Micronucleus test with colchicine given by intraperitoneal injection and oral gavage

doi:10.1016/0165-1218(89)90093-1

Mutation Research/Genetic Toxicology

Volume 223, Issue 4, August 1989, Pages 391-394

https://doi.org/10.1016/0165-1218(89)90093-1 Get rights and content

Abstract

The effects of micronucleus induction in mouse bone marrow cells by intraperitoneal injection (i.p.) and oral administration (p.o.) were studied with the spindle poison colchicine in 2 strains of mice (MS/Ae and CD-1).

The final micronucleus test was performed with a 24-h sampling time at doses of 0.25–2.0 mg/kg by the i.p. route and 2–16 mg/kg by the p.o. route based on a simple acute toxicity test and a pilot micronucleus experiment.

Colchicine induced micronuclei in both strains and at all doses tested. Micronuclei were induced more effectively by the i.p. route.

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Cited by (12)

In vivo erythrocyte micronucleus assay. III. Validation and regulatory acceptance of automated scoring and the use of rat peripheral blood reticulocytes, with discussion of non-hematopoietic target cells and a single dose-level limit test
2007, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
The in vivo micronucleus assay working group of the International Workshop on Genotoxicity Testing (IWGT) discussed new aspects in the in vivo micronucleus (MN) test, including the regulatory acceptance of data derived from automated scoring, especially with regard to the use of flow cytometry, the suitability of rat peripheral blood reticulocytes to serve as the principal cell population for analysis, the establishment of in vivo MN assays in tissues other than bone marrow and blood (for example liver, skin, colon, germ cells), and the biological relevance of the single-dose-level test.
Our group members agreed that flow cytometric systems to detect induction of micronucleated immature erythrocytes have advantages based on the presented data, e.g., they give good reproducibility compared to manual scoring, are rapid, and require only small quantities of peripheral blood. Flow cytometric analysis of peripheral blood reticulocytes has the potential to allow monitoring of chromosome damage in rodents and also other species as part of routine toxicology studies. It appears that it will be applicable to humans as well, although in this case the possible confounding effects of splenic activity will need to be considered closely. Also, the consensus of the group was that any system that meets the validation criteria recommended by the IWGT (2000) should be acceptable. A number of different flow cytometric-based micronucleus assays have been developed, but at the present time the validation data are most extensive for the flow cytometric method using anti-CD71 fluorescent staining especially in terms of inter-laboratory collaborative data. Whichever method is chosen, it is desirable that each laboratory should determine the minimum sample size required to ensure that scoring error is maintained below the level of animal-to-animal variation.
In the second IWGT, the potential to use rat peripheral blood reticulocytes as target cells for the micronucleus assay was discussed, but a consensus regarding acceptability for regulatory purposes could not be reached at that time. Subsequent validation efforts, combined with accumulated published data, demonstrate that blood-derived reticulocytes from rats as well as mice are acceptable when young reticulocytes are analyzed under proper assay protocol and sample size.
The working group reviewed the results of micronucleus assays using target cells/tissues other than hematopoietic cells. We also discussed the relevance of the liver micronucleus assay using young rats, and the importance of understanding the maturation of enzyme systems involved in the processes of metabolic activation in the liver of young rats. Although the consensus of the group was that the more information with regard to the metabolic capabilities of young rats would be useful, the published literature shows that young rats have sufficient metabolic capacity for the purposes of this assay. The use of young rats as a model for detecting MN induction in the liver offers a good alternative methodology to the use of partial hepatectomy or mitogenic stimulation. Additional data obtained from colon and skin MN models have been integrated into the data bases, enhancing confidence in the utility of these models.
A fourth topic discussed by the working group was the regulatory acceptance of the single-dose-level assay. There was no consensus regarding the acceptability of a single dose level protocol when dose-limiting toxicity occurs. The use of a single dose level can lead to problems in data interpretation or to the loss of animals due to unexpected toxicity, making it necessary to repeat the study with additional doses. A limit test at a single dose level is currently accepted when toxicity is not dose-limiting.
In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals
2007, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
The use of flow cytometry with rat peripheral blood erythrocytes is expected to increase the sensitivity of the in vivo micronucleus test and allows assessment of the genotoxic effects at doses that may be equal or close to those relevant to human exposure. However, there was a limitation to the use of rat peripheral blood erythrocytes since the spleen selectively removes micronucleated erythrocytes from circulation. In the present study, the selective analysis by flow cytometry of young MN-PCEs (micronucleated polychromatic erythrocytes or reticulocytes) by use of anti-CD71 antibodies was intended to compensate for the splenic clearance of micronucleated erythrocytes. The young polychromatic erythrocytes have on their surface a specific marker (CD71 antigen) that decreases in density during the maturation process. To investigate the usefulness of the flow cytometric micronucleus analysis combined with anti-CD71 staining of reticulocytes several compounds were tested in acute or sub-chronic treatment regimens. Furthermore, an evaluation was conducted in comparison with the standard rat bone-marrow micronucleus test with additional compounds. The results of acute studies with intraperitoneal application of ethyl methanesulfonate (EMS) (50, 100 and 200 mg/kg) and mitomycin C (MMC) (0.5, 1 and 2 mg/kg), were comparable to data published in the literature. Sub-chronic experiments were performed with cyclophosphamide (CP) (1, 2, 4 and 8 mg/(kg day)), colchicine (6, 8 mg/(kg day)) and mitomycin C (0.1 mg/(kg day)) and showed dose- and time-dependent accumulation of MN-PCEs. Parallel analysis of micronucleus induction in peripheral blood and bone marrow performed with Novartis compounds up to the highest tested dose (5 mg/kg of compound A, 200 mg/kg of compound B and 1250 mg/kg of compound C) showed concordant results. Furthermore, we performed kinetic studies of micronucleus induction in peripheral blood samples obtained at various times after a single treatment with 10 mg/kg CP and with 6 or 8 mg/kg of colchicine. Such experiments gave important supplementary information about the time course of micronucleus induction. Our data suggest that the peripheral blood flow-cytometry micronucleus test can be used for the assessment of micronucleus induction after acute and chronic exposures of rats to chemicals.
Induction of micronuclei in mouse polychromatic erythrocytes by the administration of non-radioactive CsCl by the oral and intraperitoneal route
2001, Mutation Research - Genetic Toxicology and Environmental Mutagenesis
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 500 mg/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 1000 mg/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.
Achievements by CSGMT/JEMS · MMS: The Collaborative Study Group for the Micronucleus Test in the Mammalian Mutagenesis Study Group of the Environmental Mutagen Society of Japan
1996, Mutation Research - Reviews in Genetic Toxicology
The Collaborative Study Group for the Micronucleus Test (CSGMT) is one of the task groups in the Mammalian Mutagenesis Study Group (MMS) of the Environmental Mutagen Society of Japan (JEMS). It was established in 1982 and has made efforts to understand what the micronucleus test is, what are the advantages and disadvantages of the test as an in vivo detection system for mutagens/carcinogens, and to establish a standard protocol applicable to numerous chemicals. Members of the CSGMT have published more than 75 papers as part of collaborative studies and have contributed to the understanding of the nature of the micronucleus test and to setting guidelines for testing of medicinal and other chemicals. The CSGMT held some workshops to share up-to-date knowledge and techniques on the micronucleus test. Through workshops and collaborative studies, the CSGMT contributed to the maintaining of a high standard of knowledge and techniques among Japanese researchers of the micronucleus test. This paper reviews achievements made by the CSGMT until now.
Micronucleus test with mouse peripheral blood erythrocytes by acridine orange supravital staining: The summary report of the 5th collaborative study by CSGMT/JEMS · MMS
1992, Mutation Research/Genetic Toxicology
The main goal of the Collaborative Study Group for the Micronucleus Test (CSGMT) was to validate a new method for the micronucleus test, recently introduced by Hayashi et al. (1990), using mouse peripheral blood cells stained supravitally with acridine orange (AO). The micronucleus tests were performed on CD-1 mice using 23 chemicals with various modes of action. As a rule, one chemical was studied by two participants. Peripheral blood sampled from the same animal was examined 0, 24, 48, and 72 h (or longer) after treatment. The frequencies of micronucleated peripheral reticulocytes (MNRETs) were recorded based on observation of 1000 reticulocytes per mouse.
All chemicals induced MNRETs dose-dependently. Interlaboratory differences in the induction of MNRETs were in an acceptable range for most chemicals tested. Although differences were observed with some chemicals, there were no discrepancies in qualitative judgment. Most chemicals gave the greatest response 48 h after treatment, which was less variable than in the bone marrow assay (greatest response, 24–48 h). These results suggest that the peripheral blood assay using the AO supravital staining technique generates reproducible and reliable data to evaluate the clastogenicity of chemicals. This makes the peripheral blood micronucleus assay an attractive alternative to the conventional bone marrow assay.
Micronucleus test with vincristine sulfate and colchicine in peripheral blood reticulocytes of mice using acridine orange supravital staining
1992, Mutation Research/Genetic Toxicology
The induction of micronuclei in mouse peripheral blood reticulocytes (RETs) was studied with the spindle poisons vincristine sulfate (VINO and colchicine (COL) using acridine orange (AO) supravital staining. Each chemical was studied independently in two laboratories using the same protocol. Blood samples were prepared at 0, 24, 48, and 72 h after a single intraperitoneal treatment with VINC (0.0625, 0.125, and 0.25 mg/kg) or COL (0.25, 0.5, 1.0, and 2.0 mg/kg). Both VINC and COL induced micronucleated RETs (MNRETs) significantly and dose-dependently with a peak at 48 h after treatment. Maximum frequencies of micronucleated polychromatic erythrocytes (MNPCEs) were observed 24 h after treatment with VINC; thus, the transition time from MNPCEs to MNRETs was about 24 h. Both spindle poisons gave comparable results in the paired laboratories, indicating that the present AO supravital staining method is highly reproducible.

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Research paperMicronucleus test with colchicine given by intraperitoneal injection and oral gavage

Abstract

Mutation Res.

Mutation Res.

Mutation Res.

Mutation Res.

Research paper
Micronucleus test with colchicine given by intraperitoneal injection and oral gavage