Effects of multiple dosing of phenacetin in the micronucleus test

As a part of the international cooperative study to identify the most sensitive regimen in the micronucleus test, phenacetin was given i.p. to male CD-1 mice at doses of 37.5, 75, 150, 300, 400, and 600 mg/kg once, twice, thrice or four times and the bone marrow cells were harvested 24 h after the final dosing. Positive responses were seen at 600 mg/kg after single and triple dosing and at 400 and 600 mg/kg after double dosing. No dose level gave a positive response after quadruple dosing. A repeated-dosing effect was detected at double and triple dosing. Although triple dosing gave the highest magnitude of micronuclei at 600 mg/kg, double dosing showed a sufficient sensitivity and was more convenient from the viewpoint of selecting a suitable test dose and carrying out the micronucleus test.     

Cytogenetic effects of ribavirin on mouse bone marrow

The micronucleus test and mitotic chromosome analysis were used to study the in vivo mutagenic activity of ribavirin on bone marrow cells of Swiss albino mice. To determine the incidence of micronuclei, mice were injected i.p. twice, at an interval of 24 h. with the drug at doses of 20, 100 and 200 mg/kg. Animals were killed 6 h after the second dose and bone marrow was examined for the presence of micronuclei in developing erythrocytes. Ribavirin significantly (P less than 0.05) induced micronuclei in polychromatic erythrocytes at all doses. A study was conducted to investigate the cytogenetic effect of the drug on mitotic chromosomes. Ribavirin at 200 mg/kg/day was administered to mice for 3 and 5 days. Repeated treatment with the high dose of ribavirin produced a highly significant (P less than 0.02) increase in abnormal metaphase spreads. The results indicate that ribavirin is mutagenic to bone marrow cells of mice as evaluated by the micronucleus test and by chromosome analysis.     

Caffeine concentrations in mice plasma and testicular tissue and the effect of caffeine on the dominant lethal test.

Large groups of male Swiss mice received per os on average 100 mg caffeine per kg body weight per day for 1 or 8 weeks. The dominant lethal test was designed to achieve maximum sensitivity in order to detect any possible mutagenic effect. No mutagenic induction of dominant lethals, pre-implantation egg loss or depression of the fertility of females, caused by caffeine at the dose levels administered were observed. The half life of caffeine, which was between 2.5 and 3 h, was similar in plasma and testicular tissue. It was concluded that caffeine did not accumulate in the testicular tissue of mice. The maximum concentration of caffeine found was below 10 microgram/g testicular tissue, which is about a 100 times lower than concentrations that cause chromosome aberrations in cultured mammalian cells.     

Multiple-dosing effects of benzo[a]pyrene in the mouse bone marrow micronucleus test

Multiple-dosing effects of benzo[a]pyrene (B[a]P) in the micronucleus test were studied using CD-1 male mice. Mice were treated orally once, twice or 3 times with 250, 500, 1000 or 2000 mg/kg, at 24-h intervals. Bone marrow cells were sampled 24 h after the last administration. The present study indicated that the incidence of polychromatic erythrocytes with micronuclei significantly increased more in the group of animals that received B[a]P twice than in those receiving it one or 3 times. The dose of 500 mg/kg B[a]P yielded the greatest response of any dose regimen.     

Sex differences in micronucleus induction with hycanthone methanesulfonate in bone marrow cells of mice.

The clastogenic effect of the antischistosomal drug hycanthone methanesulfonate was studied with the micronucleus test in mouse bone marrow cells. Male and female (102/El x C3H/El)F1 mice were treated with single i.p. injections. Bone marrow was sampled 18, 24 and 30 h after treatment with 100 mg/kg. The highest micronucleus yield occurred at 24 h. The dose response for micronucleus induction at 24 h after treatment was non-linear for doses between 5 and 300 mg/kg. The lowest effective dose was 5 mg/kg for females and 10 mg/kg for males. The experiments revealed a significantly higher sensitivity of female mice for the induction of micronuclei in polychromatic erythrocytes by hycanthone methanesulfonate. This result supports the recommendation to use both sexes for quantitative assessment of genotoxicity in the micronucleus test.     

Experiences with the dominant lethal test in female mice: effects of alkylating agents and artificial sweeteners on pre-ovulatory oocyte stages.

Pre-ovulatory oocytes are especially sensitive to mutagenic influences. Since post-dictyotene oocytes are not subject to selection and elimination before fertilization, they may reveal mutagenic effects directly and unrestrictedly. Presuming that a chemical is administered at pro-estrus to female mice one can conclude that the substance or its active metabolite has the chance to reach the gamete during the sensitive pre-fertilization stages. We proved the usefulness of the test system by investigating the effects of alkylating agents. A second step was to investigate other substances. The following treatments induced dominant lethal effects: methyl methanesulfonate 100 mg/kg i.m., cyclophosphamide 200 mg/kg per os, triaziquone 0.25 mg/kg i.p. In contrast, the following agents were ineffective and can be classified as not mutagenic in this method: sodium cyclamate 10 000 mg/kg per os, saccharine sodium, 10 000 mg/kg per os, cyclohexamine sulfate 150 mg/kg per os, ethanol 5 ml/kg per os.     

Genotoxic effects of deltamethrin in the mouse bone marrow micronucleus assay

The genotoxicity of deltamethrin was studied in Swiss albino male mice (five animals/group) using the bone marrow micronucleus assay. Deltamethrin (two i.p. injections, 30 h and 6 h before sample collection) was found to induce micronuclei at 162.5 and 300.0 mg/kg body weight. A lower dose (32.5 mg/kg body weight) failed to induce a significant increase in micronuclei over the control level.     

Relationship between experimental results in mammals and man. I. Cytogenetic analysis of bone marrow injury induced by a single dose of cyclophosphamide.

The extrapolation of experimental results to man was studied by cytogenetic bone marrow analysis and micronucleus test in mice, rats and Chinese hamsters. Furthermore, the frequency of chromosomal aberrations was compared with the frequencies of polychromatic erythrocytes containing micronuclei. Cyclophosphamide (CY) was given intraperitoneally at the doses of 5, 10, 20, 40 and 80 mg/kg b.w. to ICR mice and Wistar rats and at the doses of 10, 20, 40, 80, 120 and 160 mg/kg b.w. to Chinese hamsters. Five patients with various types of malignancies until then medically untreated, were i.v. administered 40 mg CY/kg b.w. Bone marrow cells were examined 24 h after the administration. CY induced in all rodents a clear-cut dose-effect relationship in the frequency of breaks, abnormal metaphases as well as in the frequency of micronuclei in polychromatic erythrocytes. When comparing the results in rodents and man at the dose of 40 mg CY/kg b.w., the sensitivity pattern of species was mice greater than rats greater than Chinese hamsters greater than man. From this aspect the possible differences in the metabolism of CY in analysed species are discussed. The presented results tend to a conclusion that micronucleus testing may be a very suitable method used for screening purpose, however, the method of classical cytogenetic analysis, especially the evaluation of breaks, still remains the most exact and reliable technique.     

Micronucleus test and bone-marrow chromosome analysis: A comparison of 2 methods in vivo for evaluating chemically induced chromosomal alterations

The sensitivities of 2 cytogenetic tests, chromosome analysis and the micronucleus test, were compared by using mice exposed to the substances methyl methanesulfonate (MMS), mitomycin C (MC) and procarbazine (Natulan®). The lowest dose at which a significant effect could be observed in bone-marrow cells of mice was determined. Both test systems proved equally sensitive for MC and procarbazine. Doses as low as 0.16 mg of MC per kg and 3.12 mg of Natulan® per kg significantly increased both the aberration rates and the micronucleus rates above those of the controls. In contrast, after exposure to MMS, chromosomal aberrations were elevated above control levels at 5 mg/kg, and the micronucleus rate differed significantly from that of the controls after a dose of 10 mg/kg. With the present protocol and sample size one can conclude that the micronucleus test is generally comparable in sensitivity to the chromosome analysis. However, the MMS data indicate that there might be chemicals for which the resolution of the chromosome analysis is higher.

When the mutagens were given in 2 single i.p. injections separated by 24 h, the polychromatic erythrocytes were analyzed for the presence of micronuclei 6 or 24 h after the second injection. The double treatment did not increase the micronucleus rates above the single-treatment results at either sampling interval.     

Investigation of soy sauce treated with nitrite in the chromosomal aberration test in vitro and the micronucleus test in vivo

Soy sauce pretreated with 2300 ppm nitrite caused no more aberrations than did untreated soy sauce in the chromosomal aberration test in vitro using a Chinese hamster fibroblast cell line with or without S9 mixture. The aberration induction by soy sauce is likely to be caused by the 17% sodium chloride it contains. Soy sauce with or without pretreatment with 2300 ppm nitrite was orally given to ICR mice at a dose of 14 ml/kg body weight once or 6 ml/kg body weight/day for 5 consecutive days. This oral administration did not induce any significant increase in micronuclei in the micronucleus test in vivo.     

Mutagenic effect of pesticides fastac 10 EK and durs ban 4E studied in a micronucleus test iin mouse bone marrow cells

The mutagenic activity of vastak and durs ban pesticides was studied by the micronucleus test in mouse bone marrow. The frequency of micronuclei in polychromatic erythrocytes was tested at 24, 36 and 42 h after oral administration of 50% LD50 dose of vastak (14 mg/kg) and durs ban (30.5 mg/kg). Significantly different increase in micronucleated polychromatic erythrocytes was established at 24, 36 and 48 h after vastak administration, and at 24 and 36 h after durs ban treatment. Doses of 25% LD50 for both pesticides showed no mutagenic activity, as judged by the induction of micronuclei in polychromatic erythrocytes.     

2-Hydroxy-1,4-naphthoquinone, the natural dye of Henna, is non-genotoxic in the mouse bone marrow micronucleus test and does not produce oxidative DNA damage in Chinese hamster ovary cells

2-Hydroxy-1,4-naphthoquinone (HNQ) has been found positive in a previous chromosome aberration test in Chinese hamster ovary (CHO) cells and in a mouse bone marrow micronucleus test at 72h after oral administration (vehicle: DMSO). However it was negative at 24 and 48h sampling times, and in subsequent micronucleus tests that used 0.5% aqueous methyl cellulose (MC) as vehicle. We performed a bone marrow micronucleus test in male and female NMRI BRL/BR mice at oral doses of 75, 150 and 300mg/kg in two vehicles (DMSO and 0.5% aqueous MC), evaluated micronuclei at 24, 48 and 72h, plasma levels of HNQ at 0.5, 1 and 4h, and haematology parameters at 72h after administration. The mechanism of in vitro clastogenic activity of HNQ was investigated by evaluation of the potential of HNQ to produce oxidative DNA damage after treatment of CHO with 10mM HNQ, followed by quantification of DNA fragments using the comet assay. In the micronucleus test, HNQ at 300mg/kg produced mortality and clinical signs at similar incidence and severity for both vehicles. Levels of HNQ in the plasma of treated mice were dose-related, of similar magnitude for both vehicles, but higher in females than in males. Maximum concentrations were found at 0.5 or 1h. At 300mg/kg, HNQ slightly affected RBC parameters suggesting haematotoxicity. No increase in the frequency of micronuclei was observed for any dose, vehicle or time point, whereas the positive control substance (CPA) produced a clear positive response. No evidence of HNQ-induced oxidative DNA damage was found at clastogenic concentrations in vitro, whereas the positive control substance (H(2)O(2)) produced a clear increase. In conclusion, HNQ was negative for induction of bone marrow micronuclei in mice up to 72h after administration in two different vehicles, and its in vitro clastogenicity was not due to oxidative damage. These results confirm that HNQ poses no or negligible genotoxic risk.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

In vitro and in vivo mutagenicity studies with airborne particulate extracts

The contribution of nitro compounds to airborne particulate mutagenicity was studied with Salmonella typhimurium strains TA98, TA98NR, TA98/1,8DNP6. The results obtained indicate that nitropyrenes play a minor role in air particulate mutagenicity. Seasonal variations indicate a relatively greater contribution of nitro compounds to the mutagenicity of spring and summer samples. Fractionation of extracts into acidic, neutral and basic components shows that neutral compounds account for about two-thirds of the total mutagenic activity. Attempts to extract mutagens adsorbed onto particulate matter with aqueous media were almost completely negative. No significant mutagenicity was detected in urine and faecal extracts and in plasma samples of Sprague-Dawley rats treated with air particulate extracts at 80 mg/kg either per os or by i.p. injection. Negative results were obtained in the micronucleus test with Swiss mice treated at 200 and 400 mg/kg (twice by i.p. injection). A significant decrease in liver aminopyrine-N-demethylase was observed in Swiss mice injected with air particulate extracts or its basic and neutral fractions. In vitro experiments suggest a direct interaction of test materials with microsomal cytochrome P-450.     

Mutagenic evaluation of tromaril (N-beta-phenylethylanthranilic acid), a novel anti-inflammatory drug, using mammalian test systems

The mutagenic effect of tromaril, a new anti-inflammatory drug, was assessed in Swiss male mice employing bone-marrow micronucleus induction, abnormal sperm formation, and the induction of meiotic chromosome anomalies in spermatocytes as the test parameters. Administration of tromaril induced significantly higher percentages of MN and abnormal sperm at 600 and 900 mg/kg body wt., whereas chromosomal anomalies in spermatocytes were significantly higher at all the 3 doses of 300, 600 and 900 mg/kg body wt. at time intervals of 7, 15 and 30 days as compared to parallel controls.     

Mutagenic effects of dimethyl terephthalate on mouse somatic cells in vivo

The mutagenic activity of dimethyl terephthalate (DMtP) was evaluated in the micronucleus test in mice. A clear clastogenic effect was obtained at all concentrations studied (0.2-1.0 mmole/kg body weight). The maximum number of micronuclei occurred 24 h after a single intraperitoneal (i.p.) injection. The time-course for the DMtP-induced micronuclei was in agreement with the available data on the rapid excretion of phthalates from the mammalian body. The dose-effect response was best described by a linear equation with a logarithmic component. The emergence of the latter term was related to the toxic effects of DMtP at higher concentrations on bone marrow erythropoietic function. A comparison of the effects induced by DMtP and by methyl nitrosourea indicated that DMtP cannot be considered a strong mutagenic compound. We have compared the sensitivity of the mouse micronucleus test and that of Drosophila dominant-lethal test by contrasting the effects obtained at similar exposure doses. This comparison leads to the conclusion that the micronucleus test is capable of responding to far lower phthalate concentrations than the Drosophila dominant-lethal mutation test. Our results testify to the ability of dimethyl terephthalate to cause genotoxic damages in vivo in both somatic and germinal cells of higher organisms. Thus, the chemical in question may be of potential genetic hazard to man.     

Micronuclei in mouse bone-marrow cells. A simple in vivo model for the evaluation of drug-induced chromosomal aberrations

For studying, in vivo, chromosomal damage in bone-marrow cells of CD mice the following compounds were used: Trenimon®; Endoxanm® (cyclophosphamide); triethylenemelamine (TEM); methyl methanesulfonate (MMS); ethyl methanesulfonate (EMS); mitomycin C; colchicine; N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and caffeine. In a first set of experiments the compounds were given twice intraperitoneally with an interval of 24 h. In a second set, effects on bone marrow were studied after 2 i.v. or p.o. administrations of TEM or EMS. All compounds except MNNG and caffeine produced bone-marrow depression and micronuclei, depending on the dose. For the active compounds an interesting difference was revealed by a comparison of the lowest effective dose (as measured by micronuclei formation) with the lethal dose. Trenimon, TEM, cyclophosphamide and MMS (some of which are used in human chemotherapy in similar mg/kg doses) were active on mouse bone-marrow at very low doses compared with their lethal doses. On the other hand, colchicine, mitomycin C and EMS exhibited an effect only at doses very close to, or within, the toxic range. Different routes of administration of either TEM or EMS produced similar effects.The results indicate that the test is especially suitable for initial large-scale screening of suspected chromosomal mutagens and spindle poisons. In addition, the use of the relationship between doses required to induce micronuclei and lethal doses in mice provides a practical measure of the relative potencies of such compounds.     

Mutagenicity evaluation of HC Blue No. 1 and HC Blue No. 2, I. Effect on the induction of micronuclei in mouse bone marrow cells

Two hair-dye chemicals, HC Blue No. 1 and HC Blue No. 2, were assessed for the ability to produce chromosome breakage and/or spindle malformation in vivo by evaluating the capacity of these compounds to induce micronuclei in polychromatic erythrocytes of mouse bone marrow. Initial studies were conducted in ICR male and female mice given a single intraperitoneal dose of 1000, 500 or 250 mg/kg body weight and examined for micronucleus induction 24 or 48 h later. Activity was observed in female mice given 1000 mg/kg of HC Blue No. 1 at the 24-h harvest time. A questionable response was noted with HC Blue No. 2 in males at the 1000 mg/kg, 24-h time point. No activity was observed in either sex at the 48-h harvest time. In a second set of studies, mice from two strains, ICR and CD-1, were administered a single intraperitoneal dose of 1000 mg/kg of each chemical and the bone marrow was extracted 24 h later. In these experiments, HC Blue No. 1 again produced a statistically significant elevation of micronuclei in female ICR mice. No significant effect was observed in CD-1 mice of either sex. HC Blue No. 2 did not produce any significant elevation of micronuclei in either sex of ICR or CD-1 mice.     

Genotoxicity studies with pure trans-capsaicin

Both positive and negative effects have been found in classical genetic toxicology assays with capsaicin. However, the capsaicin tested in most studies has been derived from pepper plant extracts, which is likely to display varying degrees of purity and possibly diverse impurity profiles. Therefore, the objective of the series of studies reported here was to test the genotoxic potential of pure, synthetic trans-capsaicin (the only naturally occurring geometric isomer of capsaicin), using four genotoxicity assays widely used to evaluate drug substances. These included the Ames, mouse lymphoma cell mutation, mouse in vivo bone marrow micronucleus and chromosomal aberration in human peripheral blood lymphocytes (HPBL) assays. In the Ames assay, pure trans-capsaicin was not mutagenic to Salmonella typhimurium or Escherichia coli when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. trans-Capsaicin was weakly mutagenic in mouse lymphoma L5178Y cells, in the presence of S9 mix, when dissolved in dimethylsulfoxide and tested at concentrations extending into the toxic range. Limited evidence for very weak activity was also obtained in the absence of S9 mix. trans-Capsaicin did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 800 mg/kg per day in male and 200 mg/kg per day in female CD-1 mice using a 0 h plus 24 h oral dosing and 48 h sampling regimen. Finally, trans-capsaicin did not induce structural or numerical chromosomal aberrations when evaluated for its ability to induce clastogenicity in blood lymphocytes. Taken together, these data suggest that the genotoxic potential of pure trans-capsaicin is very low, especially as the clinical significance of weak mutagenicity in the mouse lymphoma assay for catechol-moiety containing compounds is unclear. Moreover, the different genotoxicity profiles of pure trans-capsaicin and purified chili pepper extracts suggest that the purity and source of capsaicin should always be an important consideration for toxicological evaluations.     

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.