Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Nossan s.r.l., Corezzana, Italy,
- Age at study initiation: 5-6 weeks
- Weight at study initiation: 26-34 grams for males and 22-27 grams for females
- Assigned to test groups randomly: no data
- Housing: 5/polycarbonate cage, by sexes
- Diet: Altromin MT diet
- Water: tap water
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle: no data
Duration of treatment / exposure:
Single administration
Post exposure period:
24 and 48 hour sampling time
Doses / concentrations
Remarks:
Doses / Concentrations:
0 (vehicle), 500, 1000 and 2000 mg/kg
Basis:

No. of animals per sex per dose:
Each group consisted of five male and five female animals with the exception of the control and high-dose groups, which included an additional five animals of each sex per group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Route of administration: intraperitoneal
- Doses / concentrations: 2.0 mg/kg bw

Examinations

Tissues and cell types examined:
5 animals/sex/group were sacrificed at the 24-hour sampling time. The  additional animals were sacrificed at the 48-hour sampling time. At least  2000 polychromatic cells per animal were examined for the presence of micronuclei. The ratio of mature to polychromatic erythrocytes was also  determined.  
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary toxicity study was conducted in which groups of two male and two female mice were dosed once (i.p. injection) with n-butyl  methacrylate at 2000, 1500 and 1000 mg/kg. Clinical signs were observed  in all animals on the day of dosing, but all animals recovered by the  following day. No bone marrow cytotoxicity, as measured by increases in  the NCE/PCE ratio, was observed. The doses for the definitive study were  selected based on this preliminary toxicity study. 

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Five animals per sex from each group were sacrificed at the 24 hour sampling time. The additional animals were sacrificed at the 48 hour sampling time.

DETAILS OF SLIDE PREPARATION:
The femurs were removed and bone marrow cells obtained by flushing with foetal calf serum. The cells were centrifuged and a concentrated suspension prepared to make smears on slides. These slides were air-dried and then stained with May-Gruenwald and Giemsa, and mounted with Eukitt. Three slides were made from each animal.

METHOD OF ANALYSIS:
The slides were randomly coded by a person not involved in the subsequent microscope scoring. The slides were examined under low power (x 16 objective) and one slide from each animal was selected according to staining and quality of smears. At least 2000 polychromatic cells per animal were examined for the presence of micronuclei at high power (x 100 objective, oil immersion). At the same time the numbers of normal and micronucleated normochromatic erythrocytes were also recorded.
Evaluation criteria:
The test substance is considered to induce micronuclei if a statistically significant increase in the micronucleus incidence in polychromatic erythrocytes (at P<0.05) is observed in any treatment group, in the pooled data for both sexes, or for either sex considered separately.
Statistics:
Only counts obtained from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified Chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where this was significant it was taken into account in the comparison between groups. Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
but tested up to the limit dose
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No statistically significant increase in the incidence of micronucleated PCE's over the control value was observed at any dose-level. Slight increases in the ratio of mature to polychromatic erythrocytes, compared to the vehicle control, were seen at the 48 hour sampling time for both male and female animals from the high-dose group.

Any other information on results incl. tables

Following are the results for the male and female animals combined:

                        Incidence of         NCE/PCE
            Dose        micronucleated        Mean
Treatment   Level              PCE              Ratio
           (mg/kg)        Mean      S.E.
-----------------------------------------------------------        
24 hr. Sampling time
-----------------------------------------------------------        
Vehicle (corn oil)
            10           0.5      0.2         1.12
-----------------------------------------------------------
           (ml/kg)
n-butyl methacrylate
           500           0.6      0.2         1.19
n-butyl methacrylate        
          1000           0.9      0.2         0.93
n-butyl methacrylate
          2000           0.6      0.2         1.15
-----------------------------------------------------------
Mitomycin-C  2.0        33.6***   6.5         1.32
-----------------------------------------------------------
48 hr. Sampling time
-----------------------------------------------------------        Vehicle (corn  oil)
           10            0.8      0.3         1.10
-----------------------------------------------------------
          (ml/kg)
n-butyl methacrylate
         2000            1.2      0.3         1.48
-----------------------------------------------------------
*** Incidence significantly greater than control value at p < 0.0001

Applicant's summary and conclusion