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:
key study
Study period:
September 26, 1988 – October 26, 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test was conducted according to OECD Test Guideline No. 474, 1983, under GLP Standards, and QA. Chemical identity and purity of the test substance are not reported.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
Deviations:
yes
Remarks:
Limit test dosis: 5000 mg/kg bw
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1984
Deviations:
yes
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Remarks:
Statement of Compliance
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 3-[(diphenoxyphosphoryl)oxy]phenyl triphenyl 1,3-phenylene bis(phosphate) and tetraphenyl 1,3-phenylene bis(phosphate)
EC Number:
701-337-2
Cas Number:
not available
Molecular formula:
C30H24O8P2
IUPAC Name:
Reaction mass of 3-[(diphenoxyphosphoryl)oxy]phenyl triphenyl 1,3-phenylene bis(phosphate) and tetraphenyl 1,3-phenylene bis(phosphate)
Details on test material:
- Name of test material (as cited in study report): CR 733-S
- Physical state: No data
- Lot/batch No.: Confidential
- Stability under test conditions: No data
- Storage condition of test material: At room temperature in the dark

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, F.R.G.
- Age at study initiation: Approx. 8 weeks
- Weight at study initiation: Males: 28.0 to 31.8 g; Females: 21.7 to 23.4 g
- Assigned to test groups randomly: yes
- Fasting period before study: Feed was withheld overnight before dosing until about 4 hours after administration of the test substance.
- Housing: In groups of 5 in polycarbonate cages. The bedding material, purified saw-dust (Woody Clean), was obtained from the Broekman Institute, Someren, The Netherlands. The cage with females of the corresponding group was placed next to the cage with males.
- Diet: Standard laboratory animal diet (RMH-B, pellet diameter 10 mm), obtained from Hope Farms, Woerden, The Netherlands, ad libitum.
- Water: Tap-water, ad libitum.
- Acclimation period: Combined quarantine/acclimatisation period of 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2
- Humidity (%): 54 - 72
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: No data

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Concentration of test material in vehicle: (volume) 10 ml/kg bw
Details on exposure:
No data
Duration of treatment / exposure:
24, 48, and 72 hours (acute)
Frequency of treatment:
once (single dose treatment)
Post exposure period:
At 24, 48 and 72 hours after dosing of the test substance and the vehicle, and at 48 hours after dosing of the positive control, the animals were sacrificed by cervical dislocation.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
stomach intubation
No. of animals per sex per dose:
5 males and 5 females per sampling time and treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: orally, gavage
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone-marrow; micronucleated polychromatic erythrocytes, and ratio polychromatic erythrocytes (PCE)/normochromatic erythrocytes (NCE)
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: In a preliminary study 12 animals (3 males and 3 females per group) were dosed orally with 5000 and 2000 mg/kg bw. None of the animals showed any signs of reaction to treatment. Based on the results of this pilot study 5000 mg/kg bw was selected as an appropriate dose for the Micronucleus Test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): three different sampling times (24, 48, 72 hours) after oral administration of a single dose (5000 mg/kg).

DETAILS OF SLIDE PREPARATION: A drop of the cell suspension was placed on a slide which was previously cleaned and marked (with the animal number). The preparations were then air-dried and thereafter fixed for 5 min. in 100% methanol and air-dried overnight. Two slides were prepared per animal. The slides were stained 5 min. in May-Grünwald solution. Thereafter slides were rinsed and stained for 25 min in 5% (v/v) Giemsa solution. The preparations were rinsed for 1 min in running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

METHOD OF ANALYSIS: The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were counted in polychromatic erythrocytes only.
Evaluation criteria:
A test substance is considered positive in the micronucleus test if:
a) It induces a statistically (P < 0.05) as well as biologically significant increase in the frequencies of micronuclei (at any dose or at any sampling time) in the combined data for both sexes, or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test if:
a) None of the tested concentrations or sampling times showed a statistically significant (at P < 0.05) increase in the incidence of micronuclei either in the combined data for both sexes or in the data for male or female groups alone.
Statistics:
The Wilcoxon rank-sum test was used to assess significant differences between the numbers of micronuclei in the treatment and control groups, in which P < 0.05 was used as the lowest level of significance. Averages and standard deviations were calculated.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 and 5000 mg/kg bw
- Clinical signs of toxicity in test animals: None of the animals showed any signs of reaction to treatment.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the frequency of micronuclei was observed.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the treated groups showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of the test substance on the erythropoiesis.
- Appropriateness of dose levels and route: In the preliminary dose range finding study, oral administration of 2000 and 5000 mg kg bw did not cause any effects in male and female mice. 5000 mg kg bw was therefore the regularly limit dose, selected for the main study.
- Statistical evaluation: 5000 mg/kg bw did not show a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes from corresponding control group.

Any other information on results incl. tables

Not relevant

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
As no increase in the frequency of micronuclei was observed, it is concluded that CR 733-S can be considered as not mutagenic in this valid Micronucleus Test, under the experimental conditions described in this report.
Executive summary:

CR 733-5 was tested in the Micronucleus Test in mice, according to OECD guideline 474, 1983. Three groups, each comprising 5 males and 5 females, received a single oral dose of 5000 mg/kg bw. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle (Corn oil) treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of Cyclophosphamide (CP) at 50 mg/kg bw, was harvested at 48 hours after dosing only.

The test substance was found to respond negatively in the Micronucleus Test, as no increase in the frequency of micronuclei was observed, whereas the positive control substance (CP) produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes. It is concluded that CR 733-S can be considered as not mutagenic in the Mouse Micronucleus Test, under the experimental conditions described in this report.