UKEMS genotoxicity trial 1981: In vivo assay systems

4CMB was tested in 7 mouse micronucleus assays (3 i.p., 2 oral, 1 s.c., 1 foetal), 2 mouse sperm-head morphology assays (1 i.p., 1 s.c.), 1 dominant lethal test in mouse (oral), 3 SLRL tests in Drosophila (2 feeding, 1 injection) and one somatic cell segregation assay in Drosophila (feeding).4HMB was tested in 4 mouse micronucleus assays (2 i.p., 1 oral, 1 s.c.), 2 mouse sperm morphology assays (1 i.p. and 1 s.c.), 1 SLRL test and 1 somatic cell segregation assay in Drosophila (both feeding).BC was tested in 4 mouse micronucleus assays (2 i.p., 2 oral, 1 s.c.), 2 sperm-morphology assays (1 i.p., 1 s.c.), 2 SLRL tests and 1 somatic cell segregation assay in Drosophila (all feeding).All the mouse assays gave negative results with all 3 compounds. The response of 4CMB and BC was not reproducible when tested under similar conditions in the SLRL test in Drosophila. All 3 compounds gave a positive response in the somatic cell segregation assay in Drosophila.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c, DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

Micronuclei in human bone-marrow cells: Evaluation of the micronucleus test using human leukemia patients treated with antileukemic agents

The relevance of the micronucleus test to human studies was investigated by using bone marrow from leukemic patients treated with antileukemic drugs.The median incidence of micronucleated erythrocytes and erythroblasts, respectively, increased from control values of 0.04 and 0.72% to 0.29 and 25.3% in leukemic cases; and the frequency of micronucleated erythroblasts was inevitably higher than the control value in cases that showed a higher frequency of micronucleated erythrocytes, but the reverse was not true.These results indicate that almost the same changes of micronucleus formation that are observed in the mouse micronucleus test are produced in human bone marrow by antileukemic drugs — mutagenic compounds — and, if the micronuclei were scored restrictively in erythroblasts, the application of the micronucleus test to human bone marrow would be reasonable.     

Evaluation of nongenotoxic and genotoxic factors modulating the frequency of micronucleated erythrocytes in the peripheral blood of mice

The hematological micronucleus test is regarded as an indicator of the clastogenic effect of chemicals and acute cytogenetic damage. The test can be carried out in red blood cells of the bone marrow and of the spleen, as well as in peripheral erythrocytes. We have determined the precise background values of micronucleated red blood cells for the peripheral blood of BALB/c DBA/2, and NMRI mice. Bleeding, phenylhydrazine-induced hemolysis, and splenectomy generated an increase of micronucleated erythrocytes in the peripheral blood of mice. Our data thus demonstrate that such factors should be taken into consideration when the micronucleus test is used for screening the genotoxic potential of chemicals. Furthermore, the micronucleus-inducing effect of cyclophosphamide was studied in normal and splenectomized mice and, in addition, a comparison of the sensitivity of the micronucleus test was carried out in peripheral blood and bone marrow after cyclophosphamide treatment. Our data demonstrate that the kinetics of micronucleus formation were similar in normal and in splenectomized mice in which the micronucleus levels had returned to normal. The comparison of micronucleus formation in bone marrow and peripheral blood after cyclophosphamide treatment revealed the generation of similar quantities of micronucleated red blood cells in both tissues. The physiological mechanisms of micronucleus formation and removal and the potential role of chemically induced spleen damage during this process are discussed; the usefulness of the peripheral micronucleus test as a simple, rapid, and animal-saving modification of the standard bone marrow test is evaluated.Abbreviations CP cyclophosphamide - MN micronuclei - MNCE micronucleated normochromatic erythrocytes - MNPCE micronucleated polychromatic erythrocytes - MNRBC micronucleated red blood cells - NCE normochromatic erythrocytes - PCE polychromatic erythrocytes     

In vivo micronucleus test with flow cytometry after acute and chronic exposures of rats to chemicals

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.     

An in vivo mouse micronucleus assay on musk ketone

Musk ketone (3,5-dinitro-2,6-dimethyl-4-tert-butyl-acetophenone) was evaluated in an in vivo mouse micronucleus assay. Male and female mice were dosed with 250, 500 or 1000 mg musk ketone/kg body weight by a single intraperitoneal injection in corn oil. Results of the assay showed that under the conditions of this test evaluated at 24, 48 and 72 h after dosing, musk ketone did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice at any dose or any time period. Musk ketone was considered to be negative in the mouse in vivo micronucleus test as well as in a battery of previously published in vitro genotoxicity tests. Based on the total weight of evidence available, it was concluded that musk ketone does not have significant potential to act as a genotoxic carcinogen.     

Cytogenetic effects of diethylstilbestrol-diphosphate (DES-dp) on mouse bone marrow monitored by the micronucleus test.

6 dosages of diethylstilbestrol-diphosphate (DES-dp), ranging from 0.01 to 500 mg per kg of body weight were compared to saline and phosphate buffered saline (negative controls) and two dosages of cyclophosphamide (positive control) in the micronucleus test with 115 ICR mice. DES-dp failed to generate a significant increase in micronucleated polychromatic erythrocytes over negative controls. Cyclophosphamide produced a dose-related increase in micronuclei similar to previously published reports. Iit was therefore determined that the micronucleus test did not detect the types of chromosomal changes known to be generated by DES-dp and DES.     

A new micronucleus test using peripheral blood erythrocytes of the newt Pleurodeles waltl to detect mutagens in fresh-water pollution

A model micronucleus test system using peripheral blood erythrocytes from larvae of Pleurodeles waltl is described. The most suitable larval stage for testing chemical treatments was determined. Larvae were reared in water containing one of the 4 compounds: benzo[a]pyrene (BaP), ethyl methanesulphonate (EMS), diethyl sulphate (DES) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). Response curves as a function of treatment duration over a period of 16 days were plotted for 3 different concentrations of the 4 compounds in order to optimize conditions for a low dose micronucleus test. This model can be used as a monitoring system for the detection of fresh water pollution and can also be employed for clastogen screening of chemical compounds. The test is sensitive, reliable and easy to use.     

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.     

The micronucleus test in pigs: induction of micronuclei in polychromatic erythrocytes by various doses of X-rays

This paper describes the results of a study in which pigs were used in the bone marrow micronucleus assay. In a first experiment the spontaneous frequency of micronucleated polychromatic erythrocytes (MPE) among polychromatic erythrocytes (PE) was investigated in 78 animals. It was found that it is low with individual values of 0-4 MPE/1000 PE and a group average of 1.76 +/- 1.06% (mean +/- SD). In a second set of investigations animals were exposed to 0.25, 0.5, 1.0, 1.5, 2.25 and 2.75 Gy of 9-MeV X-irradiation performed as a single whole-body exposure. Time- and dose-dependent changes in micronucleus incidence were observed. Maximal group averages appeared nearly uniform 36 h post irradiation (p.i.). Considering the 36-h values in the dose range of 0-2.25 Gy there is a marked dose-effect relationship (r = 0.971). The data yield best to a regression curve of a third-grade polynomial indicating a complex interaction between dose and micronucleus formation. In conclusion, the results demonstrate that it appears feasible to use swine as target organisms in the micronucleus test to estimate the cytogenetic damage caused by ionizing radiations or, potentially, chemical compounds.     

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.     

The biological activity of 4-chloromethylbiphenyl, benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity. A report of an individual study in the UKEMS genotoxicity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial 'fluctuation' assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.     

The biological activity of 4-chloromethylbiphenyl,benzyl chloride and 4-hydroxymethylbiphenyl in 4 short-term tests for carcinogenicity: A report of an individual study in the UKEMS genotoxity trial 1981

4-Chloromethylbiphenyl (4CMB), benzyl chloride (BC) and 4-hydroxymethyl-biphenyl (4HMB) were tested for biological activity in the following assays: (i) the Salmonella/microsome assay; (ii) a bacterial ‘fluctuation’ assays; (iii) a DNA repair assay in Hela cells, and (iv) a mouse lymphoma mutation assay. 4CMB was active in assays (i), (ii) and (iii) but not in (iv); BC was active in assays (i), (ii), (iii) but not in (iv) while 4HMB was inactive in all assays. Where biological activity was seen this did not require addition of a liver S9 preparation. 4CMB was more active than BC in all the test systems in which a positive response was obtained. The implication of these results for a test battery approach to in vitro testing is discussed.     

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.     

The effects of 4CMB, 4HMB and BC on SCE,chromosome aberration and point mutation in cultured Chinese hamster ovary cells

Cultured Chinese hamster ovary cells (CHO-KI-BH₄) were treated for 2 h with 4CMB, 4HMB and BC, in the absence of any exogenous metabolic activation system. The cells were subjected to tests for survival, sister-chromatid exchange, chromosome aberration and mutation to thioguanine resistance.4HMB had no effect in any test at concentrations up to 100 μg/ml. 4CMB was slightly more toxic than BC. Both 4CMB and BC induced SCE and chromosome aberrations, but the effects were more marked with BC. With 4CMB, SCE increased with dose only up to about 7 μg/ml and then levelled off. A weak mutagenic effect was observed with both BC and 4CMB, but in each case, the response reached a peak and was not evident at higher doses.     

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.     

Simplified mouse peripheral reticulocyte micronucleus test with dimethylnitrosamine.

The induction of micronuclei by treatment with dimethylnitrosamine was evaluated and compared in peripheral blood and bone marrow cells of male CD-1 mice. Peripheral blood preparations were made on acridine orange (AO)-coated slides and scanned by fluorescence microscopy. A significant increase in micronuclei was observed 24 h after treatment in bone marrow polychromatic erythrocytes, and 24-48 h after treatment in peripheral reticulocytes. The peak frequency of micronuclei in peripheral reticulocytes was delayed by about 24 h relative to bone marrow polychromatic erythrocytes. This micronucleus test using peripheral blood was shown to be easy to do and as sensitive as the test using bone marrow cells. From this result, it is concluded that the method with AO-coated slides and peripheral blood is as suitable as bone marrow cells for the micronucleus assay.     

The micronucleus test. Methodological aspects

Changes in the cellular compositiion of bone marrow were studied in relation to dose of Trenimon® and time after treatment. Strong mutagenic effects caused a partial depletion of the marrow cavities of nucleated blood cell precursors, with subsequent retention of newly formed erythrocytes and inundation with peripheral blood. The influence of these changes on the results of micronucleus scoring was investigated in a time-effect and a dose-effect study using three different methods of evaluation, relating the incidence of micronuclei to (a) nucleated cells, (b) all erythrocytes, and (c) polychromatic erythrocytes. Conclusions are drawn on the practical use of the micronucleus test system. The simplest scoring procedure, namely relating micronucleated polychromatic erythrocytes to a given total number of erythrocytes, was very efficient in the range of low mutagenic effects and therefore well suited for safety screening wherease dose-effect studies comprising very high mutagenic effects required the application of a modified method of scoring.     

1-Methyl-2-pyrrolidinone (NMP) does not induce structural and numerical chromosomal aberrations in vivo

1-Methyl-2-pyrrolidinone induces aneuploidy in yeast, but only under special treatment conditions. Other genotoxic effects have not been found in vitro, and in vivo no data are available in the literature. Therefore, NMP was investigated in the mouse micronucleus test and the Chinese hamster bone marrow test for structural and numerical chromosomal aberrations. These tests can detect both types of alterations as demonstrated by appropriate positive control substances (cyclophosphamide, vincristine sulfate and benomyl). NMP at single oral doses up to 3800 mg/kg body weight (∼ 80% of the LD₅₀) did not lead to an increase either in micronucleated erythrocytes or in structural or numerical chromosomal aberrations when bone marrow was sampled 16, 24 and 48 h after treatment in the micronucleus test or after 24 and 48 h for karyotype analysis.     

The effects of 4CMB, 4HMB and BC in the micronucleus test

Male Tuck To (outbred mice) were exposed to doses of up to 100 mg/kg 4CMB, 400 mg/kg 4HMB and 300 mg/kg BC by i.p. injection with 2 treatment times. No increase in micronuclei in polychromatic erythrocytes was observed at any dose.