Activity of the human carcinogens benzidine and 2-naphthylamine in triple- and single-dose mouse bone marrow micronucleus assays: results for a combined test protocol

The activities of the human bladder carcinogens benzidine and 2-naphthylamine in the mouse bone marrow micronucleus assays using a limited test protocol (oral dosing to male mice, sampling 24 h later) have recently been established. As a contribution to the International Collaborative Study on the evaluation of the sensitivity of the triple-dose micronucleus test protocol it was decided to re-evaluate benzidine and 2-naphthylamine using a combined triple- and single-dose test protocol. Benzidine gave a clear positive response in male mice 24 h after 3 daily doses of 150 and 300 mg/kg. A single dose of 900 mg/kg of benzidine gave a weaker response 24 h after dosing. In the case of 2-naphthylamine a stronger positive response was observed 24 h after a single dose of 600 mg/kg as compared to 3 daily doses of 200 or 400 mg/kg. There was no significant difference in the increased positive response observed for a single dose of 30 mg/kg of cyclophosphamide compared with 3 successive daily doses of 10 mg/kg. Based on the present data the combined triple/single-dose micronucleus test protocol is strongly supported.     

The micronucleus test with peripheral reticulocytes from phenacetin-treated mice.

The in vivo micronucleus test is conventionally performed using mouse bone marrow cells (BM assay). Using phenacetin as a test chemical, an alternative method using reticulocytes (RET assay) was examined to determine if this could be substituted for the BM assay. Single doses of 400, 600, and 800 mg/kg gave negative results 24 h after i.p. administration, but positive results were obtained with 600 and 800 mg/kg after 48 h. Responses were weak at 72 h. Double treatment enhanced the responses; 400 mg/kg gave a positive result. Maximum responses were generally reached 24 h after the second treatment, 48 h if doses were highly toxic. When the BM and RET assays were compared, the BM assay seemed to be slightly more sensitive than the RET assay; double treatment was superior to a single treatment in both BM and RET assays. Both assays can be used routinely but in the RET assay, sequential samples can be obtained from the same individuals without killing them, providing a firm basis to substitute it for the BM assay. Taking advantage of this characteristic of the RET assay, a regimen of double treatments and double sampling at 24 and 48 h is recommended for a wide range of doses. These data were obtained with CD-1 mice; MS/Ae mice gave a higher incidence of micronuclei than did the CD-1 strain.     

Induction of micronuclei in bone marrow of mice exposed to 1, 2 or 3 daily doses of urethane

Urethane was studied for its potential to induce micronuclei in bone marrow of CD-1 mice following various dosing and sampling schedules. It was found that: (1) mice dosed for 3 days by gavage with urethane at daily doses of 300, 600 and 1200 mg.kg-1 showed no increases in micronuclei compared to normal control values; (2) mice injected with 3 daily intraperitoneal (i.p.) doses of urethane at 400 and 1200 mg.kg-1 demonstrated substantial and dose-related increases in micronuclei; (3) mice given urethane at i.p. doses of 400 mg.kg-1 for 1, 2 and 3 days showed protocol related micronucleus yields with triple greater than single greater than double dosing, while mice given urethane at i.p. doses of 1200 mg.kg-1 showed a different trend with double greater than single dosing, the triple-dosing regimen proving cytotoxic to the bone marrow; (4) mice exposed i.p. to 400 mg.kg-1 of urethane had more micronuclei when sampled at 24 h than at 48 h after 1 or 2 daily treatments, while mice dosed i.p. with 1200 mg.kg-1 of urethane showed more micronuclei at 48 h than at 24 h after a single treatment, the double-dosing protocol being toxic to the marrow; (5) female mice were more susceptible to bone-marrow micronucleus induction than males with both quantitative and qualitative sex differences noted, depending on dose, regimen and sample.     

Comparison of single/multiple-dose protocols using triethylenemelamine and procarbazine hydrochloride for the mouse bone marrow micronucleus test

Treatment of mice with a single dose of either 4.8 mg/kg of triethylenemelamine (TEM) or 348 mg/kg of procarbazine hydrochloride (PC) induced higher frequencies of micronucleated polychromatic erythrocytes (MPE) after 48 h than after 24 h. The same observation was made when animals were treated with 1.6 or 8 mg/kg of TEM or 116 or 580 mg/kg of PC for 2 consecutive days (double-dose protocol). Surprisingly, the third dose of either 1.6 or 8 mg/kg of TEM caused lower MPE frequencies at the 72-h than at the 48-h sampling time. The observation that lower MPE frequencies after 72 h were also accompanied by reduced bone marrow toxicity might have reflected a drug-related adaptive reaction of the animals, for example the induction of detoxifying enzymes. Mean MPE frequencies as well as bone marrow toxicity were also slightly decreased after the third dose of either 116 or 580 mg/kg of PC, but statistical analysis showed no differences between the 48-h and the 72-h sampling times as regards the MPE frequencies and bone marrow toxicity. In addition to the high mean MPE frequency observed after 2 doses of 116 mg/kg of PC at the 48-h sampling time, a late increase in micronucleus induction was also seen after triple dosing at the 96-h sampling time. The present experiments with TEM and PC showed similar sensitivity for the multiple-dose assays when compared with the single-dose micronucleus test. In the case of the triple-dose assay, bone marrow toxicity proved to be a critical factor for appropriate dose selection. The computerized image analysis system was a convenient and time-saving tool for the automatic scoring of large quantities of cells for micronuclei as well as for the evaluation of bone marrow depression from the entire cell population analyzed for micronuclei.     

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.     

Concomitant observations of UDS in the liver and micronuclei in the bone marrow of rats exposed to cyclophosphamide or 2-acetylaminofluorene

Oral dosing of between 5–30 mg/kg of cyclophosphamide (CP) to Alderley Park rats induced micronuclei in the bone marrow between 12 and 36 h after dosing, but failed to induce unscheduled DNA synthesis (UDS) in the liver at similar dose levels and treatment periods. Dose levels of > 30 mg/kg were toxic to the liver. In contrast, 2-acetylaminofluorene (2AAF) induced UDS in the rat liver between 4–36 h after dosing, but gave only a weak response in the bone marrow assay at dose levels between 0.5 and 2 g/kg. Selected observations were made for each chemical using both tissues of the same test animal.

It is concluded that an assessment of the genotoxicity in vivo of chemicals defined as genotoxic in vitro will contribute to an assessment of their possible mammalian carcinogenicity, and that these should involve assays conducted using both the bone marrow and the liver of rodents. Due to its relative ease of commission, the bone marrow micronucleus assay will usually be conducted first; in the case of negative results it is recommended that a liver genotoxicity assay should also be conducted. The case for employing in vivo short-term genotoxicity tests to predict the possible organotropic carcinogenicity or germ cell mutagenicity of a new in vitro genotoxin is discussed.     

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.     

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.     

Embryotoxicity of oral administered chlorothalonil in mice

BACKGROUND: Chlorothalonil (2,4,5,6-tetrachloroisophthalonitril), the nephrotoxic fungicide, was examined for its potential to produce developmental toxicity in mice after oral administration. METHODS: Pregnant ICR (CD-1) mice were given sublethal doses of 0 (corn oil), 100, 400, and 600 mg/kg/day chlorothalonil by gavage on gestation days (GD) 6-15. RESULTS: Maternal effects in 400 and 600 mg/kg/day dose groups included signs of toxicity such as weakness and depression in the maternal activity, and reduction in body weight and weight gain. No maternal toxicity was apparent in the 100 mg/kg/day dose group. Maternal exposure to chlorothalonil during organogenesis significantly affected the number of live fetuses, early resorption, and mean fetal weight in the 400 and 600 mg/kg/day dose groups. No external, visceral, and skeletal abnormalities were observed among any of the treated groups compared to the control. CONCLUSIONS: On the basis of the present results chlorothalonil can produce clinical signs of toxicity and fetotoxicity without teratogenic effects at 400 and 600 mg/kg/day dose groups.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9-9.4J/cm2 of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.     

The prevention of benzene-induced genotoxicity in mice by indomethacin

Benzene is a myelotoxin which affects hemopoietic progenitor cells leading to bone-marrow depression as well as a genotoxin which causes chromosomal abnormalities including micronucleus formation. We have demonstrated previously that benzene administered to DBA/2 or C57B1/6 mice causes bone-marrow depression and increased prostaglandin E2 levels in bone marrow; both of these effects can be prevented by the coadministration of indomethacin, a selective inhibitor of prostaglandin synthase. We report, herein, that benzene (400-600 mg/kg body weight), under conditions where it depresses bone-marrow cellularity, also induces an increase in the frequency of micronucleus formation in polychromatic erythrocytes of C57B1/6 mice which is also prevented by the coadministration of indomethacin at levels that do not inhibit cytochrome P450 or myeloperoxidase. In Swiss Webster wild-type mice doses of benzene from 400 to 1000 mg/kg were without effect on marrow cellularity, but did induce the formation of micronucleated polychromatic erythrocytes which could be prevented by indomethacin. In contrast, DBA/2 mice, a strain highly sensitive to benzene, exhibited significant bone-marrow depression at a dose of benzene of 100 mg/kg body weight. Even at this low dose, benzene is too toxic toward developing erythrocytes to allow the evaluation of micronucleus formation. The frequency of induction of micronucleated polychromatic erythrocytes by benzene thus depends on the strain of mouse used. Furthermore, micronucleus formation appears to be an early and very sensitive indicator of benzene toxicity. A possible role for prostaglandin H synthase in the geno- and myelo-toxicity of benzene is discussed.     

Activity of 1,2-dimethylhydrazine in the mouse bone marrow micronucleus assay using a triple- and a single-dosing protocol

In the present study the ability of 1,2-dimethylhydrazine (DMH) to induce micronuclei in mouse bone marrow erythrocytes was investigated using 2 different dosing regimens. DMH caused an increase in micronuclei in both male and female mice following single administration and sampling after 24 h. The effect was more pronounced in female than in male animals. Triple administration of DMH at concentrations corresponding to 80, 40 and 20% of the median lethal dose (MLD) did not increase the incidence of micronuclei in either sex. Small increases in micronucleus incidence were observed after triple dosing at 100% of the MLD value. These results suggest that a future micronucleus protocol should include animals of both sexes and single and repeated administration of the test substance.     

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.     

Effects of vitamin C on the hypobaric hypoxia-induced immune changes in male rats

Hypobaric hypoxia (HH) induces oxidative stress (OS) and is associated with the generation of reactive oxygen species (ROS). Vitamin C is an efficient antioxidant, and it is used in a high-altitude environment to reduce the OS. The present study explores the role of vitamin C on some HH-induced changes of immune parameters in rats which were exposed to HHc condition at 18,000 ft in a simulated chamber for 8 h/day for 6 days with and without vitamin C administration at three different doses (200, 400, and 600 mg/kg body wt). The phagocytic activity of circulating blood WBC was increased, and the cytotoxic activity of splenic mononuclear cell (MNC) and the delayed type of hypersensitivity (DTH) responses to bovine serum albumin (BSA) were decreased in rats exposed to HHc condition, but these immune changes were blocked after administration of vitamin C at 400 mg/kg body wt. The leukocyte adhesive inhibition index (LAI) was not altered either in HHc condition or after administration of vitamin C in HHc condition. The serum corticosterone (CORT) concentration was increased in rats exposed to HHc condition which was blocked after administration of vitamin C (400 mg/kg body wt). The immune parameters and serum CORT concentration, however, did not show any recovery after administration of vitamin C at the dose of 200 and 600 mg/kg body wt. The present study indicates that administration of vitamin C at a dose of 400 mg/kg body wt may prevent the HH-induced immunological changes but not at the lower dose (200 mg/kg body wt) or higher dose (600 mg/kg body wt) in rats.     

In vivo photochemical micronucleus induction due to certain quinolone antimicrobial agents in the skin of hairless mice

The skin micronucleus test combined with irradiation due to a sunlight simulator having a spectrum almost identical to solar irradiation was used as a novel in vivo testing method for detecting or comparing the photochemical chromosome damage of quinolone antibacterial agents (quinolones). Eight-week-old male SKH1 hairless mice were orally administered once lomefloxacin (LFLX), a strong in vitro photochemical clastogen, at 25 or 50 mg/kg, followed by light irradiation at 7.9–9.4 J/cm² of ultraviolet A (UVA). Animals were killed on Days 2, 3, 4, 5 or 8 (the dosing day was designated as Day 1), and the incidence of micronucleus in the epidermis was determined. As results, LFLX at either dose caused significant increases in the micronucleus frequency, which peaked on Day 4. These changes tended to return to the control level on Day 8. Then, the micronucleus induction potential of the quinolone derivatives levofloxacin (LVFX) and clinafloxacin (CLFX) at 10, 20 or 40 mg/kg was assessed on Day 4 under the same experimental conditions as for LFLX. Although LVFX was negative even at 40 mg/kg, CFLX dose-dependently induced significant increases in micronucleus frequency at all doses. The correlation of magnitude among the three quinolones in the skin micronucleus test with light irradiation was similar to that in our previous in vitro photochemical clastogenicity study. No significant increase in micronucleus frequency was observed in any of three quinolones employed without light irradiation. In conclusion, the experimental method presented here would be a useful tool for detecting in vivo photochemical chromosome damage and for research on photochemical carcinogenesis of chemicals.     

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.     

Effects of antioxidants on induction of micronuclei in rat peripheral blood reticulocytes by potassium bromate.

Micronucleus induction in male F344 rat peripheral blood by potassium bromate (KBrO3), a rat renal carcinogen, and its inhibition by several antioxidants were studied using the acridine orange supravital staining method. The frequency of micronucleated reticulocytes (MNRETs) peaked 32 h after a single i.p. treatment of rats with KBrO3 at a dose of 60 mg/kg. Co-treatment with glutathione (GSH) or cysteine (Cys) i.p. at doses of 800 mg/kg and 400 mg/kg, respectively, 30 min before and 30 min after the KBrO3 treatment significantly inhibited the micronucleus induction by KBrO3. Daily i.g. administration of vitamin C for 5 days at a dose of 200 mg/kg/day was also effective in protecting against micronucleus induction by KBrO3 given on the 4th day. However, co-treatment with superoxide dismutase in liposome-encapsulated form by i.p. injection at a dose of 18,000 U/kg 30 min before and 30 min after the KBrO3 application exerted no effect. The results indicate that antioxidants, especially sulfhydryl compounds, have protective potential against the clastogenicity of KBrO3, also suggesting that active oxygen species may play an important role in its clastogenicity.     

In vivo postirradiation protection by a vitamin E analog, alpha-TMG

The water-soluble vitamin E derivative alpha-TMG is an excellent radical scavenger. A dose of 600 mg/kg TMG significantly reduced radiation clastogenicity in mouse bone marrow when administered after irradiation. The present study was aimed at investigating the radioprotective effect of postirradiation treatment with alpha-TMG against a range of whole-body lethal (8.5-12 Gy) and sublethal (1-5 Gy) doses of radiation in adult Swiss albino mice. Protection against lethal irradiation was evaluated from 30-day mouse survival and against sublethal doses was assessed from micronuclei and chromosomal aberrations in the bone marrow 24 h after irradiation. An intraperitoneal injection of 600 mg/kg TMG within 10 min of lethal irradiation increased survival, giving a dose modification factor (DMF) of 1.09. TMG at doses of 400 mg/kg and 600 mg/kg significantly reduced the percentage of aberrant metaphases, the different types of aberrations, and the number of micronucleated erythrocytes. DMFs of 1.22 and 1.48 for percentage aberrant metaphases and 1.6 and 1.98 for micronuclei were obtained for 400 mg/kg and 600 mg/kg TMG, respectively. No drug toxicity was observed at these doses. The effectiveness of TMG when administered postirradiation suggests its possible utility for protection against unplanned radiation exposures.     

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.     

Monitoring the exposure of rats to 2-acetylaminofluorene by the estimation of mutagenic activity in excreta, sister-chromatid exchanges in peripheral blood cells and DNA adducts in peripheral blood, liver and spleen

The sensitivity of various methods suitable for biomonitoring the exposure to genotoxicants was compared in an animal model. The results were related to the presence of genotoxic effects in the target organ. Groups of male Wistar rats were given one oral dose of 0, 0.1, 10 or 200 mg 2-acetylaminofluorene (2-AAF)/5 ml dimethyl sulphoxide/kg body weight. Peripheral blood cells, excreta, liver and spleen were collected at different time intervals after dosing. Mutagenicity in urine and extracts of faeces was determined using the Ames test with Salmonella typhimurium TA98 with and without S9 and with and without beta-glucuronidase. Genotoxic effects were studied by measuring DNA-adduct formation in lymphocytes, liver and spleen, and sister-chromatid exchanges (SCEs) in lymphocytes. DNA adducts were measured with immunochemical techniques and postlabelling methods. Mutagenicity in urine and faeces, collected during the first 24 h after treatment, was detected at 2-AAF doses of 1 mg/kg b.w. and higher. At these doses DNA adducts also became apparent in the liver, the main target organ for tumour induction by 2-AAF. The adduct detected appeared to be the N-(deoxyguanosin-8-yl)-2-AAF adduct. There was no evidence of the presence of any other types of DNA adducts. At doses of 1 and 10 mg/kg b.w. no mutagenicity was detected in excreta collected during the second and third day after dosing. The DNA-adduct level in liver cells of the 1 mg/kg b.w. group was maximal 24 h after dosing. At 200 mg/kg b.w. a delay in excretion of mutagenicity with urine and faeces was seen and at 10 and 200 mg/kg b.w. the amount of DNA adducts continued to increase with time after dosing. At 24 and 48 h after treatment with 10 mg, the adduct levels were of the same order of magnitude as those found after the 20-fold higher dose. This points to overloading of the metabolizing system which in combination with the enterohepatic circulation, may lead to an increased retention of 2-AAF in the body. A slightly increased incidence of SCEs of doubtful significance was seen in lymphocytes, but only at the very high dose of 200 mg/kg b.w. No DNA adducts could be detected in blood lymphocytes or spleen cells at any of the dose levels applied, either with the immunochemical or with the postlabelling method.(ABSTRACT TRUNCATED AT 400 WORDS)