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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: According to OECD Guideline 474. GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
Hydrocarbons, C10-C13, aromatics, >1% naphthalene
EC Number:
926-273-4
IUPAC Name:
Hydrocarbons, C10-C13, aromatics, >1% naphthalene

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 Breeding Laboratories, Inc.
Sex: Male (65), Female (65)
Age at study initiation: Approximately 9-10 weeks
Weight at study initiation: 23-39g
Housing: Individually
Diet (e.g. ad libitum): Purina Certified Rodent 5002 chow (pellets), ad libitum
Water (e.g. ad libitum): Automatic watering system, ad libitum
Acclimation period: 35d

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Corn oil was used. Dose volume did not exceed 1.0 ml/100 g bw.
Details on exposure:
The test material and the carrier were administered by oral gavage as a single dose. The carrier was dosed at a volume equal to the test material dose volume. The individual animal dose volumes did not exceed 1.0 ml/100 g body weight; animals were administered 0.25, 0.5, or 1.0 g test material/ kg body weight. The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.
Duration of treatment / exposure:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
Frequency of treatment:
One dose was given at either 0.25, 0.5, or 1.0 g test material/ kg body weight.
Post exposure period:
Animals were sacrificed 24, 48, and 72 hours after dose administration.
Doses / concentrations
Remarks:
Doses / Concentrations:
0.25, 0.5, 1.0g/kg/bw
Basis:
other: oral gavage
No. of animals per sex per dose:
Male (65), Female (65) ; 5 Males and 5 Females per treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control, cyclophosphamide was administered as a single intraperitoneal injection (40 mg/kg) using water as a carrier.

Examinations

Tissues and cell types examined:
Erythrocytes derived from femur bone marrow.
Details of tissue and slide preparation:
Immediately following the sacrifice of the animals, both femurs were removed and the bone marrow was removed and suspended in fetal bovine serum. After the suspension was centrifuged the pellet was resuspended and smears were prepared (two slides per animal).
Evaluation criteria:
Slides were stained using acridine orange; polychromatic erythrocytes (PCE) stained red/orange, nonchromatic erythrocytes (NCE) are unstained (dull green), and micronuclei stain bright yellow. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell’s diameter. 1000 PCE from each animal were examined for the presence of micronuclei and the ratio of PCE to NCE was determined for each animal by counting 1000 erythrocytes (PCE and NCE).
Statistics:
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 (ANOVA). When 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). Therefore nonparametric analysis was not performed.

Sexes were analyzed separately.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
2(male), 1 (female) animals at 2.5 g/kg; 1 (female) at 1.0 g/kg
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The positive control (cyclophosphamide) induces a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes, indicating that the positive control was clastogenic and was responding in an appropriate manner. Carrier control values for the mean percent of polychromatic erythrocytes and for the mean number of micronucleated polychromatic erythrocytes are within the normal range for the corn oil control.

MRD-90-884 did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. A significant regression coefficient (p<0.05) was observed in the mean micronucleated polychromatic erythrocytes for the 48 hour female group. However, the mean values were not significant when compared to the carrier control and were within the normal range of the carrier control. Therefore this statistical response is not believed to be biologically significant. MRD-90-884 was not clastogenic in mouse bone marrow under the conditions of this test.

Necropsies of the four animals that died prior to the scheduled sacrifice (2(male), 1 (female) animals at 2.5 g/kg; 1 (female) at 1.0 g/kg) revealed that most common effects were urine stains and a tan mucous like material in the small intestine. The most probable cause of death was due to the toxicity of the test material.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
These data indicate that MRD-90-884 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 1.0 g/kg of body weight.
Executive summary:

The test material, MRD-90-884 was tested in the mammalian bone marrow micronucleus assay using CD-1 mice.  MRD-90-884 was tested at 24, 48, and 72 hour intervals following exposure and did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes or an increase in the mean number of micronucleated polychromatic erythrocytes.  Both the positive (cyclophosphamide) and the negative (carrier) controls behaved in an appropriate manner.  These data indicate that MRD-90-884 is not cytotoxic and is not clastogenic in CD-1 mouse bone marrow cells at doses up to and including 1.0 g/kg.

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