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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 from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, minor restrictions in design and/or reporting but otherwise adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): MRD-91-935
- Physical state: pale yellow liquid
- Analytical purity: considered 100%
- Lot/batch No.: 1
- Expiration date of the lot/batch: 31 May 1996
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Portage, Shaver Road, Portage, Michigan 49081, USA
- Age at study initiation: Approximately 8 to 9 weeks
- Weight at study initiation: 20 - 29 g three days prior to experimental start
- Assigned to test groups randomly: yes, using a computer generated, body weight sorting program
- Housing: Individually in suspended stainless steel cages
- Diet: Purina Certified Rodent 5002 Chow (pellets) ad libitum (except during exposure) Ralston Purina Company, Checkerboard Square, St. Louis, Missouri, 63164, USA
- Water: Mains water ad libitum
- Acclimatisation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 68-76°F
- Humidity: 29-71%
- Air changes (per hr): Not reported
- Photoperiod: 12hrs dark / 12hrs light

IN-LIFE DATES: From: 5 August 1991 To: 12 September 1991

Administration / exposure

Route of administration:
inhalation
Vehicle:
- Vehicle(s)/solvent(s) used: air
Details on exposure:
TYPE OF INHALATION EXPOSURE: whole body

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
The test material was delivered to the inside of a cylindrical glass vapour generator. The test material evaporated as it flowed down the heated generator. Resultant vapours were mixed with room air as both were drawn up through the generator and into the exposure chamber. The exposure chambers (volume approximately 1000 L) operated under slight negative pressure at 200 L/min airflow. The theoretical equilibration time (T99) was 23 mins.

TEST ATMOSPHERE
- Brief description of analytical method used: Gas chromatography
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
2 days
Frequency of treatment:
6 hours/day for 2 consecutive days
Post exposure period:
Approximately 24, 48 or 72 hours after second exposure (test and negative control groups). Approximately 25.5 hours after single ip injection for cyclophosphamide (positive control).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 30, 100, 300 ppm
Basis:
other: target exposure concentrations
Remarks:
Doses / Concentrations:
0, 30, 102, 309 ppm
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
N/A, 37, 117, 337 ppm
Basis:
nominal conc.
No. of animals per sex per dose:
15/sex (i.e. 5/sex/sacrifice interval) for test and negative controls. 5/sex for positive control
Control animals:
yes, sham-exposed
Positive control(s):
cyclophosphamide
- Route of administration: single intraperitoneal injection
- Doses / concentrations: 40 mg/kg bodyweight (carrier water)

Examinations

Tissues and cell types examined:
polychromatic erythrocytes of the bone marrow of B6C3F1 mice
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES: Five animals/sex were killed approximately 24, 48 and 72 hours after second exposure period (MRD-91-935 and negative (air) controls). Positive controls were killed approximately 25.5 hours following dosing. Immediately after death, both femurs were removed. The bone marrow was then removed and suspended in foetal bovine serum. After the suspension was centrifuged, the pellet was resuspended and smears were prepared (two slides per animal).

DETAILS OF SLIDE PREPARATION: Slides were stained using acridine orange
Evaluation criteria:
Polychromatic erythrocytes (PCE), normochromatic erythrocytes (NCE) and micronuclei identified by stain. Additional criteria for scoring micronuclei are a circular appearance and a diameter between 1/20 and 1/5 of the cell's diameter. 1000 polychromatic erythrocytes (PCE) from each animal were examined for micronuclei, and the ratio of PCE's to NCE's was determined for each animal by counting 1000 erythrocytes (PCE's and NCE's).
Statistics:
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 control to dosed group means were by 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 (at the 0.01 level of significance) in more than 25% of the analyses. Therefore nonparametric analyses were not performed.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 500, 2000 ppm
- Clinical signs of toxicity in test animals: All animals at 500 and 2000 ppm died prior to second day of dosing. All animals exposed at 100 ppm survived.
Based on the range finding study results, the doses used for the mutation assay were 30, 100 and 300 ppm of test material in air.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: MRD-91-935 did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes.
- Ratio of PCE/NCE: MRD-91-935 did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes which is a measure of bone marrow toxicity.
- Positive control: Induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes which indicates that the positive control is clastogenic and is responding in an appropriate manner.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
MRD-91-935 was not toxic to mouse bone marrow under the conditions of this test. MRD-91-935 was not clastogenic in bone marrow of B6C3F1 mice.
Executive summary:

MRD-91-935 did not induce a statistically significant decrease in the mean percent of polychromatic erythrocytes which is a measure of bone marrow toxicity. Thus MRD-91-935 was not toxic to mouse bone marrow under the conditions of this test. This test material did not induce a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes. Thus MRD-91-935 was not clastogenic in bone marrow of B6C3F1 mice.