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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:
09 July 2009 - 06 September 2009
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Reference substance name:
SPP100 C2
IUPAC Name:
SPP100 C2
Constituent 2
Reference substance name:
173336-76-0
EC Number:
605-674-5
Cas Number:
173336-76-0
IUPAC Name:
173336-76-0
Details on test material:
Molecular formula C11H15BrO3
Molecular weight 275.14
CAS Number 173336-76-0
Description Nacre solid (determined at NOTOX)
Batch OP 07004
Purity 100%
Test substance storage At room temperature in the dark
Stability under storage conditions Stable
Expiry date 27 February 2010

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 32 - 36 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: - Housing: 5 animals per sex per cage, cages type MII height: 14 cm containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 - 22.4
- Humidity (%): 45 - 80
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hour light/12 hour dark cycle. Due to cleaning procedures or performance of functional observations in the room, temporary deviations from the light/dark cycle (with a maximum of 1 hour) and the maximum level for humidity (with max. 10%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used:corn oil (Roth, Karlsruhe, Germany)
- Justification for choice of solvent/vehicle: Test substance dissolved in corn oil. Corn oil is accepted by international guidelines
Duration of treatment / exposure:
Highest dose level: 24 and 48 hours
Solvent control, low and mid dose: 24 hours
Positive control: 48 hours
Frequency of treatment:
Once
Post exposure period:
none
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
250 mg/kg body weight
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
125 mg/kg body weight
Basis:
nominal conc.
No. of animals per sex per dose:
5 male mice were treated per sampling time in each treatment group
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide (CP; CAS no. 50-18-0; Endoxan, Asta-Werke, Germany) dissolved in physiological saline (B. Braun, Melsungen AG, Germany)

- Justification for choice of positive control(s): Accepted and approved by authrities and international guidelines
- Route of administration: intraperitoneal injection
- Doses / concentrations: 40 mg/kg body weight

Examinations

Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal. The staining was based on Giemsa.

METHOD OF ANALYSIS: Slides were scored at a magnification of 1000 x. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.
Evaluation criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
b) The positive control substance induced a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range
Statistics:
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals was above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals was within the historical control data range.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:1000, 750 and 500 mg/kg body weight

- Clinical signs of toxicity in test animals:

1000 mg SPP100 C2 per kilogram body weight (one male and one female): lethargy, ventral recumbency, no reaction to a stimulus and a low body temperature.

750 mg/kg body weight (one male and one female): lethargy, ataxia, ventral recumbency, no reaction to a stimulus and closed eyes.

500 mg/kg body weight (three males and three females, within 1.5 hour after dosing): lethargy, ataxia, hunched posture (1 male, 1 female) and rough coat (1 female). Within 4 hours after treatment: lethargy, ataxia, hunched posture and rough coat (3 males, 2 females).
Within 21 hours after dosing all animals had recovered from the treatment.


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of SPP100 C2 treated animals compared to the vehicle treated animals.
- Ratio of PCE/NCE (for Micronucleus assay): The animals of the groups, which were treated with SPP100 C2 showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this test substance on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with SPP100 C2.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range. Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

It is concluded that this test is valid and that SPP100 C2 is not clastogenic or aneugenic in the micronucleus test under the experimental conditions described.