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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
three-treatment, single-sample micronucleus test protocol.
- Principle of test: The mouse bone marrow micronucleus assay is a commonly used test for detecting the genetic toxicity of chemicals in laboratory rodents. Considerable effort has gone into developing protocols which maximize the likelihood of detecting genetic toxicity while minimizing the human and animal resources required to conduct a test. In studies designed to assess the relative effectiveness of protocols in which the numbers of daily treatments and sample times varied from one to three , three test chemicals were used: (1) 7, 12-dimethylbenzanthracene, which is slowly absorbed and metabolized and was the primary basis for testing guidelines requiring multiple sample times out to 72 hours posttreatment; (2) benzidine, which is an in vivo micronucleus inducer but has proven difficult to detect; and (3) mitomycin C, which is water soluble, direct acting, and has an in vivo half life of less than an hour. A protocol involving three daily treatments and a single sample time 24 hr following the final treatment was found to be the most effective for detecting all three chemicaIs.
- Short description of test conditions: Preliminary dose determination experiments were performed, after which groups of 5 male B6C3F1 mice were injected intraperitoneally three times at 24 hr intervals with Monuron dissolved in com oil. The total dosing volume per mouse was 0.4 mL. Solvent control animals were injected with 0.4 mL of solvent only. A concurrent positive control group of mice was included in each of the micronucleus tests Twenty-four hours after the final, smears of the bone marrow cells from femurs were prepared. Air-dried smears were fixed and stained with acridine orange; 2000 polychromatic erythrocytes (PCE) were scored per animal for frequency of micronucIeated cells. In addition, the percentage of PCEs among the total erythrocyte population in the bone marrow was scored for each dose group as a measure of toxicity. The results were tabulated as the mean of the pooled results from all animals within a treatment group, plus or minus the standard error of the mean. Repeat tests were performed for Monuron, based on results of the initial micronucleus test.
Type of assay:
other: in vivo micronucleus test in mouse bone marrow cells

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Monuron was received from the NTP chemical repository (Radian Corporation, Austin, TX).

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: obtained from the National Toxicology Program production facility at Taconic Farms
- Age at study initiation: common age between 9 and 14 weeks
- Weight at study initiation: weighing within a 2 g range of a mean weight between 25 and 33 g
Additional information on animal husbandry can be found in Tice et al. [Tice RR, Erexson G. Hilliard CJ. Huston JL, Boehm RM. Gulati D, Shelby MD (1990a): Effect of treatment protocol and sample time on frequencies of micronucleated cells in mouse bone marrow and peripheral blood. Mutagen 5:313-321.].

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil (for Monuron)
- Justification for choice of solvent/vehicle: corn oil was used for for water-insoluble chemicals
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Each chemical was prepared in the appropriate solvent (corn oil for water-insoluble chemicals (Monuron)) and suspended using a Tek-Mar Tissumizer® for chemicals in com oil All test chemicals were administered within 30 min of preparation. Suspended DMBA (positive control in case of Monuron) was stored at room temperature and dissolved MMC was stored at 0-5°C between treatments within an experiment. All treatments were by intraperitoneal (IP) injection at a volume of 0.4 mL per mouse. Identification numbers were randomly assigned to mice prior to euthanasia.
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
3 intraperitoneal injections at 24 h intervals (3 consecutive days)
Post exposure period:
24 hr after the third treatment
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
62.5 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
MN Induction Experiment
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Repeat study
Dose / conc.:
125 mg/kg bw/day (actual dose received)
Remarks:
Repeat study
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
Repeat study
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
7,12-dimethylbenzanthracene (DMBA) in corn oil (in case of Monuron); DMBA was purchased from Eastman Kodak (Rochester, NY)
- Route of administration: intraperitoneal
- Doses / concentrations: a weakly active dose of DMBA in corn oil (12.5 mg/kg DMBA)

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Dose Determination Studies were performed. The test doses for Monuron were based on toxicity/mortality observed in the dose determination studies. Groups of 5 mice were administered the test chemical by IP injection on three consecutive days. Animals were monitored twice daily, and 48 hr after the third treatment, the surviving mice were euthanized by CO2 asphyxiation. Bone marrow and peripheral blood smears (two slides/tissue/mouse) were prepared by a direct technique [Tice et al., 1990a]. Air-dried smears were fixed using absolute methanol and stained with acridine orange [Tice et al., 1990a). Bone marrow smears from each animal were evaluated at 1000 x magnification using epi-illuminated fluorescence microscopy (450-490 nm excitation, 520 nm emission) for determination of the percentage of PCE among 200 erythrocytes. Based on the results obtained, the maximum administered dose was estimated or additional dose determination experiments were conducted to more accurately estimate the maximum dose to be tested in the primary MN test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): For the initial MN test, groups of 5-7 mice were injected IP on three consecutive days with either the test chemical (at 1 x, 1/2 x , and 1/4 x , where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical (DMBA (7, 12-dimethylbenzanthracene) in corn oil or MMC (Mitomicyn C) in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hr after the third treatment. For Monuron also a repeat (second) bone marrow test was conducted.

DETAILS OF SLIDE PREPARATION:. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange.

METHOD OF ANALYSIS: For each animal, slides were evaluated at 1000 x magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.

OTHER:
Evaluation criteria:
The test conclusions (see Table 1) are based on the statistical analysis of trend and of pairwise comparisons of the solvent control with individual doses, the absolute increases in MN-PCE frequency, and where appropriate, the combination of results from repeat studies.
Statistics:
The data were analyzed using the Micronucleus Assay Data Management and Statistical software package (version 1.4), which was designed specifically for in vivo micronucleus data [ILS, 1990]. The level of significance was set at an alpha level of 0.05. To determine whether a specific treatment resulted in a significant increase in MN-PCE, the number of MN-PCE were pooled within each dose group and analyzed by a one-tailed trend test. In the software package used, the trend test incorporates a variance inflation factor to account for excess animal variability. In the event that the increase in the dose response curve is nonmonotonic, the software program allows for the data to be analyzed for a significant positive trend after data at the highest dose only has been excluded. However, in this event, the alpha level is adjusted to 0.01 to protect against false positives.
The %PCE data were analyzed by an analysis of variance (ANOVA) test based on pooled data. Pairwise comparisons between each group and the concurrent solvent control group was by an unadjusted one-tailed Pearson chi-squared test which incorporated the calculated variance inflation factor for the study.

Results and discussion

Test results
Sex:
male
Genotoxicity:
positive
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. . MN Data Analysis: 49 NTP Chemicals: Monuron

Chemicala

Tissueb

Trendc

P value

Dosed

(mg/kg)

MN-PCE/1000e

(no. animals)

Pair-wisef

Survivalg

%PCEh

Monuron C

Positive

-/-

150-68-5

EHRT (279574)

(CO)

BM

0.310

(0.003)

0

62.5

125

250

1.10 ± 0.10 (5)

1.75 ± 0.32 (4)

2.80 ± 0.56 (5)

1.40 ± 0.29 (5)

 

0.1223

0.0032

0.2741

5/5

5/5

5/5

5/5

56.4

38.1

47.6

48.6

BM

<0.001

0

125

250

1.20 ± 0.41 (5)

3.70 ± 0.64 (5)

6.10 ± 1.02 (5)

 

0.001

<0.001

5/5

5/5

52.0

51.6

aChemical name (rodent bioassay results, C = carcinogen; NC = non-carcinogen). MOUSE-MN CALL structural alert [Tennant and Ashby, 1991] Salmonella result. CAS No. Laboratory (Radian No.) solvent: CO = com oil, PBS = phosphate buffered saline) (ILS = Integrated Laboratory Systems, EHRT = Environmental Health Research and Testing).

bTissue used (BM = bone marrow, PB = peripheral blood.)

cValue of P for trend analysisα= .05 (value of P for trend analysis omitting the high dose valueα= .01).

dChemical concentration administered i. p. , daily, to each anima!.

eMicronucleated PCEs per 1000 PCE scored (± Standard Error of the Means) (number of animals scored).

fThe value of P for pair-wise comparisons between each treatment group and the concurrent solvent control groupα= .05.

gNo. of animals surviving treatment over number of animals treated.

hPercentage of erythrocytes that were polychromatic (i.e., [No. of PCE/No. of PCE + No. of NCE] x 100).

Table 2. Summary of Solvent Control Data From Mouse Bone Marrow Micronucleus Tests

Laboratory

Solvent

No.a

MN-PCE ± SDb

Range

EHRTc

COd

14

2.12 ± 0.70

1.10-3.70

 

PBSe

9

2.10 ± 0.40f

1.60-2.70

ILSg

CO

17

2.38 ± 0.93

1.10-4.60

 

PBS

8

2.66 ± 0.36f

2.20-3.10

 

aNo. of experimental groups (4 or 5 animals per group).

bMicronucleated PCEs per 1000 PCEs ±standard deviation.

cEnvironmental Health Research and Testing.

dCorn oil (0.4 mL)

ePhosphate buffered saline (0.4 mL).

fThe ILS-PBS control value is significantly higher than the EHRT-PBS control value (P = 0.009).

glntegrated Laboratory Systems.

 

Table 3. Summary of Positive Control Data From Mouse Bone Marrow Micronucleus Tests

Laboratory

Solvent

No.a

MN-PCE ± SDb

Range

EHRTc

DMBAd

14

6.93 ± 2.59

2.00-12.17

 

MMC

9

6.82 ± 1.24

4.55-8.50

ILSf

DMBA

17

7.93 ± 1.69

4.40-10.60

 

MMC

8

6.85 ± 2.26

3.80-11.20

 

aNo. of experimental groups (4 or 5 animals per group).

bMicronucleated PCEs per 1000 PCEs ±standard deviation.

cEnvironmental Health Research and Testing.

dDimethylbenzanthracene (12.5 mg/kg).

eMitomycin C (0.2 mg/kg).

flntegrated Laboratory Systems.

Applicant's summary and conclusion

Conclusions:
For Monuron the initial test was negative to 250 mg/kg by trend analysis but the trend test was positive if the high dose group was omitted. The repeat test was positive by trend analysis with the 125 and 250 mg/kg dose groups elevated significantly above the control. Sharma et al. [1987] reported that monuron, administered intraperitoneally once, twice, or three times at a dose of 14.4 mg/kg, increased the frequencies of micronuclei and chromosome aberrations in the bone marrow cells of Lacca strain mice.
Executive summary:

Forty-nine chemicals were tested in a mouse bone marrow micronucleus test that employed three daily exposures by intraperitoneal injection.

For the initial MN test, groups of 5-7 mice were injected IP on three consecutive days with either the test chemical (at 1 x, 1/2 x , and 1/4 x , where x is the maximum dose determined in the dose determination experiments), a weakly active dose of the positive control chemical (DMBA (7, 12-dimethylbenzanthracene) in corn oil or MMC (Mitomicyn C) in PBS), or the appropriate solvent. Mice were euthanized with CO2 24 hr after the third treatment. Bone marrow smears (two slides mouse) were prepared, fixed in absolute methanol, and stained with acridine orange. For each animal, slides were evaluated at 1000 x magnification for the number of MN-PCE among 2000 PCE and for the percentage of PCE among 200 erythrocytes.

For Monuron the initial MN test was performed at 0, 62.5, 125 and 250 mg/kg bw with corn oil as solvent control and DMBA in corn oil as positive control. For Monuron a repeat study was performed as well at 0, 125 and 250 mg/kg bw.

The initial test was negative to 250 mg/kg by trend analysis but the trend test was positive if the high dose group was omitted. The repeat test was positive by trend analysis with the 125 and 250 mg/kg dose groups elevated significantly above the control. Sharma et al. (Sharma GP. Sobti RC, Chaundhry A, Gill RK, Ahluwali KK (1987): Mutagenic potential of a substituted urea herbicide: Monuron. Cytologia 52: 841 -846 ) reported that monuron, administered intraperitoneally once, twice, or three times at a dose of 14.4 mg/kg, increased the frequencies of micronuclei and chromosome aberrations in the bone marrow cells of Lacca strain mice.

Overall results for the micronucleus tests in vivo in B6C3F1 mice with Monuron was positive.