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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2001-09-10 to 2001-10-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted 21 July 97,
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
Directive 92/69/EEC
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(2-oxoimidazolidin-1-yl)ethyl methacrylate
EC Number:
289-214-5
EC Name:
2-(2-oxoimidazolidin-1-yl)ethyl methacrylate
Cas Number:
86261-90-7
Molecular formula:
C9H14N2O3
IUPAC Name:
2-(2-oxoimidazolidin-1-yl)ethyl methacrylate
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories (Kingston, N.Y.)
- Age at study initiation: 9 weeks
- Weight at study initiation: 30.8-39.9 g for males; 26.1-30.1 g for females
- Assigned to test groups randomly: yes, animals were assigned to treatment groups through the use of a computerized randomization procedure according to sex and body weight prior to administration of the test articles.
- Fasting period before study: yes, 3 h
- Diet: Purina Certified Rodent Chow 5002 C was available ad libitum
- Water: Water filtered through a reverse osmosis system was available ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 43-62

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: water
- Justification for choice of solvent/vehicle: good solubility in water
Details on exposure:
The test article and the negative control were administered by gavage, in a single oral dose. For each treatment group and vehicle and positive control group, 5 male and 5 female animals were dosed per time point, with a volume of 10 mL/kg.
Duration of treatment / exposure:
24 and 48 hours
Frequency of treatment:
single treatment
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 200, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
For each treatment group and vehicle and positive control group, 5 male and 5 female animals were dosed per time point. In the high dose group, 2 additional animals per time point were dosed to account for the possibility of unexpected deaths.
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control MMC (at a dose of 2.0 mg/kg bw) was administered in a single dose by intraperitoneal (i.p.) injection since this is the accepted route for this substance.

Examinations

Tissues and cell types examined:
- Clinical observations:Animals were observed for the presence of clinical signs during the treatment period and prior to sacrifice
- Organs examined at necropsy: Bone marrow was examined in each treatment and control group.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The maximum concentration chosen for this study was a limit dose of 2000 mg/kg bw. In addition to this dose level, 1000 and 200 mg/kg bw of the test substance was tested.

TREATMENT AND SAMPLING TIMES:
Animals from test article and vehicle control groups were euthanized by cervical dislocation at approximately 24 and 48 hours after dosing. Animals from the positive control groups were euthanized 24 hours after dosing.

DETAILS OF SLIDE PREPARATION:
Animals were prepared for micronucleus evaluation as follows: The groin area was wetted with 70% ethanol in water. Both femurs were removed by making incisions at the hip joint and below the knee cap, and the knee cap was removed. The bone marrow was flushed into a 15-mL centrifuge tube, containing approximately 1 mL of Fetal Bovine Serum (FBS) using a 1-cc syringe fitted with a 25-gauge needle. The tubes were centrifuged at 120 x g for 5 minutes, and the supernatant was removed, leaving approximately 0.1 mL above the cell pellet. The cell pellet was re-suspended in the remaining serum until a homogeneous suspension was observed. A small drop of the cell suspension (approximately 10 µL) was placed on the unfrosted end of a clean microscope slide and spread along the length of the slide. The slides were air dried for at least 1 hour, and then fixed in methanol for 15 minutes and allowed to dry. The slides were stained with Acridirie Orange staining solution.

METHOD OF ANALYSIS:
Slides from at least five animals per sexldose group were observed when possible. Three slides were prepared per animal. Slides were coded and read blind in order to avoid bias on the part of the scorer. The slides were read using an epifluorescence microscope to illuminate the acridine orange stain. Slides were scanned for regions of suitable technical quality, where the cells were well spread, undamaged and well stained. These regions were normally located in a zone close to the middle of the smear. Staining was tan to faint grey in normochromatic erythrocytes (NCE) and bright orange in polychromatic erythrocytes (PCE). Micronuclei appeared bright green against an orange background in PCE and generally were round, although almond and ring-shaped micronuclei occasionally occur. Micronuclei have sharp borders and were usually between 1/20 and 115 the size of the PCE. The end point to be scored was the number of cells containing micronuclei (not the number of micronuclei per cell).
For each animal, a total of at least 1000 erythrocytes (polychromatic, referred to as PCE 1 and normochromatic) were recorded to calculate the PCEINCE ratio. For each animal, the remaining number of polychromatic erythrocytes were recorded to total at least 2000 (referred to as PCE 2) and were scored for the presence or absence of micronuclei. The frequency of micronucleated polychromatic erythrocytes (MN-PCE) and the PCE/NCE ratio were calculated on the basis of these data.
Evaluation criteria:
The criteria for a positive response was the detection of a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response. A test article that did not induce both of these responses was considered negative. Statistical significance was not the only determinant of a positive response; the Study Director also considered the biological relevance of the results in the final evaluation.
Statistics:
Data were analyzed separately for male and female animals using a Statistical Analysis System (SAS), version 6.09 enhanced. An arcsine square root transformation was applied to the percent of micronucleated PCE's; all subsequent analyses for this parameter were conducted on transformed data. Initially, a three-way analysis of variance model was applied to the data to determine the significance of each main effect (sex, group, and day) and all two-way and three-way interaction effects. If significant interaction effects were identified, then the data were analyzed separately for each sex and/or day. Three independent single degree of freedom contrasts of the group means were used to test for trends in the group means and included an assessment of: 1) an overall effect of the test substance treatment relative to control, and 2) a linear dose-response trend among the test substance, and 3) a quadratic dose-response trend among the test substance. Additionally, pair-wise comparisons between each of the three test substance groups and the control group were made using Dunnett's t-test (Kirkland, 1989).

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Clinical Signs
No clinical signs of toxicity were observed in any of the mice treated with the test article or control articles.

Micronucleus Evaluation
The test article did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow cells of male or female mice when compared to the vehicle control values. This was true for both the 24 and 48 hour time points. There was no statistically significant change in the polychromatic/normochromatic ratio at either 24 or 48 hours, which is indicative of the absence of cytotoxicity.
An increase in the frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow cells of male and female mice treated with 2.0 mg/kg of the positive control, mitomycin-C. When compared to the vehicle controls, the increase was greater than two-fold, indicating that the assay was sufficiently sensitive to detect induced cytogenetic damage.

CONCLUSION
Under the conditions of this study, the test substance was not mutagenic in the micronucleus assay in CD-1 mouse bone marrow cells.

Applicant's summary and conclusion

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