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.     

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 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.     

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 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.     

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.     

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.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

Micronucleus test with ethyl methanesulfonate in mouse peripheral blood reticulocytes stained supravitally using acridine orange-coated slides.

A new method for the micronucleus test using peripheral blood reticulocytes stained supravitally using acridine orange-coated slides was evaluated in male CD-1 mice treated with ethyl methanesulfonate (EMS) at doses of 100, 200, 300, and 400 mg/kg. Peripheral blood samples were taken 0, 24, 48, 72, and 96 h after treatment from each mouse without killing. The frequencies of micronucleated reticulocytes increased dose-dependently with the peak at 48 h after treatment. These results indicate that, at least for EMS, the new method used here can be an alternative to the conventional method using bone marrow polychromatic erythrocytes.     

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.     

Survival and mutagenesis of bacteriophage T7 damaged by methyl methanesulfonate and ethyl methanesulfonate

We have examined survival and mutagenesis of bacteriophage T7 after exposure to the alkylating agents methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS). It was found that although both alkylating agents caused increased reversion of specific T7 mutations, EMS caused a higher frequency of reversion than did MMS. Exposure of the host cells to ultraviolet light so as to induce the SOS system resulted in increased survival (Weigle reactivation) of T7 phage damaged with either EMS or MMS. However, after SOS induction of the host we did not detect an accompanying increase in mutation frequency measured as either reversion of specific T7 mutants or by generation of mutations in the T7 gene that codes for phage ligase. Neither mutation frequency nor survival of alkylated phage was affected by the umuD,C mutation in the Escherichia coli host nor by the presence of plasmid pKM101. This may mean that the mode of Weigle reactivation that is detected in T7 is not mutagenic in nature.     

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.     

A comparison of intraperitoneal and oral gavage administration in comet assay in mouse eight organs

One of the important advantages of the comet assay is its ability to detect genotoxicity in many different organs. Since the exposure route of the test compounds is likely to influence the genotoxicity detected in a given organ, it is an important factor to consider when conducting the assay. In this study, we compared the effects of numerous model compounds on eight organs when administered to mice by intraperitoneal (i.p.) injection and oral (p.o.) gavage.Groups of four mice were treated once i.p. or p.o. at the identical proportion of LD50 for each route, and the stomach, colon, liver, kidney, bladder, lung, brain, and bone marrow were sampled 3, 8, and 24h after treatment. For 19 of the 20 tested mutagens with various modes of action, genotoxicity in some organs varied with treatment route; only the genotoxicity of methyl methane sulfonate was not affected. Treatment route, however, did not produce a qualitative difference in the genotoxicity of promutagens at the sites of conversion to ultimate mutagens, with aromatic hydrocarbons as the exception. When chemicals with positive responses in at least one organ were considered to be comet assay-positive, the administration route made no difference. Since azo reduction is mediated by azo reductase synthesized in the gastrointestinal wall and by gut microflora and i.p.-administered azo dyes bypass their activation site (colon), the administration route is expected to make a difference in their in vivo genotoxicity. Direct-acting mutagens are expected to affect the mucosa of the gastrointestinal tract when given p.o. For those mutagens, however, the administration route did not make a qualitative difference in gastrointestinal tract genotoxicity. Moreover, although the gastrointestinal mucosa is the first site to be exposed to p.o. administered agents, the peak times in the stomach tended to be the same as in most other organs. Based on those results, we concluded that the genotoxicity at high exposures was due to a systemic effect, and that both routes are acceptable for the comet assay when the liver and gastrointestinal organs are sampled, so long as appropriate dose levels for systemic exposure are selected for each route.     

Nuclear and extranuclear mutations in yeast induced by ethyl methanesulfonate

When zero-point mutations were induced in the yeastSaccharomyces cerevisiae using ethyl methanesulfonate (EMS) no differences were found in the frequency of auxotrophic mutants formed by a short and a prolonged treatment of the agent at equal survival level. The expression of a part of the mutations induced by a prolonged EMS treatment was delayed by one or two division cycles. The total frequency of auxotrophs due to both the zero point and delayed mutations, however, is still considerably lower than the frequency of auxotrophs induced by a prolonged treatment of EMS in some bacterial species. Both the prolonged and short EMS treatment induces in yeast also extranuclear respiration-deficient (RD) mutants at a relatively high frequency; in wild strains at equal survival level the prolonged treatment produces a higher number of RD mutants than the short one. In strain which is more susceptible to the lethal EMS effect than wild strain the number of RD mutants produced by the agent is much higher than in the wild strain. The results support the assumption of the different DNA arrangement in yeast nuclei and mitochondria and indicate the possible effect of repair mechanisms during the induction of mutations causing the respiration deficiency.     

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.