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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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Acceptable, well-documented study report similar or equivalent to OECD 475. 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

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MCP 917
- Physical state: pale yellow liquid
- Lot/batch No.: 89153
- Expiration date of the lot/batch: 04/01/1994

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Portage MI
- Age at study initiation: 6 weeks
- Weight at study initiation: male: 21-29 g; female: 20-24 g
- Housing: individually housed in stainless steel cages with wire mesh bottoms
- Diet (e.g. ad libitum): Purina Certified Lab Chow, #5002, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10-12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 68-72
- Humidity (%): 42-74
- 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: Corn oil was chosen as the vehicle because it dissolved the study substance, was not mutagenic, and had been used as the vehicle for the study substance in other toxicity studies performed at this laboratory.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Purity: 100%
Duration of treatment / exposure:
3 consecutive days
Frequency of treatment:
One injection per day, approximately 24 hours apart
Doses / concentrations
Remarks:
Doses / Concentrations:
1250, 2500, 5000 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide monohydrate
- Justification for choice of positive control(s):
- Route of administration: The stability of the positive control substance was indicated by its induction of micronuclei in CP-treated animals.
- Doses / concentrations: 50 mg

Examinations

Tissues and cell types examined:
Micronucleated erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The high dose was chosen based on the results of the Preliminary Toxicity Test and is the highest dose that can be tested in this system.

DETAILS OF SLIDE PREPARATION: The slides were fixed in absolute methanol and stained with Acridine Orange.

METHOD OF ANALYSIS: All slide analyses were performed by one experienced person. Observations were made using a Leitz Orthoplan fluorescent microscope with an HBO 100 Watt mercury bulb, Ploemopak vertical fluorescent illumination, an H2 filter cube and a Leitz NPL FLUOTAR 63/0.90 objective lens.
Evaluation criteria:
The mean frequency of micronucleated PCEs in the negative (vehicle) control should not exceed 0.3%. The positive control group should have a statistically significant, elevated frequency of micronucleated PCEs about the negative (p<0.05, normal test).
Statistics:
The proportion of micronucleated PCEs for each treatment group was compared to the negative control group. Any absolute increase in the proportion of micronucleated PCEs about the negative control was evaluated for statistical significance using the normal test for equality of proportions. A one-tailed P value of 0.05 or less was considered significant. To test for dose responsiveness, the Cochran-Armitage test for trend in binomial proportions was used. The test was performed as one-tailed. A P value of 0.05 or less was considered significant.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
not specified
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: none
- Evidence of cytotoxicity in tissue analyzed: none
- High dose with and without activation: 5000 mg/kg

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): none
- Ratio of PCE/NCE (for Micronucleus assay): No decrease in the mean ratio
- Appropriateness of dose levels and route: No evidence for dose responsiveness was found for either sex or for pooled data for both sexes.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
The in vivo micronucleus assay of the study substance was negative. This finding does warrant the classification of the study substance as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.
Executive summary:

The study substance was examined for its potential to induce chromosome damages in a mammalian species using the formation of micronucleated erythrocytes as an indicator of damage. A preliminary toxicity test was performed in male and female mice of the B6C3F1 strain to determine a maximum tolerated dose for three treatments. No toxicity was observed at 5000 mg/kg/day, which is the highest dose level usually included in a micronucleus test. The study substance did not cause cytogenetic damage to bone marrow erythrocytes in mice in this assay. The study substance was not considered to be genotoxic under the conditions of the test. This finding does not warrant the classification of the study substance as a genotoxin under the new Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP) or under the Directive 67/518/EEC for dangerous substances and Directive 1999/45/EC for preparations.