Mouse Micronucleus Test Research Articles

Mouse micronucleus tests with known and suspect spindle poisons: results from two laboratories

Micronucleus assays were carried out independently in two laboratories with 10 chemicals selected for aneuploidy studies in a coordinated project supported by the CEC. All chemicals were distributed from a single batch by the project coordinator. Male and female mice were treated by single i.p. injections, bone marrow was sampled 6, 24 and 48 h after treatment. Polychromatic and normochromatic erythrocytes were evaluated for the presence of micronuclei after May--Grünwald/Giemsa or Wright staining. The results from both laboratories are in close agreement even though different stocks of mice and different sample sizes were employed in the two laboratories. Only three of the 10 compounds induced micronuclei in mouse erythrocytes: colchicine, hydroquinone and vinblastine. The other seven chemicals gave negative results: econazole, chloralhydrate, diazepam, thiabendazole, cadmium chloride, thimerosal and pyrimethamine. The negative results for econazole and chloralhydrate were unexpected in light of the fact that these two chemicals were previously shown to increase the mitotic index and to induce c-mitotic effects in mouse bone marrow cells. The need for further investigations of these compounds by chromosome counting in second post-treatment metaphases is emphasized to determine their ability to induce aneuploidy.

Final Report on the Safety Assessment of Hydroxybenzomorpholine

Hydroxybenzomorpholine (HBM) is a heterocyclic compound that is used in cosmetics as a coupler in coal tar hair dyes. No deaths were reported in a subchronic oral toxicity study in rats. Some degenerative changes in the cortical tubules of the kidneys were observed in the mid and high-dose groups. HBM was considered to be practically nonirritating to the rabbit eye and produced only slight skin irritation. HBM was neither a sensitizer nor a photoallergen. HBM was not mutagenic in either the Ames assay or in the mouse micronucleus test. On the basis of the data included in the report, Hydroxybenzomorpholine is considered to be safe as a hair dye ingredient at the current concentrations of use.

Selection and Evaluation of an Appropriate Model for Screening Adenosine Analogs for Chromosome Aberrations

As part of a preclinical safety assessment profile, the adenosine analog SC-46267, a candidate antihypertensive drug, was tested for potential genotoxicity in a series of in vitro and in vivo assays. Negative results were obtained in the Salmonella/microsome and CHO/HGPRT point mutation assays and in an acute mouse micronucleus test. However, in the presence of exogenous metabolism, positive results in the mouse lymphoma L5178Y assay and an in vitro chromosome aberration assay with purified rat lymphocytes indicated that the analog was a potential clastogen. Both adenosine and the analog were subsequently tested in rat lymphocytes using whole-blood versus purified lymphocyte cultures. High concentrations of adenosine and the adenosine analog induced chromosome aberrations in the purified lymphocytes. However, neither compound induced chromosome damage when cultured in the presence of whole blood, regardless of the presence or absence of the metabolic activation system. These results were similar to those reported in the literature that suggested that high concentrations of adenosine and other naturally occurring nucleotides can produce nonphysiological perturbations of nucleotide pools that are clastogenic in purified cultures of lymphocytes but not in whole-blood cultures. Our studies appear to support the hypothesis that adenosine deaminase, present at normal physiological levels in whole blood, is capable of detoxifying excessive levels of adenosine. Both compounds were also negative in a 10-day repeated dose mouse micronucleus test, which further ensures that the positive in vitro findings are not biologically significant. An in vitro clastogenicity assay using whole blood appears to be more appropriate than use of purified lymphocytes for screening adenosine analogs. The results from this series of experiments demonstrate the importance of considering culture conditions when designing in vitro test protocols. Furthermore, this study exemplifies the need to explore the mechanisms of activity as well as in vivo relevance of in vitro results.

Expulsion of demecolcine‐induced micronuclei from mouse bone marrow polychromatic erythrocytes

The mouse micronucleus test is a valuable tool for evaluating in vivo chromosome damage produced by test articles in polychromatic erythrocytes of bone marrow. Compounds that are clastogens, such as cyclophosphamide, induce micronuclei that are smaller than those induced by compounds that are spindle poisons, such as demecolcine. In vitro studies have previously shown that the frequency of mitomycin C- and vincristine-induced micronuclei in mouse L-929 cells was reduced due to micronuclear extrusion following treatment with cytochalasin B. The current study shows that micronuclei are also expelled in vivo, that expulsion is dependent upon micronuclear size, and that observation of these extruded micronuclei is dependent upon the method of sample preparation.

Comparison of the activity of topically applied pesticides and the herbicide 2,4-D in two short-term in vivo assays of genotoxicity in the mouse

Genotoxicity of eight topically applied compounds was determined using the bone marrow micronucleus (MN) test and hair follicle nuclear aberration (NA) assay in CD1 mice. Twenty-four hours after a single treatment, cyclophosphamide (CY), applied at doses corresponding to 1 4 , 1 8 , 1 16 , and 1 32 of the published dermal LD50, and N-methyl- N-nitrosourea (MNU), applied at 1 4 , 1 8 , and 1 16 of the published dermal LD50, were found to increase the incidence of NA in a dose-dependent manner. The frequency of MN was significantly increased only at the highest dose of CY. Using the same protocol, six pesticides applied in dimethyl sulfoxide (DMSO) at doses of 1 8 , 1 16 , and 1 32 of the dermal LD50 were investigated. Aminocarb and chlordane induced a dose-dependent increase in the frequency of NA, while there was an observed increase in NA incidence at only the highest doses of dichlorvos (DDVP), 4,4′-DDT (DDT), and 2,4-dichlorophenoxyacetic acid (2,4-D). No effect was observed with fenitrothion on nuclear aberrations in hair follicles. Except for the highest dose of chlordane, none of the pesticides tested positive in the bone marrow micronucleus test. Serum cholinesterase levels were reduced to 70 ± 4.7% of the DMSO control level with DDVP, 57 ± 8.2% with aminocarb, and 60.3 ± 4.8% with fenitrothion, indicating some systemic activity with these topically applied agents. The data suggest that aminocarb, chlordane, DDVP, DDT, and 2,4-D are genotoxic as determined by the NA assay and that this assay may be more useful in detecting topically applied genotoxic agents than the more often used bone marrow micronucleus test.

Evaluation of genotoxicity of clofazimine, an antileprosy drug, in mice in vivo. II. Micronucleus test in bone marrow and hepatocytes

The antileprosy drug, clofazimine, was tested for its possible genotoxicity using micronucleus (MN) tests in mice. A significantly higher incidence of MN in bone marrow erythrocytes, particularly in polychromatic erythrocytes, as well as in regenerated hepatocytes revealed a positive clastogenic effect of the drug. The drug also had a marked antimitotic effect as indicated by a negative correlation with the dose.

In vivo anticlastogenic and antimutagenic effects of tannic acid in mice

The anticlastogenic effect of tannic acid was studied in vivo in the mouse micronucleus test. The frequencies of micronuclei induced by mitomycin C, ethyl nitrosourea (ENU) or 4-nitroquinoline 1-oxide in mouse bone marrow cells were decreased by the oral administration of tannic acid 6 h before the mutagen injection. The observed suppressing effect was not a reflection of a delay in the formation of micronuclei by the cytotoxic effect of tannic acid. The antimutagenic effect of tannic acid was also investigated in vivo in the mouse spot test using male PW and female C57BL/10 mice. Tannic acid was given orally to pregnant females 6 h before the intraperitoneal injection of ENU on the 10th day of pregnancy. The frequency of pups with recessive color spots induced by ENU was decreased by the administration of tannic acid. The observed decrease was not due to toxic effects on the embryo. These results indicate that tannic acid acts as an anticlastogen and antimutagen in vivo.

9 Final Report on the Safety Assessment of Phenoxyethanol

Phenoxyethanol is an aromatic ether which is used in cosmetics as a preservative at concentrations below 1% and as a fixative for perfumes. According to the classification scheme of Hodge and Sterner,(1) Phenoxyethanol is practically nontoxic when administered orally or dermally to rats. In a subchronic oral toxicity study in rats of Phenoxyethanol, signs of toxicity included reduced body weights and an impaired ability to utilize feed. Increased liver, kidney, and thyroid weights were noted at necropsy in surviving rats. Undiluted Phenoxyethanol was a strong eye irritant, but was nonirritating when tested at 2.2%. Phenoxyethanol at 2.0% was a slight irritant to rabbit skin, but was neither an irritant nor sensitizer to guinea pig skin. In dermal treatment studies, Phenoxyethanol was neither teratogenic, embryotoxic, or fetotoxic at doses which were maternally toxic. Phenoxyethanol was nonmutagenic in the Ames test, with and without metabolic activition, and in the mouse micronucleus test. In clinical studies, Phenoxyethanol was neither a primary irritant nor sensitizer. Phenoxyethanol was not phototoxic in clinical studies. It is concluded that Phenoxyethanol is safe as a cosmetic ingredient in the present practices of use and concentration.

Genotoxic activity of nickel subsulphide α-Ni 3S2

Four mutagenicity tests of alpha-Ni3S2 were performed: the Ames test on five Salmonella typhimurium strains, HPRT test on V79 cells, in vitro chromosomal aberrations on human lymphocytes and the in vivo micronucleus test in mice. The in vitro tests were carried out without metabolic activation. (i) The Ames test (5-1500 micrograms/plate) demonstrated no mutagenic activity, thus confirming previous observations by other authors. (ii) The HPRT test was carried out under standard conditions (3 h exposure) with alpha-Ni3S2 concentrations from 30 to 1000 micrograms/ml. No significant difference was observed with the control cells. Ultrastructural examination revealed alpha-Ni3S2 binding to the cell membrane. Very few particles were found in the cytoplasm but not in the nucleus. (iii) In vitro metaphase analysis in human lymphocytes were performed after exposure to total alpha-Ni3S2 suspension and to the soluble fraction in culture medium with or without fetal calf serum (FCS) (3-100 micrograms/ml, 24 h exposure). Nickel concentrations in the soluble fraction were determined by electrothermal atomic absorption spectrometry. A clastogenic effect of alpha-Ni3S2 became evident under all experimental conditions with a significant additional increase of chromosomal aberrations in 20% FCS complemented medium. No difference was observed between total suspension and soluble fraction. (iv) The micronucleus test confirmed the clastogenic effect of alpha-Ni3S2 in vivo after administration of 250 mg/kg (i.p.). This test revealed a clear increase of micronuclei frequency in polychromatic erythrocytes 24, 48 and 72 h after the treatment. In addition, we observed a statistically significant decrease in the number (%) of polychromatic erythrocytes after 24 and 48 h exposure. The soluble, i.e. cell-entering, fraction seems to play a great part in the clastogenic effect, as has also been shown with other test methods by other authors.

Summary of Toxicity Data on Methyl Isothiocyanate (MITC)

In order to investigate the toxicological properties of MITC and its formulations, a number of animal toxicity studies were conducted. In conclusion MITC is a toxic substance and shows severe corrosive effects and inflammation to skin and mucous membranes. In addition, it's vapours irritate mucous membranes and are lachrymatory.In a sensitization test in guinea pig, the test substance showed a mild skin sensitizing potential. In sub-chronic studies administered by oral gavage to rats and mice, MITC gave considerable dose-related toxic effects (e. g. stomach lesions, small round cell infiltration of the liver, slight spermatogenic disorder, decreased body weight gain, decreased ovary weight in mice, etc.) particularly at higher doses. A repeat study on mice to assess the effect on ovaries did not give any treatmentrelated effects in these organs. Long term chronic oral toxicity/oncogenicity studies in which MITC was administered to rats or mice in the drinking water, did not show any of the dose-dependent or substance-related effects that had been found in the sub-chronic studies. The only significant changes in the long term studies were a decreased body weight gain and water consumption in the high dose groups.MITC showed no oncogenic activity and had no adverse effect on reproductive capacity or in teratogenicity studies. In a number of mutagenicity studies, Rec-assay, reverse mutation, mouse micronucleus test, unscheduled DNA synthesis, sister chromatid exchange and forward mutation, MITC was negative although a in vitro chromosome aberration in Chinese hamster's cells was positive. Whilst in human lymphocytes it was negative. Di-Trapex and Trapexide were initially registered as soil fumigants in 1976 and 1982, respectively.Withholding values for registration have been set at 0.05ppm on fruits, 0.2ppm on vegetables and tea, and 0.5ppm on potatoes. MITC is a toxic substance with severe irritant effects on skin and mucous membranes, therefore, proper application in accordance with the recommended handling precautions including the use of full protective clothing must be followed. Then products containing MITC may not only be useful but also safe to users and applicators.

An evaluation of caprolactam and benzoin in the mouse micronucleus test

Caprolactam (CAP) and benzoin (ZOIN) were tested in the mouse micronucleus test at two dose levels, one of which was the maximum tolerated dose. The compounds were administered by the oral route to groups of 5 male and 5 female mice. No statistical significant increase over control values of the frequency of PCE-containing micronuclei was observed at any dose level or sampling time, with the exception of CAP at a dose level of 700 mg/kg at the 24-h sampling time, where a small statistically significant effect was observed both when the sexes were analysed combined and separately. Due to this observation a limited repeat was carried out on CAP at the 700 mg/kg dose level at the 24-h sampling time. In the repeat study similar trends were observed even following the analysis of 5000 cells per animal. However, when these data were compared with historical control data no such effects were observed, the effects were therefore considered to be of questionable validity. Throughout the study the positive control (cyclophosphamide) gave an elevated biologically and statistically significant increase at all sampling times, thus verifying the sensitivity of the test system. It was therefore concluded that caprolactam and benzoin are not clastogenic in the mouse micronucleus test.

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 K 2CrO 4 in 2 mouse strains (MS/Ae and CD-1). A simplified acute toxicity test to estimate the toxic dose levels of K 2CrO 4 showed that the LD 50s 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. K 2CrO 4 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 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 LD 50.

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 LD 50 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 LD 50. Thus both i.p. and p.o. routes are acceptable in the micronucleus test of this chemical.

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 LD 50 values and when the maximum responses were neglected.

Studies on the genotoxicity of asataf (acephate), an organophosphate insecticide, in a mammalian in vivo system

The genotoxic potential of asataf (acephate) was evaluated by a battery of in vivo tests: bone marrow chromosome aberrations, micronucleus, sperm-shape abnormality and dominant lethal tests in mice. A significant enhancement in the percentage of chromosome aberrations was noticed in 3 doses, 3 routes and 3 h after asataf treatment of groups of mice as well as in chronic (sub-acute) treatment. A significant difference in the occurrence of micronuclei was found only at the highest dose whereas all the results of the sperm-shape abnormality test were highly significant. In the dominant lethal mutagenicity assay only the result (dead implants) of a single week (3rd) with the higher dose differed significantly from control. On the basis of the present in vivo results in mouse test systems asataf may be considered to be a potential mutagen.

In vivo antimutagenic effect of ascorbic acid against mutagenicity of the common antiamebic drug diiodohydroxyquinoline

We have previously shown that the common antiamebic drug diiodohydroxyquinoline (DIHQ) exhibits mutagenic activity in the in vivo micronucleus test in Swiss albino mice. Results of experiments undertaken to study the influence of ascorbic acid (vitamin C) on the mutagenicity of DIHQ in this model system showed that ascorbic acid acts as an antimutagen against DIHQ. The effective antimutagenic doses of ascorbic acid themselves do not show any genotoxic effects in this in vivo system. It will be necessary, however, to elucidate the mechanism of action of ascorbic acid as well as its effects on the therapeutic properties of DIHQ before a practical use of ascorbic acid is contemplated for this purpose.

Micronucleus tests in mice on four chrome-containing pigments.

Micronucleus tests in mice on four chrome-containing pigments.

Effects of indomethacin on the micronucleus test in mice

Effects of indomethacin on the micronucleus test in mice

Cytogenetic activity of methyl isocyanate in vivo in the mouse micronucleus test

The ability of methyl isocyanate (MIC) to induce mutagenic and cytotoxic effects in vivo in the mouse micronucleus test was evaluated by assessing the induction of micronuclei and depression of polychromatic erythrocytes in bone marrow and peripheral blood smears. Animals were exposed to MIC through intraperitoneal injection for 2 and 5 days in separate experiments, and bone marrow and peripheral blood were sampled 6 and 48 h after the last injection, respectively. MIC did not significantly increase the frequencies of micronucleated polychromatic erythrocytes (MN-PCE) and micronucleated normochromatic erythrocytes (MN-NCE) in bone marrow and peripheral blood samples respectively in either twice or multiply treated mice. However, a dose-dependent depression in percentage PCE observed was significant. This indicates that MIC exposure led to the cytotoxic effect by inhibition of bone marrow cell proliferation.