Registration Dossier

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:
supporting study
Study period:
January 12 - July 24, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (GLP)

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
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:
- Test Article Lot No.: 12376-64 (Sample II)
- Test Article Purity: 99%
- Test Article Description: Clear, colorless liquid
- Storage Conditions: Frozen (-20+/-5°C), protected from light

Test animals

Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Males: 29.7 to 37.3 g; Females: 22.3 to 27.6 g
- Assigned to test groups randomly: yes, based on distribution according to body weight
- Housing: group housing with up to five mice per cage in plastic autoclavable cages with filter tops
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-27
- Humidity (%): 50±20
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle used: deionized water
- Justification for choice of vehicle: solubility
- Concentration of test material in vehicle: 1.9, 2.9, 3.8, 5.8, 7.5 and 11.5 mg/ml
Details on exposure:
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by IP injection at a constant volume of 10 ml/kg body weight. All mice in the experimental and control groups were weighed immediately prior to dose administration and the dose volume was based on individual body weights. Animals were observed after dose administration for clinical signs of chemical effect.
Duration of treatment / exposure:
Observation period: 7 days
Frequency of treatment:
single
Doses / concentrations
Remarks:
Doses / Concentrations:
males: 0, 19, 38, 75 mg/kg bw; females: 0, 29, 58, 115 mg/kg bw
Basis:

No. of animals per sex per dose:
0 mg/kg: 5 males and 5 females per sampling time (24, 48 or 72 hours): control
19 mg/kg: 5 males per sampling time: low test dose
29 mg/kg: 5 females per sampling time: low test dose
38 mg/kg 5 males per sampling time: mid tetst dose
58 mg/kg: 5 females per sampling time: mid test dose
75 mg/kg: 5 males per sampling time: high test dose
115 mg/kg 5 females per sampling time: high test dose

Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: intraperitoneal
- Dose: 30 mg/kg bw
- 5 males and 5 females, 24 hours

Examinations

Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The high dose levels of 75 or 115 mg/kg were calculated to be approximately 80% of the LD50 (7 days) for males or females, respectively (as determined in a pilot study).

SAMPLING TIMES:
24, 48 and 72 hours

DETAILS OF SLIDE PREPARATION:
At the scheduled sacrifice time, if available, five mice per sex were sacrificed by CO2 asphyxiation. Immediately following sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a capped centrifuge tube containing approximately 1 ml fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended by aspiration with a capillary pipet and a small drop of bone marrow suspension was spread onto a clean glass slide. Two to four slides were prepared from each animal. The slides were fixed in methanol, stained with May-Gruenwald-Giemsa and permanently mounted.

METHOD OF ANALYSIS:
Slides were coded using a random number table by an individual not involved with the scoring process. Using medium magnification, an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion, 1000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normocytes in the field of 1000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes counted was also recorded.
Evaluation criteria:
The incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each animal and treatment group. All analyses were performed separately for each sex. In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group. The test article was considered negative if no statistically significant increase in micronucleated polychromatic erythrocytes above the concurrent control was observed at any sampling time. The test article was considered to induce a positive response if a treatment-related increase in micronucleated polychromatic erythrocytes was observed relative to the vehicle control (P<=0.05). The positive response must be dose-dependent or must be observed at a single dose level at adjacent sacrifice times. If a single treatment group is significantly elevated at one sacrifice time with no evidence of a dose-response, the assay is considered a suspect or unconfirmed positive and a repeat assay will be recommended.
Statistics:
Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Mortality occured at the high dose level in both male and female mice: 5/10, 8/10 and 6/10 animals were found dead in the 24, 48, and 72 hour sacrifices, respectively. Clinical signs of toxicity included lethargy and irregular breathing in males receiving 75 mg/kg and irregular breathing, crusty eyes, piloerection, ataxia and lethargy in females receiving 115 mg/kg. No reduction in the ratio of polychromatic erythrocytes (PCE) to total erythrocytes was observed in the test article-treated groups relative to the vehicle control suggesting that the TS did not induce bone marrow toxicity. No significant increases in micronucleated polychromatic erythrocytes were observed.

Any other information on results incl. tables

Micronucleated polychromatic erythrocytes (PCE’s) regarding 24, 48 and 72 h sacrifices:

 

Dose (mg/kg)

Mean number per 1000 PCE’s

males

females

Solvent control

 

0

0.0-0.2

0.0-0.4

Treatments

Low test dose

19

0.0

 

29

 

0.0-0.2

Mid test dose

38

0.0-0.6

 

58

 

0.2-1.0

High test dose

75

0.0-0.3

 

115

 

0.0

Positive control (24 hours)

 

30

17.2

16.8

Result

 

 

Negative

Negative

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of this study, the test substance did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.
Executive summary:

The study was conducted according to OECD Guideline 474, GLP standards were fulfilled.

Male ICR mice were exposed to 19, 38, or 75 mg/kg and female ICR mice to 29, 58, or 115 mg/kg of the test substance which was administered in a total volume of 10 ml/kg as a single i.p. injection. The high dose levels of 75 or 115 mg/kg were calculated to be approximately 80% of the LD50 (7 days) for males or females, respectively. The vehicle used to prepare test substance dosing stocks was deionized water. Mortality occured at the high dose level in both male and female mice: 5/10, 8/10 and 6/10 animals were found dead in the 24, 48, and 72 hour sacrifices, respectively. Clinical signs of toxicity included lethargy and irregular breathing in males receiving 75 mg/kg and irregular breathing, crusty eyes, piloerection, ataxia and lethargy in females receiving 115 mg/kg. No reduction in the ratio of polychromatic erythrocytes (PCE) to total erythrocytes was observed in the test article-treated groups relative to the vehicle control suggesting that the test substance did not induce bone marrow toxicity. No significant increases in micronucleated polychromatic erythrocytes were observed. The negative and positive controls fulfilled the requirements for determination of a valid test.The results of the study indicate that the test substance did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female ICR mice.

Conclusions: Under the conditions of this study, the test substance did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the micronucleus test using male and female ICR mice.