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:
13.05. - 29.07.1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
no
Remarks:
but performed under GLP-like quality control with QAU statement included.
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Appearance: yellow liquid
- Storage condition of test material: room temperature

Test animals

Species:
hamster, Chinese
Strain:
other: random outbred strain
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln
- Age at study initiation: females: 6-10 weeks; males: 4-9 weeks
- Weight at study initiation: females 22-35 g, males 23-35 g (tolerability test), females 28-34 g, males 28-33 g (mutagenicity test)
- Assigned to test groups randomly: no data
- Fasting period before study: no data
- Housing: not specified
- Diet: standard diet NAFAG No.924, ad libitum
- Water: tap water, ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 52-56
- Air changes: no data
- Photoperiod: 12 hrs dark /12 hrs light

IN-LIFE DATES: from 24.02. - 13.05.1986

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: arachis oil
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was formulated in arachis oil.
Duration of treatment / exposure:
Single treatment.
Frequency of treatment:
The animals received the test item, the vehicle or the positive control substance once. The dosing volume was 10 mL/kg bw.
Post exposure period:
16 h, 24 h or 48 h
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
24 at 5000 mg/kg and for negative control (8 per sampling time).
8 for positive control.
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Justification for choice of positive control(s): as recommended by the guideline
- Route of administration: oral gavage
- Doses / concentrations: 64 mg/kg bw in 10 mL/kg bw arachis oil

Examinations

Tissues and cell types examined:
Bone marrow smears from femur.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary test was performed to determine the highest dosage of the test item to be applied in the mutagenicity assay. In this experiment the dose of 5000 mg/kg was determined as the highest applicable in the mutagenicity assay.

TREATMENT AND SAMPLING TIMES
The preparation was administered orally to groups of 24 female and 24 male animals each in the negative and in the 5000 mg/kg bw dose group. The positive control group consisted of 8 female and 8 male animals. Treatment consisted of a single application. 16, 24 and 48h after application 8 female and 8 male animals per sampling time were sacrificed. Bone marrow was harvested from the shafts of both femurs.

DETAILS OF SLIDE PREPARATION:
Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approx. 0.5 µl rat serum the bone marrow was drawn up. In order to receive a homogenous suspension the content of pipette was aspirated gently about three times. Small drops of the homogenous bone marrow suspension in rat serum were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grünwald solution for 2 min then in May-Grünwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approx. 2 min. After rinsing with distilled water and air-drying, the slides were cleared in Xylene and mounted in Eukitt.

METHOD OF ANALYSIS:
The slides of five female and five male animals each of the negative control group and of the dosage group sacrificed at 16, 24 and 48 hours post-treatment were examined. The slides of five female and five male animals of the positive control group were scored at 24 hours post-treatment only. 1000 polychromatic erythrocytes each were scored for the incidence of micronuclei per animal. The ratio of polychromatic to normochromatic erythrocytes was determined for each animal by counting a total of 1000 erythrocytes.
Evaluation criteria:
Not specified.
Statistics:
The significance of difference was assessed by X² -test.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Three groups of four Chinese hamsters (2 females and 2 males) were treated with three different doses, one receiving the maximum dose of 5000 mg/kg bw, and the other two doses of 1/5 and 1/25 of that amount respectively.
- Solubility: no data
- Mortality: no mortality within the observation period of 24, 48 or 72 hours.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used.
- Ratio of PCE/NCE (for Micronucleus assay): After treatment with the test item the number of PCEs was not substantially decreased as compared to the mean value of PCEs of the vehicle control thus indicating that the test item did not exert any cytotoxic effects in the bone marrow.
- Appropriateness of dose levels and route: yes
- Statistical evaluation: No statistically significant differences in the frequency of erythrocytes containing micronuclei between the solvent control and the dose groups were observed.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.
Executive summary:

In a micronucleus test performed under GLP-like quality control, the test article in arachis oil was given by gavage to three groups of 8 male and 8 female Chinese hamsters. The animals were treated once with the highest applicable dose of 5000 mg/kg and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. The negative control animals received the vehicle alone. The bone marrow smears from animals treated with the test substance showed no statistically significant increase (p>0.05) in the number of micronucleated polychromatic erythrocytes compared to the negative control animals at all three sampling times. The respective "positive control" experiments with cyclophosphamide (64 mg/kg) yielded an average of 2.9% polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.06%) treated with the vehicle (arachis oil) alone. It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the limit dose of 5000 mg/kg bw of the test article.