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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 7, 1989 - October 16, 1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
other: micronucleus assay in vivo

Test material

Constituent 1
Reference substance name:
O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate
EC Number:
406-940-1
EC Name:
O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate
Cas Number:
126019-82-7
Molecular formula:
C30-54H47-87O3PS
IUPAC Name:
O,O,O-tris(2(or 4)-C9-10-isoalkylphenyl) phosphorothioate
Details on test material:
- Physical state: viscous liquid, insoluble in water; pH 7
- Storage condition of test material: room temperature
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

Test animals

Species:
hamster, Chinese
Strain:
other: Cricetulus griseus
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: CIBA-GEIGY Tierfarm, Sisseln, Switzerland
- Weight at study initiation: (tolerability test) females: 26-30 g, males: 26-33 g; (mutagenicity test) females: 24-33 g, males: 24-35 g
- Housing: induvidually caged
- Diet: standard diet (NAFAG No. 924)
- Water: ad libitum (tap water)
- Acclimation period: at least 3 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 43-49
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: no data
Maximum dosing volume: 10 mL/kg bw
Duration of treatment / exposure:
The animals of the test group and the negative control group were sacrificed 16, 24 and 48 h after application.
Frequency of treatment:
single application
Post exposure period:
16, 24 and 48 hours
Doses / concentrations
Dose / conc.:
5 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
8/sex/dose/sacrifice time
Control animals:
yes, concurrent vehicle
Positive control(s):
- Cyclophosphamide in arachis oil
- Justification for choice of positive control: Applied dose yielded an average of 1.38 % polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.17 %) treated with the vehicle (arachis oil) alone.
- Route of administration: per os
- Doses / concentrations: 64 mg/kg bw cyclophosphamide
- Sacrificed: 24 h after administration
- Dosing volume: 10 mL/kg bw
- Vehicle: arachis oil

Examinations

Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on preliminary toxicity test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sacrifice time: 16, 24 and 48 h after treatment

DETAILS OF SLIDE PREPARATION:
Bone marrow is harvested from the shafts of both femurs with fetal calf serum. After centrifugation small drops of the sediment mixture are transferred on the end of a slide, spread out with the aid of a polished cover glass and the preparations are air-dried. Within 24 hours, the slides are stained in undiluted May-Gruenwald solution for 3 min then in May-Gruenwald solution/water 1/1 for 2 min. After being rinsed in distilled water, the slides are left immersed in diluted Giemsa solution (16.6%), for 10 min. After rinsing with distilled water and air-drying, the slides are cleared in Xylene and mounted.

METHOD OF ANALYSIS:
No. of animals: 5 sex/dose/sacrifice time
1000 polychromatic erythrocytes per animal each are scored for the incidence of micronuclei.
Cytotoxicity: (mitotic activity) ratio PCE/NCE, performed in 1000 erythrocytes
Evaluation criteria:
A test substance is considered to be active in this test system if a statistically significant increase in the number of polychromatic erythrocytes with micronuclei in comparison with the negative control occurs at any sampling time.
Statistics:
The significance of difference is assessed by x²-test.

Results and discussion

Test results
Key result
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:
PRELIMINARY TOXICITY TEST:
- No. of animals/dose/sex: 2
- Doses: 5000, 1000, 200 mg/kg bw
- Result: no mortality or signs of toxicity were obseved up to 5000 mg/kg bw. Hence, 5000 mg/kg bw was determined as the highest applicable dose in the mutagenicity assay.

RESULTS OF DEFINITIVE STUDY: see attachment

Any other information on results incl. tables

There was no significant increase in the number of micronucleated polychromatic erythrocytes in the animals treated with the dose of 5000 mg/kg of test material as compared with the negative control animals at all three sampling times. By contrast, the positive control (cyclophosphamide, 64 mg/kg, sampling time 24 hours) yielded a marked increase of the percentage of micronucleated cells.

Applicant's summary and conclusion

Conclusions:
It is concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test item.
Executive summary:

A GLP-compliant Mammalian Erythrocyte Micronucleus Test was performed folloing OECD guideline 474 to evaluate any mutagenic effect on polychromatic erythrocytes in bone marrow cells in vivo.In this study the animals were treated once with the highest applicable dose of 5000 mg/kg by gavagge and sacrificed 16, 24 and 48 hours thereafter. From the bone marrow smears were made. The bone marrow smears showed no statistically significant increase (p >0.05) in the number of micronucleated polychromatic erythrocytes in comparison with the negative control animals at all three sampling times. The respective "positive control" experiment with cyclophosphamide (64 mg/kg) yielded an average of 1.38 % polychromatic erythrocytes with micronuclei. This is significantly different from the controls (0.17 %) 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 test item.

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