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Diss Factsheets

Administrative data

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report equivalent or similar to OECD guideline 474: GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
According to US EPA Guideline 84-2
GLP compliance:
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics
EC Number:
Molecular formula:
Combination of mainly CnH2n+2 and CnH2n structures comprised mainly within a carbon number range from C10 to C13.
Hydrocarbons, C10-C13, n-alkanes, isoalkanes, cyclics, < 2% aromatics
Details on test material:
- Name of test material (as cited in study report): MRD-89-582

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Breeding Laboratories
- Age at study initiation: ca. 8-9 weeks
- Weight at study initiation: 21-40 grams
- Assigned to test groups randomly: [no/yes, under following basis: computer generated, body weight sorting program
- Housing: individual
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: 28 days

- Temperature (°F): 68-76
- Humidity (%): 40-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
- Vehicle(s)/solvent(s) used: corn oil
- Amount of vehicle (if gavage or dermal): not to exceed 1ml/100 grams bw
- Purity: assumed to be 100% pure
Details on exposure:
The test material was weighted out and on the day of dosing, mixed with the carrier to provide stock solutions such that individual animal dose volumes did not exceed 1ml/100grams body weight. The mice were administered 1.25, 2.5, or 5.0 grams of test material/kg of body weight. Corn oil served as the carrier for the test material and was dosed at the same volume as the test material.

Duration of treatment / exposure:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing.
Positive control animals were sacrificed 24 hours after injection
Frequency of treatment:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing
Post exposure period:
Animals were treated once by oral gavage and sacrificed 24h, 48h or 72h after dosing
Doses / concentrationsopen allclose all
Doses / Concentrations:
5.0 g/kg/bw
nominal conc.
Doses / Concentrations:
nominal conc.
Doses / Concentrations:
1.25 g/kg/bw
nominal conc.
No. of animals per sex per dose:
30 animals (5 male; 5 female)/dose; 10/timepoint
Positive control(s):

- Route of administration: intraperitoneal injection
- Doses / concentrations:40 mg/kg using water as the carrier


Tissues and cell types examined:
Bone marrows were collected and extracted, smear preparations made and stained. Polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE) were scored for each animal.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Range finding study was performed using 5.0, 2.5, and 1.0 gram of test material per kg of body weight. Two males and two females were used for each dose group. All animals survived and were sacrificed 24 hours after dosing. bome marrow was removed and slides were prepared. Slides were evaluated for percent of polychromatic erythrocytes in 1000 erythrocytes and for number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes.

DETAILS OF SLIDE PREPARATION: After sacrifice, both femurs were removed. The bone marrow was then removed and suspended in fetal bovine serum. After the suspension was centrifuged, the pellet was resuspended and smears were prepared (two slides per animal). Slides were labeled with blind coding. Slides were stained using acridine orange. 1000 polychromatic erythrocytes from each animal were examined for the presence of micronuclei, and the ratio of PCE’s to NCE’s determined

METHOD OF ANALYSIS: staining color, and circular appearance and a diameter between 1/20 and 1/5 of the cell's diameter

Statistical analysis included calculation of means and standard deviations of the micronuclei data and a test of equality of group means by a standard one way analysis of variance at each time period. When the ANOVA was significant, comparisons of carrier control to dosed group means were made according to Duncan’s Multiple Range Test. A standard regression analysis was performed to test for a dose response. Residuals from the ANOVA were analyzed for normality by Wilk’s Criterion. The residuals were normally distributed (values were greater than 0.01 level of significance) in more than 25% of the analyses. Therefore nonparametric analysis were not performed. Sexes were analyzed separately

Results and discussion

Test results
Key result
no effects
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Additional information on results:
No deaths or clinical signs of toxicity were observed in animals dosed with the test material up to the maximum recommended dose of 5g/kg

Applicant's summary and conclusion

Interpretation of results: negative
The in vivo micronucleus assay of MRD-89-582 in mice was negative. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations.
Executive summary:

MRD-89-582 was examined for its potential to induce chromosomal damage in bone marrow erythrocytes in mice dosed by oral gavage at concentrations of 5.0,2.5, and 1.25 g/kg. Vehicle and positive control animals received corn oil and cyclophosphamide, respectively.  Bone marrow samples were collected and evaluated for micronucleus formation 24, 48 and 72 hours after dosing.  MRD-89-582 did not induce a statistically significant change in the PCE/NCE ratio in any of the test material dose groups when compared to their concurrent vehicle control groups. The positive control material (cyclophosphamide) produced a marked increase in the frequency of micronucleated PCE when compared to the concurrent vehicle control group The test material was considered to be non-genotoxic and non-clastogenic under the conditions of the test. This finding does not warrant the classification of the test material as a genotoxin under EU GHS guidelines and does not warrant classification under the EU requirements for dangerous substances and preparations guidelines.