of administration in the micronucleus test with potassium bromate Madoka Nakajima 1, Michiyo Kitazawa 1, Kousuke Oba 1, Yoshinori Kitagawa 2 and Yoshiko Toyoda 2 1 Biosafety Research Center, Foods, Drugs, and Pesticides (An-Pyo Center), 582-2, Arahama, Shioshinden, Fukude-cho, lwata-gun, Shizuoka-ken 437-12 (Japan) and 2 Suntory Co. Ltd., 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618 (Japan)
(Received 8 September1988) (Revision received2 February 1989) (Accepted 2 February 1989)
Keyword: Potassium bromate
Summary The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice ( M S / A e and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 m g / k g in the i.p. test and of 37.5, 75, 150, and 300 m g / k g in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with M S / A e mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.
The aim of the third collaborative study organized by the Collaborative Study Group for the Micronucleus Test (CSGMT) was to detect administration-route-related differences in the micronucleus test. Potassium bromate, a flour-bleaching agent, was used in the present study. The studies using M S / A e mice were performed at the An-Pyo Center and those using CD-1 mice were done at Suntory Co. Ltd.
Correspondence: Dr. Madoka Nakajima, Biosafety Research Center, Foods, Drugs, and Pesticides (An-Pyo Center), 582-2, Arahama, Shioshinden, Fukude-cho, Iwata-gun, Shizuoka-ken 437-12 (Japan).
Materials and methods
Male M S / A e and CD-1 mice used in this experiment were purchased from Hitachi Medical Animal Laboratories (Sanwa, Japan) and Charles River Japan Inc. (Atsugi, Japan), respectively. Animals were 7 weeks old when received from the supplier. They were used for the assay after an acclimation period of I week. The body weights at the time of dosing were between 27.5 and 29.8 g for M S / A e and 32.1-38.5 g for CD-1. The animals were housed in a temperature- and humidity-controlled room, and were given M F pellets (Oriental Yeast Co. Ltd., Japan) and water ad libitum. Potassium bromate (KBrO3; CAS No. 7758-012), obtained from Wako Pure Chemical Industries
0165-1218/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (Biomedical Division)
40O
Ltd. (Osaka, J a p a n ) w a s dissolved in physiological saline immediately before use. The acute toxicity study was performed by a method consisting of 2 parts as described by Lorke (1983), to estimate the approximate LDs0 value. As a second step, a pilot study for the micronucleus test was carried out using 2 mice per group, 4 dose levels and 3 sampling times to find the optimal dose range and sampling time for the full-scale micronucleus study (Hayashi et al., 1984). The micronucleus test was performed using the procedure described in the summary report of this collaborative study (Hayashi et al., 1989).
4
ip
3
o fit_
89 (1/21 1/4.1 ,"1" 22 11/81 11 (1/161
m g / k O 11-0so1
72h
4
Results and discussion
130
w
3
The results of the acute toxicity test are shown TABLE 1 ACUTE TOXICITY OF POTASSIUM BROMATE IN MS/Ae AND CD-1 MICE
24.1 11/21 120 [1/4,1
Strain
Route
Dose (mg/kg)
Mortality
MS/Ae
i.p.
I0 100 1000 140 225 370 600
0/3 0/3 3/3 0/1 1/I 1/1 1/1
10 100 1000
0/3 0/3 3/3
p.o.
CD-1
i.p.
i4o
o/i
225 370 600
0/1 0/1 1/1
30 11/161 72h
mo/kO [LDnI
Fig. 1. Time course and dose responses for micronucleus induction by potassium bromate in the bone marrow of MS/Ae mice by i.p. and p.o. administration.
177
471
10
0/3
]oo
0/3
1000 225 370 600
3/3 0/1 1/1 1/1 1/1
177
10 100 1000 140 225 370 600
0/3 0/3 3/3 0/1 0/1 1/1 1/1
289
140
p.o.
LDs0 (mg/kg)
in Table 1. In both strains, all 3 mice died after receiving potassium bromate at 1000 mg/kg, but none died at 100 mg/kg by either route of administration. According to Lorke's table, the second trial was performed using 4 doses between 140 and 600 mg/kg. The approximate LDsos were estimated to be 177 mg/kg for the i.p. administration in both strains and 471 and 289 mg/kg for p.o. in MS/Ae and CD-1, respectively. Following the acute toxicity tests, 4 dose levels were used in the pilot micronucleus test, i.e., approximately 1/2, 1/4, 1/8, and 1/16 of the LDs0. The results of the pilot test are shown in Figs. 1 and 2. A dose-related increase in MNPCEs was observed in MS/Ae mice 24 h after i.p. treatment; however, at 48 and 72 h after exposure there were no definite dose responses. With p.o. treatment, the incidence of MNPCEs increased markedly after 24 h treatment up to and including the dose of 60
401
mg/kg. At higher doses there were decreases of the frequency of MNPCEs due to overdosing. At other sampling times, however, lower incidences of MNPCEs were observed. On the basis of the results of the pilot experiments, we selected doses of 18.8, 37.5, 75, and 150 mg/kg for i.p. treatment and 37.5, 75, 150, and 300 mg/kg for p.o. treatment. All mice were killed 24 h after a single administration. The highest dose used was slightly above the LDs0 values in order to determine the maximum response in each experiment. The peak incidence of MNPCEs was 2.08% after i.p. treatment of MS/Ae mice with 150 mg/kg (about 85% of LDso ) and 2.28% after a p.o. dose of 300 m g / k g (about 64% of LDs0). A definite route difference was observed in CD-1 mice: more MNPCEs were induced by the i.p. than the p.o. route. The present results confirm the results in ddY mice reported by Hayashi et al. (1988) that potassium bromate induced micronuclei clearly in both routes of administration. This chemical was also evaluated in an investigation for the purpose of explaining sex-related differences in the micronucleus test by CSGMT (1986), and no dif-
ip in, ILl
Z
90 1112)
24.h
1/41 7
22.5 11181
11.25 11/161
mO/Ico ILDsol
72h
4
p0
3 m
f~ z
1 0 24h
145 11/21
11/41 ,,~
36.25 I1/81
18.13 (1/161
mg/kll ( l ~ n l
72h
Fig. 2. Time course and dose responses for micronucleus induction by potassium bromate in the bone marrow of CD-1 mice by i.p. and p.o. administration.
TABLE 2 RESULTS O F T H E M I C R O N U C L E U S TEST W I T H P O T A S S I U M B R O M A T E W I T H i.p. A N D p.o. S I N G L E T R E A T M E N T IN M S / A e A N D CD-1 M I C E ( M E A N ± SD) Dose
i.p.
(mg/kg)
MNPCEs (%)
PCEs (%)
MS/Ae
0 18.8 37.5 75.0 150 300
0.08 + 0.10 0.23+0.19 0.60+0.12 0.83+0.29 2.08 + 0.80 n.t.
53.5 + 55.4+ 57.3+ 50.2+ 38.9 + n.t.
CD-1
0 18.8 37.5 75.0 150 300
0.30+0.14 0.50+0.12 0.78 ± 0.44 1.45+0.38 3.78+0.97 n.t.
41.5+ 44.4± 43.2 + 49.7+ 30.8+ n.t.
Strain
n.t., not tested.
p.o. MNPCEs (%)
PCEs (%)
5.0 3.4 4.6 2.2 16.6
0.23 + 0.22 n.t. 0.53+0.28 0.75+0.25 1.63 + 0.82 2.28 + 1.23
56.5 + 6.4 n.t. 58.6-t- 4.8 55.8+ 2.0 49.4 + 8.8 44.1 + 10.6
3.9 6.6 7.1 3.1 4.8
0.13±0.13 n.t. 0.28 ± 0.22 0.43+0.17 0.58±0.21 1.284-0.49
53.9+ 5.1 n.t. 48.0 ± 14.1 53.4± 3.6 40.8+18.9 47.94- 6.7
402
ferences between male and female ddY mice were observed. From these results, we conclude that potassium bromate induced micronuclei through both routes of administration in both mouse strains. I.p. administration was more effective in inducing micronuclei than p.o. administration especially in CD-1 mice. Micronucleus induction was observed at a lower dose level by the i.p. route than the p.o. route; this might be explained by the fact that the LDs0 value was low in the i.p. route. References Collaborative Study Group for the Micronucleus Test (1986) Sex difference in the micronucleus test, Mutation Res., 172, 151-163.
Hayashi, M., T. Sofuni and M. Ishidate Jr. (1984) A pilot experiment for the micronucleus test: the multi-sampling at multi-dose levels method, Mutation Res., 141, 165-169. Hayashi, M., M. Kishi, T. Sofuni and M. Ishidate Jr. (1988) Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals, Fd. Chem. Toxicol., 26, 487-500. Hayashi, M., S. Sutou, H. Shimada, S. Sato, Y.F. Sasaki and A. Wakata (1989) Difference between intraperitoneal and oral gavage application in the micronucleus test: the 3rd collaborative study by CSGMT/JEMS. MMS, Mutation Res., 223, 329-344. Lorke, D. (1983) New approach to practical acute toxicity testing, Arch. Toxicol., 54, 275-287.
399
Elsevier MUTGEN 01441
Effect of route of administration in the micronucleus test with potassium bromate Madoka Nakajima 1, Michiyo Kitazawa 1, Kousuke Oba 1, Yoshinori Kitagawa 2 and Yoshiko Toyoda 2 1 Biosafety Research Center, Foods, Drugs, and Pesticides (An-Pyo Center), 582-2, Arahama, Shioshinden, Fukude-cho, lwata-gun, Shizuoka-ken 437-12 (Japan) and 2 Suntory Co. Ltd., 1-1-1, Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka 618 (Japan)
(Received 8 September1988) (Revision received2 February 1989) (Accepted 2 February 1989)
Keyword: Potassium bromate
Summary The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice ( M S / A e and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 m g / k g in the i.p. test and of 37.5, 75, 150, and 300 m g / k g in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with M S / A e mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.
The aim of the third collaborative study organized by the Collaborative Study Group for the Micronucleus Test (CSGMT) was to detect administration-route-related differences in the micronucleus test. Potassium bromate, a flour-bleaching agent, was used in the present study. The studies using M S / A e mice were performed at the An-Pyo Center and those using CD-1 mice were done at Suntory Co. Ltd.
Correspondence: Dr. Madoka Nakajima, Biosafety Research Center, Foods, Drugs, and Pesticides (An-Pyo Center), 582-2, Arahama, Shioshinden, Fukude-cho, Iwata-gun, Shizuoka-ken 437-12 (Japan).
Materials and methods
Male M S / A e and CD-1 mice used in this experiment were purchased from Hitachi Medical Animal Laboratories (Sanwa, Japan) and Charles River Japan Inc. (Atsugi, Japan), respectively. Animals were 7 weeks old when received from the supplier. They were used for the assay after an acclimation period of I week. The body weights at the time of dosing were between 27.5 and 29.8 g for M S / A e and 32.1-38.5 g for CD-1. The animals were housed in a temperature- and humidity-controlled room, and were given M F pellets (Oriental Yeast Co. Ltd., Japan) and water ad libitum. Potassium bromate (KBrO3; CAS No. 7758-012), obtained from Wako Pure Chemical Industries
0165-1218/89/$03.50 © 1989 ElsevierSciencePublishers B.V. (Biomedical Division)
40O
Ltd. (Osaka, J a p a n ) w a s dissolved in physiological saline immediately before use. The acute toxicity study was performed by a method consisting of 2 parts as described by Lorke (1983), to estimate the approximate LDs0 value. As a second step, a pilot study for the micronucleus test was carried out using 2 mice per group, 4 dose levels and 3 sampling times to find the optimal dose range and sampling time for the full-scale micronucleus study (Hayashi et al., 1984). The micronucleus test was performed using the procedure described in the summary report of this collaborative study (Hayashi et al., 1989).
4
ip
3
o fit_
89 (1/21 1/4.1 ,"1" 22 11/81 11 (1/161
m g / k O 11-0so1
72h
4
Results and discussion
130
w
3
The results of the acute toxicity test are shown TABLE 1 ACUTE TOXICITY OF POTASSIUM BROMATE IN MS/Ae AND CD-1 MICE
24.1 11/21 120 [1/4,1
Strain
Route
Dose (mg/kg)
Mortality
MS/Ae
i.p.
I0 100 1000 140 225 370 600
0/3 0/3 3/3 0/1 1/I 1/1 1/1
10 100 1000
0/3 0/3 3/3
p.o.
CD-1
i.p.
i4o
o/i
225 370 600
0/1 0/1 1/1
30 11/161 72h
mo/kO [LDnI
Fig. 1. Time course and dose responses for micronucleus induction by potassium bromate in the bone marrow of MS/Ae mice by i.p. and p.o. administration.
177
471
10
0/3
]oo
0/3
1000 225 370 600
3/3 0/1 1/1 1/1 1/1
177
10 100 1000 140 225 370 600
0/3 0/3 3/3 0/1 0/1 1/1 1/1
289
140
p.o.
LDs0 (mg/kg)
in Table 1. In both strains, all 3 mice died after receiving potassium bromate at 1000 mg/kg, but none died at 100 mg/kg by either route of administration. According to Lorke's table, the second trial was performed using 4 doses between 140 and 600 mg/kg. The approximate LDsos were estimated to be 177 mg/kg for the i.p. administration in both strains and 471 and 289 mg/kg for p.o. in MS/Ae and CD-1, respectively. Following the acute toxicity tests, 4 dose levels were used in the pilot micronucleus test, i.e., approximately 1/2, 1/4, 1/8, and 1/16 of the LDs0. The results of the pilot test are shown in Figs. 1 and 2. A dose-related increase in MNPCEs was observed in MS/Ae mice 24 h after i.p. treatment; however, at 48 and 72 h after exposure there were no definite dose responses. With p.o. treatment, the incidence of MNPCEs increased markedly after 24 h treatment up to and including the dose of 60
401
mg/kg. At higher doses there were decreases of the frequency of MNPCEs due to overdosing. At other sampling times, however, lower incidences of MNPCEs were observed. On the basis of the results of the pilot experiments, we selected doses of 18.8, 37.5, 75, and 150 mg/kg for i.p. treatment and 37.5, 75, 150, and 300 mg/kg for p.o. treatment. All mice were killed 24 h after a single administration. The highest dose used was slightly above the LDs0 values in order to determine the maximum response in each experiment. The peak incidence of MNPCEs was 2.08% after i.p. treatment of MS/Ae mice with 150 mg/kg (about 85% of LDso ) and 2.28% after a p.o. dose of 300 m g / k g (about 64% of LDs0). A definite route difference was observed in CD-1 mice: more MNPCEs were induced by the i.p. than the p.o. route. The present results confirm the results in ddY mice reported by Hayashi et al. (1988) that potassium bromate induced micronuclei clearly in both routes of administration. This chemical was also evaluated in an investigation for the purpose of explaining sex-related differences in the micronucleus test by CSGMT (1986), and no dif-
ip in, ILl
Z
90 1112)
24.h
1/41 7
22.5 11181
11.25 11/161
mO/Ico ILDsol
72h
4
p0
3 m
f~ z
1 0 24h
145 11/21
11/41 ,,~
36.25 I1/81
18.13 (1/161
mg/kll ( l ~ n l
72h
Fig. 2. Time course and dose responses for micronucleus induction by potassium bromate in the bone marrow of CD-1 mice by i.p. and p.o. administration.
TABLE 2 RESULTS O F T H E M I C R O N U C L E U S TEST W I T H P O T A S S I U M B R O M A T E W I T H i.p. A N D p.o. S I N G L E T R E A T M E N T IN M S / A e A N D CD-1 M I C E ( M E A N ± SD) Dose
i.p.
(mg/kg)
MNPCEs (%)
PCEs (%)
MS/Ae
0 18.8 37.5 75.0 150 300
0.08 + 0.10 0.23+0.19 0.60+0.12 0.83+0.29 2.08 + 0.80 n.t.
53.5 + 55.4+ 57.3+ 50.2+ 38.9 + n.t.
CD-1
0 18.8 37.5 75.0 150 300
0.30+0.14 0.50+0.12 0.78 ± 0.44 1.45+0.38 3.78+0.97 n.t.
41.5+ 44.4± 43.2 + 49.7+ 30.8+ n.t.
Strain
n.t., not tested.
p.o. MNPCEs (%)
PCEs (%)
5.0 3.4 4.6 2.2 16.6
0.23 + 0.22 n.t. 0.53+0.28 0.75+0.25 1.63 + 0.82 2.28 + 1.23
56.5 + 6.4 n.t. 58.6-t- 4.8 55.8+ 2.0 49.4 + 8.8 44.1 + 10.6
3.9 6.6 7.1 3.1 4.8
0.13±0.13 n.t. 0.28 ± 0.22 0.43+0.17 0.58±0.21 1.284-0.49
53.9+ 5.1 n.t. 48.0 ± 14.1 53.4± 3.6 40.8+18.9 47.94- 6.7
402
ferences between male and female ddY mice were observed. From these results, we conclude that potassium bromate induced micronuclei through both routes of administration in both mouse strains. I.p. administration was more effective in inducing micronuclei than p.o. administration especially in CD-1 mice. Micronucleus induction was observed at a lower dose level by the i.p. route than the p.o. route; this might be explained by the fact that the LDs0 value was low in the i.p. route. References Collaborative Study Group for the Micronucleus Test (1986) Sex difference in the micronucleus test, Mutation Res., 172, 151-163.
Hayashi, M., T. Sofuni and M. Ishidate Jr. (1984) A pilot experiment for the micronucleus test: the multi-sampling at multi-dose levels method, Mutation Res., 141, 165-169. Hayashi, M., M. Kishi, T. Sofuni and M. Ishidate Jr. (1988) Micronucleus tests in mice on 39 food additives and eight miscellaneous chemicals, Fd. Chem. Toxicol., 26, 487-500. Hayashi, M., S. Sutou, H. Shimada, S. Sato, Y.F. Sasaki and A. Wakata (1989) Difference between intraperitoneal and oral gavage application in the micronucleus test: the 3rd collaborative study by CSGMT/JEMS. MMS, Mutation Res., 223, 329-344. Lorke, D. (1983) New approach to practical acute toxicity testing, Arch. Toxicol., 54, 275-287.