An evaluation of the mutagenicity of the cutting oil preservative Grotan BK.

The micronucleus test in rats was used to investigate the mutagenic potential of Grotan BK, a preserving agent used in industrial cutting oils. The test compound was administered either by intragastric intubation, dermal application or subcutaneous injection. CFHB (Wistar) rats were given two equal dosages separated by 24 h to provide total dosages of 15,60,240 or 960 mg/kg. In addition, as a positive control, benzidine at a total dosage of 409.6 mg/kg was administered similarly by the dermal and subcutaneous routes. Bone marrow preparations were screened for the presence of micronucleated cells in 2000 polychromatic erythrocytes. No increase in the incidence of micronucleated erythrocytes was observed for any group given Grotan BK by any of the three administration routes, or at any dose level. Benzidine induced high incidences of microcucleated erythrocytes following both dermal application and subcutaneous injection.     

Micronucleus test with ethyl methanesulfonate administered by intraperitoneal injection and oral gavage

The effect of route of administration on the outcome of the micronucleus test was studied by administering ethyl methanesulfonate (EMS) by oral gavage (p.o.) and intraperitoneal injection (i.p.) to males of 2 mouse strains, MS/Ae and CD-1. Based on preliminary studies, consisting of a small-scale acute toxicity test and a pilot experiment to determine the optimal sampling time and the appropriate dosages, a micronucleus test was conducted with a 24-h sampling time and doses of 50-400 mg/kg i.p. and p.o. EMS significantly induced micronucleated polychromatic erythrocytes (MNPCEs) with a clear positive dose response by both routes in both strains. Moreover, both routes showed almost the same induction rate of MNPCEs at each dose level tested in both strains.     

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.     

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 LD50 values and when the maximum responses were neglected.     

The persistence of micronucleated erythrocytes in the peripheral circulation of normal and splenectomized Fischer 344 rats: implications for cytogenetic screening

Micronucleated erythrocytes are selectively removed from the peripheral circulation of normal rats. Splenectomy prevents this selective removal. In normal rats treated daily for 20 days with 0.2 mg/kg triethylenemelamine (TEM), micronucleated normochromatic (mature) erythrocytes did not accumulate in peripheral blood. In these same animals, the frequencies of micronucleated cells among polychromatic (newly formed) erythrocytes increased from 0.21 to 5.25 per thousand in peripheral blood and from 1.75 to 31.5 per thousand in bone marrow. Since both control and induced frequencies in peripheral blood were approximately 15% of those in bone marrow, the removal appears to be equally efficient for cells containing either spontaneously occurring or clastogen-induced micronuclei. In splenectomized rats treated daily for 11 days with 0.2 mg/kg TEM, the frequency of micronucleated normochromatic erythrocytes (NCEs) in the peripheral blood rose rapidly to 9 times the control value and remained elevated for 50-55 days, indicating a life span approximately equivalent to that of normal erythrocytes. Among splenectomized rats exposed to either 0.15 mg/kg triethylenemelamine, 6.5 mg/kg cyclophosphamide, or 300 mg/kg urethane for periods exceeding the erythrocyte life span, the incidences of micronucleated NCEs in the peripheral blood rose steadily from a control value of 1.0 per thousand to maximum values of 15.0, 12.7 and 8.9 per thousand, respectively. During these extended exposures, the mean frequencies of micronucleated polychromatic erythrocytes (PCEs) in peripheral blood increased from a spontaneous value of 0.9 per thousand to 23.0, 13.0 and 6.6 per thousand, respectively, reflecting the frequencies among PCEs in the bone marrow and approximating the maximum values among NCEs in the peripheral blood. Thus, the frequency of micronucleated erythrocytes in the peripheral blood of splenectomized rats can be used as an index of both acute and cumulative chromosomal damage, while in normal rats the use of peripheral blood for cytogenetic monitoring is restricted by the selective removal of these micronucleated cells.     

Evaluation of dosages and routes of administration of tramadol analgesia in rats using hot-plate and tail-flick tests

Tramadol is an opioid-like analgesic with relatively mild side effects. Because it is inexpensive and is not classified as a controlled substance by the US federal government, the authors wanted to evaluate its applicability as a practical and effective analgesic in male Sprague Dawley rats. They measured the efficacy of four dosages (4, 12.5, 25 or 50 mg tramadol per kg body weight) and three routes of administration (per os (p.o.) in a flavored gelatin cube, subcutaneous (s.c.) or intraperitoneal (i.p.)) using the hot-plate test and the tail-flick test, which were carried out 1 week apart. Rats that were dosed p.o. were given flavored gelatin cubes without tramadol on the 2 d before testing to help them become acclimated to the gelatin, in an effort to increase the likelihood that they would consume the gelatin on the testing day. Results from the hot-plate and tail-flick tests for rats that were given tramadol p.o. were similar before and after administration, regardless of tramadol dosage, suggesting that this route of administration was not effective. The s.c. route of administration was effective at dosages of 25 mg and 50 mg tramadol per kg body weight, although these dosages also resulted in sedation and skin lesions. The i.p. route of administration was also effective at dosages of 12.5 mg, 25 mg and 50 mg tramadol per kg body weight, though sedation was observed at dosages of 25 mg and 50 mg per kg body weight. Intraperitoneal administration of 12.5 mg tramadol per kg body weight had no notable side effects, and the authors plan to further study this dosage and route of administration in a rodent surgical model of pain.     

Determination of micronucleus frequency by acridine orange fluorescent staining in peripheral blood reticulocytes of mice treated topically with different lubricant oils and cyclophosphamide

To ascertain whether used and re-refined lubricant oil absorbed through the skin can produce a genotoxic effect or cytotoxicity in mouse bone marrow cells, we examined the induction of micronucleated erythrocytes of peripheral blood after cutaneous application. Both re-refined and used lubricant oils showed a weak but significant induction of micronucleated polychromatic erythrocytes compared with control, while virgin oil did not show micronucleus induction. Cyclophosphamide (CP) was used not only as positive control but also to compare the sensitivity between intraperitoneal and dermal routes of administration of the test compounds in mice. The efficacy of intraperitoneal injection of CP is well known. On the other hand, dermal exposure is not so common and when CP was diluted in glycerin statistically significant values (P = 0.0036) of micronuclei were also found. Topically applied lubricant oils (virgin, re-refined and used) have the capacity to interfere with mouse bone marrow hematopoiesis evidenced by a statistically significant decrease in the proportion of polychromatic erythrocytes in the peripheral blood. Physical and chemical analysis revealed that used oil is more viscous than other lubricants, suggesting the presence of insoluble compounds, oxidized products and water as well as aromatic hydrocarbons. Used oil differs from other lubricant oils in metal and polyaromatic hydrocarbon content. Re-refined oil revealed a neutral value typical of pure mineral oil. This assay is an important tool to evaluate environmental pollutants that cause genotoxicity and/or cytotoxicity through skin exposure.     

Administration-route-related differences in the micronucleus test with N-ethyl-N-nitrosourea

Administration-route-related differences in the micronucleus test were examined by giving N-ethyl-N-nitrosourea (ENU) to male mice of the MS/Ae and CD-1 strains by 2 different routes, intraperitoneally (i.p.) and orally (p.o.). The experiments consisted of 3 parts: (1) a simplified acute toxicity study, which gave LD50s of 490 (i.p.) and 840 mg/kg (p.o.) in MS/Ae and 640 (i.p.) and 960 mg/kg (p.o.) in CD-1 mice: (2) a pilot experiment for the full-scale micronucleus test to determine appropriate dosages and sampling time: and (3) the micronucleus test at doses of 12.5, 25, 50, and 100 mg/kg with a sampling time of 24 h. The results indicated that no route-related differences existed at the 2 lowest doses. At 50 mg/kg, markedly higher numbers of micronucleated polychromatic erythrocytes (MNPCEs) were induced in both mouse strains by the i.p. route. At 100 mg/kg, the difference between the routes decreased in strain CD-1 and even reversed in MS/Ae. Thus, route-related differences appeared to depend on the dose. Such differences became small, however, in both strains when the comparison was made on the basis of LD50 values.     

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.     

4CMB: Investigation of activity in a mouse micronucleus test

To study possible clastogenic effects of 4-chloromethylbiphenyl (4CMB), a mouse micronucleus test was performed (as part of the UKEMS genetic toxicology trial). Mice were given 4CMB by intraperitoneal injection, once daily for 5 consecutive days. Although the range of dosages employed included highly toxic or lethal doses, the frequency of micronucleated polychromatic erythrocytes in the bone marrow was not significantly greater in 4CMB-treated groups than in control-group animals treated with the Tween 80 vehicle alone. Thus, in this test, 4CMB did not induce chromosomal damage resulting in micronucleus formation, in bone-marrow erythrocytes.     

Genotoxic and cytotoxic effects in the bone marrow of rats exposed to a low dose of paraquat via the dermal route

The genotoxic effect of the herbicide paraquat was studied in rat bone-marrow by means of the micronucleus assay. Paraquat at dose levels of 6, 15 and 30 mg/kg body weight was given to rats in a single application via the dermal route. Marrow was collected at 24, 48 and 72 h after the application. The micronucleus assay was done as recommended by standard procedures. Paraquat gave rise to an increase in the number of micronuclei in a dose-dependent manner. The number of micronucleated polychromatic erythrocytes showed a maximum at 48 h and the toxicity was further prolonged, as there was no complete recovery at 72 h. These findings suggest a genotoxic effect of paraquat even after exposure via dermal application.     

Cytogenetic effects of pesticides. III. Induction of micronuclei in mouse bone marrow by the insecticides cypermethrin and rotenone

The production of micronuclei in mouse bone marrow by the pyrethroid insecticide, cypermethrin and the botanical insecticide, rotenone was examined. Three routes of administration were used for the insecticides: intraperitoneal, oral and dermal. The different routes of treatment with cypermethrin and rotenone caused toxicity of marrow as indicated by a significant increase in the percentage of polychromatic erythrocytes (PEs) over that of the control. Cypermethrin showed mutagenic potential as evidenced by a positive response in the micronucleus assay. Oral administration of the insecticide at a dietary level of 900 ppm for 7 and 14 consecutive days as well as double and multiple (total 4) dermal treatments (360 mg/kg body wt.) induced a statistically significant increase in the frequency of PEs with micronuclei. The conducted intraperitoneal (i.p.) treatments with cypermethrin: single injection at 60 and 180 mg/kg body wt., double and multiple injections (total 3) at 60 mg/kg body wt. did not affect the percentage of PEs with micronuclei. The different treatments with rotenone: single, double and multiple (i.p.) injections (total 3) at 2 and 3 mg/kg body wt., oral administration for 14 consecutive days at dietary level of 225 ppm and multiple dermal treatments (total 4) with 135 mg/kg body wt. showed no effect on the frequency of micronuclei in PEs.     

Effect of route of administration in the micronucleus test with potassium bromate

The effect of intraperitoneal injection (i.p.) versus oral gavage administration (p.o.) of potassium bromate was examined using the micronucleus test in 2 strains of male mice (MS/Ae and CD-1). First, a small acute toxicity test and a pilot micronucleus experiment were performed to determine the appropriate dose range and sampling time for the full-scale micronucleus test. The full-scale test was carried out using doses of 18.8, 37.5, 75, and 150 mg/kg in the i.p. test and of 37.5, 75, 150, and 300 mg/kg in the p.o. test. The sampling time was 24 h for both mouse strains. Potassium bromate induced micronucleated polychromatic erythrocytes (MNPCEs) dose-dependently by both routes of administration in both mouse strains. No distinct difference in route of administration was observed in the test with MS/Ae mice. In CD-1 mice more MNPCEs were induced by the i.p. route than by the p.o. route.     

The evaluation of a multiple dosing protocol for the mouse bone-marrow micronucleus assay using benzidine and 2,6-xylidine

Male ICR mice were treated with 1, 2 or 3 daily doses of either benzidine or 2,6-xylidine. Groups of 5 animals were sacrificed 24 h after the last dose and the bone marrow examined for micronuclei. Benzidine was given at dose levels of 40 and 200 mg/kg and 2,6-xylidine was given at dose levels of 75 and 375 mg/kg. These doses represent 10 and 50% of the respective median lethal doses. Benzidine produced a significant (p less than 0.001) dose related increase in the incidence of micronucleated polychromatic erythrocytes (MPE), while 2,6-xylidine had no effect on the frequency of micronucleated cells. Statistical analyses of the data indicated that the incidence of MPE was independent of the number of doses administered prior to bone marrow harvest.     

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.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxic and cytotoxic effects of the antiviral drug, ribavirin, was studied in rat bone marrow by employing the micronucleus assay. Ribavirin in doses of 10, 15, 20, 30, 50, 75, 100 and 200 mg/kg, and cyclophosphamide (CP) 40 mg/kg (only for sex-difference study) were injected intraperitoneally. Bone marrow was collected at 24 h and 48 h following the injection. To evaluate the recovery, the bone marrow was also sampled at 72 h from 20, 100 and 200 mg/kg treated rats. The micronucleus assay was conducted according to the standard procedure. Ribavirin elevated the incidence of micronuclei (except 10 mg/kg) in erythrocytes (P<0.01). The micronucleated polychromatic erythrocytes showed the initial steep increase at 15 and 20 mg/kg dose level, then with the gradual increase, possibly due to the limited metabolism and action of higher doses. The incidence of micronucleated normochromatic erythrocytes was not dose dependent. The effect was more at 48 h than 24 h due to prolonged toxicity of the drug or its metabolites, and by 72 h, recovery was observed eventhough the genotoxicity was significant. The PCE% decreased as the dose was increased up to 75 mg/kg, then without much difference between two higher doses. Only 100 mg/kg ribavirin and CP showed more toxicity on male rats. Cytotoxicity was seen due to hindered erythropoiesis or cell destruction. Our findings suggest that ribavirin is genotoxic and cytotoxic agent for rat bone marrow.     

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.     

环磷酰胺诱导小鼠血小板减少症模型的建立(英文）

比较由环磷酰胺两种不同给药方式诱导小鼠血小板减少症模型的效果,并对效果较稳定的一种给药方式进行最佳造模剂量摸索,以期确定一个造模效果较好,毒副作用较低,利于观察治疗药物疗效的血小板减少症模型。模型A组,第1天尾静脉注射环磷酰胺200 mg/kg,然后连续6 d,每天1次以维持剂量30 mg/kg腹腔注射环磷酰胺。模型B组,按150 mg/kg皮下注射环磷酰胺,每天1次,连续3 d。结果显示模型B组造模效果较好,故以模型B组给药方法进行剂量摸索实验。由第7天的血小板计数可知环磷酰胺低（100 mg/kg）、中（120 mg/kg）、高（140 mg/kg）剂量均可引起血小板减少症,而低剂量组与其他组比较有高效低毒的特点,更有利于观察治疗药物的作用,可用于具有升血小板作用药物的药效学研究     

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.     

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.