Effect of route of administration in the micronucleus test with potassium bromate

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 (MS/Ae 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 mg/kg in the i.p. test and of 37.5, 75, 150, and 300 mg/kg 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 MS/Ae mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.     

Micronucleus test with methyl methanesulfonate administered by intraperitoneal injection and oral gavage

The effects of 2 routes of administration, intraperitoneal injection (i.p.) and oral gavage (p.o.), in the micronucleus test were evaluated using methyl methanesulfonate (MMS) and 2 strains of mice (MS/Ae and CD-1). A small-scale acute toxicity study and a pilot micronucleus experiment were carried out first. On the basis of the results obtained, a final micronucleus test was performed at doses of 20, 40, 80, and 160 mg/kg (i.p.) and 40, 80, 160, and 320 mg/kg (p.o.), with a 24-h sampling time. MMS induced micronucleated polychromatic erythrocytes (MNPCEs) in both routes in both mouse strains under the conditions used. At 40 and 80 mg/kg, MMS induced a higher number of MNPCEs by the i.p. route in both strains. A 160 mg/kg MMS dose induced higher numbers of MNPCEs by the p.o. route in MS/Ae mice. The route-related difference with MMS on the basis of mg/kg disappeared when the difference was determined on the basis of a ratio of the LD50. In practice, both i.p. and p.o. routes are acceptable as routes of administration in the micronucleus test using this chemical.     

A comparison of intraperitoneal injection and oral gavage in the micronucleus test with mitomycin C in mice

Intraperitoneal (i.p.) injection and oral (p.o.) gavage were evaluated in the mouse micronucleus test with mitomycin C (MMC). The tests were carried out in 2 laboratories with the MS/Ae and CD-1 mouse strains. On the basis of a small-scale acute toxicity study and a pilot experiment, the full-scale micronucleus test was performed with a 24-h sampling time at doses of 1, 2, 4, and 8 mg/kg for both treatment routes. In both strains, a clear positive dose-response relation was shown by both routes. Although the frequency of micronucleated polychromatic erythrocytes (MNPCEs) was higher with i.p. on a mg/kg basis, this tendency was reversed when dose was expressed as a percentage of the LD50.     

Administration-route-related differences in the micronucleus test with N-ethyl-N-nitrosourea

Administration-route-related differences in the micronucleus test were examined by giving N-ethyl-N-nitrosourea (ENU) to male mice of the MS/Ae and CD-1 strains by 2 different routes, intraperitoneally (i.p.) and orally (p.o.). The experiments consisted of 3 parts: (1) a simplified acute toxicity study, which gave LD50s of 490 (i.p.) and 840 mg/kg (p.o.) in MS/Ae and 640 (i.p.) and 960 mg/kg (p.o.) in CD-1 mice: (2) a pilot experiment for the full-scale micronucleus test to determine appropriate dosages and sampling time: and (3) the micronucleus test at doses of 12.5, 25, 50, and 100 mg/kg with a sampling time of 24 h. The results indicated that no route-related differences existed at the 2 lowest doses. At 50 mg/kg, markedly higher numbers of micronucleated polychromatic erythrocytes (MNPCEs) were induced in both mouse strains by the i.p. route. At 100 mg/kg, the difference between the routes decreased in strain CD-1 and even reversed in MS/Ae. Thus, route-related differences appeared to depend on the dose. Such differences became small, however, in both strains when the comparison was made on the basis of LD50 values.     

Micronucleus test with 1-beta-D-arabinofuranosylcytosine administered by intraperitoneal injection and oral gavage

The effect of route of administration on the outcome of the micronucleus test was evaluated in 2 laboratories by administering the model chemical, 1-beta-D-arabinofuranosylcytosine (Ara-C), by intraperitoneal injection (i.p.) and oral gavage (p.o.) to 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot experiment for the micronucleus test, a full-scale test was performed with a 24-h sampling time at doses of 12.5, 25, 50, and 100 mg/kg i.p. and 25, 50, 100, and 200 mg/kg p.o. In both strains, MNPCEs were induced at lower dose levels by the i.p. treatment, as determined not only on the basis of mg/kg but also as a ratio of the LD50. When compared with other chemicals tested in this collaborative study, the effective dose levels of this chemical based on the LD50s were exceptionally low by both routes and in both strains, e.g., less than 0.3% of the LD50 by the i.p. treatment. The maximum frequencies of MNPCEs induced were, however, identical (MS/Ae) or even higher (CD-1) by the p.o. treatment.     

Micronucleus test with potassium chromate(VI) administered intraperitoneally and orally to mice

The effect of route of administration, intraperitoneal (i.p.) or oral gavage (p.o.), in the mouse micronucleus test was studied with K2CrO4 in 2 mouse strains (MS/Ae and CD-1). A simplified acute toxicity test to estimate the toxic dose levels of K2CrO4 showed that the LD50S were 50 mg/kg i.p. and 300 mg/kg p.o. for MS/Ae and 32 mg/kg i.p. and 180 mg/kg p.o. for CD-1. Based on results of a pilot micronucleus test to determine appropriate dose levels and the optimal sampling time, it was decided to sample bone marrow cells of both strains of mice 24 h after i.p. doses of 10-80 mg/kg and p.o. doses ranging from 20 to 320 mg/kg. K2CrO4 administered i.p. induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently in both strains. In contrast, when administered p.o. the chemical failed to induce MNPCEs. These results suggest that this difference between i.p. and p.o. routes is related to a difference of absorption or metabolic fate of chromate in vivo.     

Micronucleus test with 2-acetylaminofluorene by intraperitoneal injection and oral administration

The effect of route of administration on the outcome of the mouse micronucleus test was evaluated in 2 laboratories by administering 2-acetylaminofluorene (2-AAF) by intraperitoneal injection (i.p.) and oral gavage (p.o.) to 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus test, the full-scale experiment was performed with a 24-h sampling time at doses ranging from 75 to 600 mg/kg by both routes. The results indicated that 2-AAF induced micronucleated polychromatic erythrocytes (MNPCEs) at all doses tested by both routes. In the MS/Ae strain, higher doses were required by p.o. than by i.p. to reach a similar level of MNPCE incidence. On the other hand, similar responses were recorded by both administration routes with CD-1 mice. Since the LD50 for the p.o. route was higher than that for the i.p. route in both strains, the route-related difference with MS/Ae mice became small when the comparison between i.p. and p.o. was made on the basis of the LD50. Thus both i.p. and p.o. routes are acceptable in the micronucleus test of this chemical.     

Micronucleus test with 6-mercaptopurine monohydrate administered intraperitoneally and orally

The effect of route of administration on the induction of micronucleated polychromatic erythrocytes (MNPCEs) was examined. 6-Mercaptopurine monohydrate (6-MP) was administered intraperitoneally (i.p.) or orally (p.o.) to 2 strains of mice, MS/Ae and CD-1. From the results of an acute toxicity test and a pilot micronucleus test, the doses selected for the final micronucleus test were 12.5-100 mg/kg for the i.p. route and 25-200 mg/kg for the p.o. route. The sampling time was 48 h. Frequencies of MNPCEs increased dose-dependently by the i.p. route but peaked at 50 or 100 mg/kg for the p.o. route. 6-MP induced MNPCEs more efficiently after p.o. administration than after i.p. treatment in both strains.     

Administration-route-related difference in the micronucleus test with 7,12-dimethylbenz[a]anthracene

The effect of route of administration on the outcome of the mouse micronucleus test was evaluated in 2 laboratories by administering a model chemical, 7,12-dimethylbenz[a]anthracene (DMBA) by intraperitoneal injection (i.p.) and oral gavage administration (p.o.) to males of 2 mouse strains, MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus test, a full-scale micronucleus test was performed with a 48-h sampling time at doses of 25, 50, 100, and 200 mg/kg by both administration routes in the 2 strains. At each dose level and in both strains, higher frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were found after use of the i.p. route. In the MS/Ae strain, a linear, positive dose response was obtained by both routes. In the CD-1 strain, the maximum response was reached at 100 mg/kg and a downturn occurred at 200 mg/kg by both routes. The comparison of maximum responses indicated that MS/Ae was the higher responder for both routes of application. Although DMBA induced micronuclei more efficiently by the i.p. route than after oral administration on a mg/kg base, this route-related difference was reversed in both strains when the comparison was made on the basis of LD50 values and when the maximum responses were neglected.     

Micronucleus test with procarbazine hydrochloride administered by intraperitoneal injection and oral gavage

The difference in effect of route of administration of procarbazine hydrochloride (PCZ) in the mouse was investigated in the micronucleus test. PCZ was administered by intraperitoneal injection (i.p.) and oral administration (p.o.) to 2 strains of male mice (MS/Ae and CD-1). On the basis of a small-scale acute toxicity test and a pilot micronucleus test, bone marrow preparations were prepared 24 h after the administration by the i.p. and p.o. routes of 50-400 mg/kg and 200-1600 mg/kg, respectively. The maximum incidence of polychromatic erythrocytes with micronuclei (MNPCEs) was somewhat higher after p.o. treatment in MS/Ae mice and the same with both routes in CD-1 mice. Thus, the clastogenicity of PCZ in mouse bone marrow was revealed by both routes.     

Administration-route-related differences in the micronucleus test with benzene

The effect of route of administration on the outcome of the micronucleus test was studied in 2 laboratories by administering the model chemical benzene intraperitoneally (i.p.) and orally (p.o.) to 2 strains of mice: MS/Ae and CD-1. On the basis of results obtained in a small-scale acute toxicity study and in a pilot micronucleus test, full-scale micronucleus tests were performed with a 24-h sampling time at doses of 250, 500, 1000, and 2000 mg/kg i.p. and 500, 1000, 2000, and 4000 mg/kg p.o. In both strains of mice, a higher incidence of micronucleated polychromatic erythrocytes (MNPCEs) was observed after p.o. administration. The ratio of polychromatic erythrocytes (PCEs) to total erythrocytes decreased more markedly at higher doses i.p. in both strains. Thus, benzene induced more micronuclei via the p.o. route, while inhibitory effects on bone marrow cells were stronger after i.p. administration.     

Micronucleus induction in mouse bone marrow by phenacetin administered intraperitoneally or orally

The extent and time course of induction of micronucleated polychromatic erythrocytes (MNPCEs) in mouse bone marrow were examined after administration of phenacetin as an insoluble suspension in olive oil by intraperitoneal injection (i.p.) or gastric intubation (p.o.) to 2 strains of mice, MS/Ae and CD-1, at doses up to 1200 mg/kg. The toxicity of phenacetin and the sensitivity of micronucleus induction differed in the 2 strains, but there was little difference in the extent of MNPCEs induced by the 2 administration routes.     

Micronucleus test with cyclophosphamide administered by intraperitoneal injection and oral gavage

The effect of route of administration on the micronucleus test was examined in 2 laboratories: cyclophosphamide (CYP) was administered by intraperitoneal injection (i.p.) or oral gavage (p.o.) to 2 strains of mice. MS/Ae and CD-1. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the final micronucleus test was performed with a 48-h sampling time at doses of 25-200 mg/kg i.p. and 50-400 mg/kg p.o. CYP via the i.p. route was more toxic and induced more micronucleated polychromatic erythrocytes (MNPCEs) in MS/Ae mice than in CD-1 mice. Administration-route-related differences were not distinctly shown in the MS/Ae strain. In CD-1, however, higher doses were required for the p.o. route than for the i.p. route to induce about equal amounts of clastogenic damage.     

Difference between intraperitoneal and oral gavage application in the micronucleus test. The 3rd collaborative study by CSGMT/JEMS.MMS. Collaborative Study Group for the Micronucleus Test/Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan

In the third collaborative study organized by the Collaborative Study Group for the Micronucleus Test (CSGMT), a task group belonging to the Mammalian Mutagenesis Study subgroup of the Environmental Mutagen Society of Japan (JEMS.MMS), intraperitoneal (i.p.) injection and oral (p.o.) gavage were compared as routes of administration of test chemicals. Two mouse strains, MS/Ae and CD-1, and 17 chemicals with various modes of action were used. The chemicals were 1-beta-D-arabinofuranosylcytosine, 6-mercaptopurine monohydrate, benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 2-acetylaminofluorene, phenacetin, cyclophosphamide, ethyl methanesulfonate, N-ethyl-N-nitrosourea, methyl methanesulfonate, mitomycin C, colchicine, vincristine sulfate, potassium bromate, potassium chromate(VI), benzene, and procarbazine hydrochloride. On the basis of the findings of an acute toxicity test and a pilot experiment for dose and sampling time, a full-scale micronucleus test was performed on each chemical. Almost all the chemicals showed a positive response in micronucleus induction by both routes of administration in both mouse strains. Contradictory outcomes were obtained between the i.p. and p.o. routes on potassium chromate in both strains (i.p.: positive, p.o.: negative). In the CD-1 mice, benzene potently induced micronuclei when administered p.o., but gave only a marginal response when administered i.p. Generally, the chemicals induced micronuclei at lower dose levels (mg/kg) when administered i.p. This tendency, however, was decreased or even reversed when the dose was expressed as a percentage of the LD50. Although the i.p. route, an artificial exposure route, is useful to detect the inducibility of micronuclei of a test chemical per se at a small dose, the p.o. route seemed sensitive and valuable enough to evaluate the test chemicals. When the dose levels of chemicals are adjusted on the basis of the LD50, both i.p. injection and p.o. gavage are acceptable as routes of administration in the micronucleus test.     

Micronucleus test with benzo[a]pyrene using a single peroral administration and intraperitoneal injection in males of the MS/Ae and CD-1 mouse strains

The effect of route of administration on the outcome of the micronucleus test was examined by administering benzo[a]pyrene (B[a]P) perorally (p.o.) and intraperitoneally (i.p.) to males of the MS/Ae and CD-1 mouse strains. This study consisted of 3 parts. First, an acute toxicity study lasting 3 days was done to estimate LD50s. The LD50 was larger than 1600 mg/kg for both routes in the 2 strains. Second, pilot micronucleus tests were carried out, on the basis of which an appropriate sampling time (48 h) and dose levels (62.5, 125, 250, and 500 mg/kg) were chosen for both routes and both strains. Third, full-scale micronucleus tests were done, which indicated that (1) B[a]P induced micronuclei dose-dependently by each administration route in each strain, (2) the i.p. route induced frequencies of micronuclei almost equal to or slightly higher than did the p.o. route, and (3) the MS/Ae strain was the higher responder.     

Micronucleus test with vincristine administered by intraperitoneal injection and oral gavage

The effects of vincristine sulfate (VINC) on micronucleus induction were studied in 2 strains of mice (MS/Ae: CD-1) following intraperitoneal (i.p.) or oral administration (p.o.) of the chemical. On the basis of a small-scale acute toxicity study and a pilot micronucleus experiment, the full-scale micronucleus test was performed with a sampling time of 24 h at doses of 0.063, 0.125, 0.25 and 0.5 mg/kg (i.p.) and 1.25, 2.5, 5.0 and 10 mg/kg (p.o.). The maximum frequency of micronucleated polychromatic erythrocytes was 7.15% in MS/Ae mice and 4.98% in CD-1 mice at 5.0 mg/kg p.o. in both cases. The maximum frequencies by the i.p. route (9.93% in MS/Ae mice; 11.68% in CD-1 mice) occurred at 0.25 mg/kg and 0.125 mg/kg, respectively. Although the doses showing a positive response were different between the 2 routes, VINC induced micronuclei very efficiently at all doses tested by both administration routes in both strains.     

Micronucleus test with cyclophosphamide using mouse peripheral blood reticulocytes.

The usefulness of the micronucleus test using supravital staining of peripheral blood reticulocytes with acridine orange was evaluated in two laboratories after administering cyclophosphamide (CYP) as a model chemical by intraperitoneal injection (i.p.) to CD-1 mice. The frequencies of micronucleated peripheral reticulocytes (MNRETs) increased dose-dependently at each sampling time. There were no significant differences in the results obtained with this new method by the two laboratories. Although the induction of MNRETs was delayed by about 24 h compared to that of micronucleated polychromatic erythrocytes (MNPCEs) in the bone marrow, the frequencies of MNRETs and MNPCEs were almost identical at each optical sampling time, 24 h for MNPCEs and 48 h for MNRETs. Therefore, it is concluded that this method is a suitable alternative to that using femoral marrow cells.     

Anticlastogenicity of two derivatives of 1,4-dihydroisonicotinic acid in mouse micronucleus test

Effects of two derivatives of 1,4-dihydroisonicotinic acid (1,4-DHINA) against the monofunctional alkylating agent ethyl methanesulfonate (EMS) were studied in the micronucleus test in (CBA x C57Bl/6(j)) mice. Adult males and pregnant females were treated with an antimutagen (i.p.) and 12h later they were exposed to EMS (i.p.). The frequencies of micronucleated (MN) polychromatic erythrocytes (PCEs) in mouse bone marrow and foetal liver were analysed 6, 12, 18, 24, 30, 36, 48 or 24, 48 and 72 h after the mutagen injection. In adults, the maximum number of MNPCEs was observed 36 or 24h after the EMS administration. In foetuses, which were treated in a maternal organism, such peak was found at 24h. Pre-treatment of mice with the antimutagens 2,6-dimethyl-3,5-diethoxycarbonyl-4-(Na carboxylate)-1,4-dihydropyridine (DHP) and glutapyrone (GP) decreased the yield of MNPCEs in male bone marrow. Having been observed at a peak of MN induction, the anticlastogenic effect of DHP (1/10 LD(50) or 340 mg/kg) reached 30%. DHP at the doses of 0.5-1mM/kg did not affect the EMS-induced frequency of MNPCEs in bone marrow, whereas GP inhibited it at the similar millimolar concentrations. Simultaneously with maternal bone marrow, foetal liver cells were analysed for MNs in the transplacental test. The anticlastogenic effect of DHP (1/10 LD(50)) was found to be more prolonged and higher in females than in males and to average 50%, but this antimutagen was not efficient in foetuses. Both antimutagens did not change the polychromatic/normochromatic erythrocyte (PCE/NCE) ratio as compared with EMS action.Results presented indicate a peak of EMS-induced micronucleated cells in mouse bone marrow 24 or 36 h and in foetal liver 24h after animal treatment. Two 1,4-DHINA derivatives exhibited anticlastogenic activity in adults, but not in foetuses.     

The micronucleus test of methyl methanesulfonate with mouse peripheral blood reticulocytes using acridine orange-coated slides.

The usefulness of the micronucleus assay using mouse peripheral blood erythrocytes and acridine orange (AO)-coated slides was evaluated with methyl methanesulfonate (MMS). The micronucleus test was carried out at doses ranging from 20 to 80 mg/kg body weight in CD-1 mice by intraperitoneal injection. Peripheral blood cells were examined from 0 to 72 h after treatment at 12- or 24-h intervals. Bone marrow cells from other mice treated with 80 mg/kg MMS were also sampled at the same times. The frequency of micronucleated reticulocytes (MNRETs) increased dose-dependently at every sampling time except 72 h, and the maximum frequency of MNRETs was observed at about 36 h after treatment. Micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow after a dose of 80 mg/kg were significantly induced at 12 h to 36 h, and the maximum frequency of MNPCEs was observed at 24 h after treatment. The induction of MNRETs was delayed by about 12 h compared to that of MNPCEs in bone marrow, and the maximum frequencies of MNRETs were lower than those of MNPCEs, but the induction of MNRETs by MMS was significant and dose-dependent. It is concluded, therefore, that bone marrow cells could be replaced by peripheral blood cells as material for the micronucleus assay using AO-coated slides.     

Induction of micronuclei in mouse polychromatic erythrocytes by the administration of non-radioactive CsCl by the oral and intraperitoneal route

In the present study, we describe the effects of the concentration and route of administration of non-radioactive cesium chloride (CsCl) in inducing micronuclei in mouse bone marrow polychromatic erythrocytes (PCEs). When the dose of 500mg/kg body weight was administered perorally (p.o.), no significant incidence of micronuclei was detected. However, when the same dose was administered intraperitoneally (i.p.), a significant induction of micronuclei in PCEs was observed compared to control. At the dose of 1000mg/kg, both routes were efficient, with no significant difference in micronucleus frequencies. We conclude that both the p.o. and i.p. routes are efficient in inducing micronuclei, with the i.p. route being more efficient when lower CsCl doses are used.