Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1985

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: EEC Directive 79/831, Annex V, Method No. 431
Deviations:
no
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
This is formulation of 49.2% of the test substance in water.
Specific details on test material used for the study:
- Analytical purity: 80.2% w/w s. in ethanol solution
- Impurities (identity and concentrations): 0.2% free amine and 0.1% amine hydrochloride
- Lot/batch No.: L- 5383
- pH (1% water): 6.3

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
Test animals
- Source: Breeders were purchased from G1. Bomholtgard Ltd., but the mice used were born in the Scantox Laboratories
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 25-30g
- Assigned to test groups randomly: Yes, in groups of 5
- Fasting period before study: No
- Housing: 5 animals/cage, males and females separately in type III Macrolone cages, bedding used was special softwood sawdust "Spanvall Special White " from Spanvall Ltd., DK-4535 Vallekilde
- Diet: Complete rodent diet "Altromin 1314" from Chr. Petersen Ltd., DK-4100 Ringsted, ad libitum
- Water: Drinking water adjusted to pH 2.5 with hydrochloric acid, ad libitum
- Acclimation period:

Environmental conditions
- Temperature: 21 ±2°C
- Humidity: 55 ± 15%
- Air changes: 10/h
- Photoperiod: 12h dark /12h light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: Distilled water
- Concentration of test material in vehicle: 400 mg/10 mL distilled water for the dose level of 400 mg/kg bw.
- Amount of vehicle: The vehicle was administered orally at a volume of 10 mL/kg bw.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dilution of the test substance in distilled water

Frequency of treatment:
Once
Post exposure period:
24, 48 and 72 h


Doses / concentrations
Dose / conc.:
400 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (positive control):
- Route of administration: Oral
- Doses / concentrations: 30 mg/10 mL equivalent to 30 mg/kg bw


Examinations

Tissues and cell types examined:
Bone marrow erythrocytes




Details of tissue and slide preparation:
Dose selection:
Preliminary investigations:
- A few mice were treated orally with various concentrations of the test substance diluted with distilled water. Thereby the maximum tolerated dose was estimated at 400 mg/kg bw. At this dosage bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).

Details of slide preparations: Immediately after sacrifice, femurs of a mouse were dissected free of muscle, and by a 1 mL syringe with needle the bone marrow was flushed out into 5 mL of fetal calf serum. After thorough shaking, the mixture was centrifuged for 10 min. at about 1000 rpm. Thereafter, smears were made after removal of the supernatant. The specimens were fixed in methanol and stained with May-Grunwald/Giemsa.

Method of analysis: Prior to microscopic assessment, all slides were furnished with code numbers, so that the counting was blind.
The following counts were made:
Number of normochromatic erythrocytes (NCE) per 1000 erythrocytes
Number of polychromatic erythrocytes (PCE) per 1000 erythrocytes
Number of micronuclei (MN) in 1000 normochromatic erythrocytes
Number of micronuclei (MN) in 1000 polychromatic erythrocytes.
Evaluation criteria:
Increase in the frequency of micronucleated polychromatic erythrocytes in treated animals as compared to controls


Statistics:
The statistical difference was analysed by one-way ANOVA. In the case of PCE (%) the test was performed on the values observed, and for the MN (per thousand) the test was done on computed rank values transformed to normal scores according to Blom's method (Blom, 1958).

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
one mortality in the 72h test group on Day 3 after treatment
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results of the dose-range finding study
- Dose range: Up to 400 mg/kg bw
- Clinical signs of toxicity in test animals: Maximum tolerated dose was estimated at 400 mg/kg bw. At a dose exceeding 400 mg/kg bw, the mortality was too high.
- Evidence of cytotoxicity in tissue analyzed: At 400 mg/kg bw, bone marrow smears showed a reduced number of polychromatic erythrocytes (PCE) as compared with normochromatic erythrocytes (NCE).
Results of the main test
- Induction of micronuclei: No significant difference as compared to controls.
- Appropriateness of dose levels and route: Yes
- Statistical evaluation: Yes

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice.
Executive summary:

A study was conducted to determine the in vivo toxicity of the test substance according to OECD Guideline 474, in compliance with GLP. This study was performed to evaluate the chromosome-damaging effect of the test substance in mice. The experimental animals were 50 NMRI mice, divided into 5 groups. Of the 5 groups, three were test groups, one negative control group and one positive control group. The test groups were treated with 400 mg test substance/kg bw, the negative control group with distilled water and the positive control group with 30 mg cyclophosphamide/kg bw. The mice were killed 24, 48 and 72 h, respectively after treatment. From bone marrow smears micronucleus counts were made per 1000 polychromatic erythrocytes. Under the test conditions, the test substance did not induce an increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood samples from both male and female mice (Kallesen, 1985).