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:
2007-01-09 to 2007-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of assay:
other: micronucleus assay, chromosome aberration

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
additive
Test material form:
liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Weight at study initiation: within 20 % of the mean
- Fasting period before study: 3-4 h prior to dosing
- Housing: The animals were group housed (5 animals per sex per cage) in labelled polycarbonate cages (type Mil height: 14 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as nest material (Enviro-dri, TecniLab-BMI BV, Someren, The Netherlands).
- Diet: Free access to standard pelleted laboratory animal diet (SM R/M-Z from SSNIFF® SpezialdiSten GmbH, Soest, Germany)
- Water: Free access to tap water.
- Acclimation period: At least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle/solvent used: corn oil
Details on exposure:
Dosing volume: 10 mL/kg bw
Duration of treatment / exposure:
Single oral intubation
Frequency of treatment:
Animals were dosed once
Post exposure period:
24 and 48 h
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
375 mg/kg bw/day (nominal)
Dose / conc.:
750 mg/kg bw/day (nominal)
Dose / conc.:
1 500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5 males per group
Control animals:
yes, concurrent vehicle
Positive control(s):
Yes: Cyclophosphamide
- Route of administration: oral intubation
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow, eryhtrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study.

TREATMENT AND SAMPLING TIMES: Single treatment, Sampling time: 24 and 48 h

DETAILS OF SLIDE PREPARATION: The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether (Merck, Darmstadt, Germany) and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a coverslip.

METHOD OF ANALYSIS: All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. 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. Averages and standard deviations were calculated.
Evaluation criteria:
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, onesided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time)

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000, 1500, 2000 mg/kg bw
- Clinical signs of toxicity in test animals: In the dose range finding test, one male and one female animal dosed with 2000 mg/kg b.w. died within 20 and 44 hours after dosing, respectively. One male and one female animal dosed with 1000 mg/kg b.w. showed no reaction to treatment. Three male and three female mice dosed with 1500 mg/kg b.w. showed the following clinical signs within 1.5 hours after dosing: ataxia (3 male and 2 female animals), lethargy and hunched posture (2 male and 3 female animals). Within 3 hours after dosing all animals were still lethargic, had a hunched posture and two male and one female animal had a rough coat. Within 25 hours after dosing two female animals recovered from the treatment and all males and one female still had a hunched posture. Within 49 hours after dosing all animals had recovered from the treatment, except for one male animal that still had a hunched posture.

Any other information on results incl. tables

The animals of the groups treated with 750 and 375 mg/kg b.w. and the animals of the negative and positive control groups showed no abnormalities.

The following clinical observations were made in the groups treated with 1500 mg test item/kg b.w.

During the first 1.5 hours after dosing all animals of the groups treated with 1500 mg/kg b.w. were lethargic and had a hunched posture. Four animals also showed ataxia.

Within 4 hours after dosing all animals were still lethargic and had a hunched posture. Five animals also had a rough coat and one of.these animals also had showed the following toxic signs: eyes closed, ataxia and ventral recumbency.

Within 21 hours after dosing two animals died. Two animals had a hunched posture and a rough coat. One of these animals also showed the following toxic signs: lethargy, closed eyes, tremors and slow breathing. Six animals recovered from the treatment.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of test item treated animals compared to the vehicle treated animals.

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, the acceptability criteria of the test were met.

The animals of the groups, which were treated with test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound 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.

Within 45 hours after dosing all surviving animals had recovered from the treatment.

Applicant's summary and conclusion

Conclusions:
The test item was tested in the Micronucleus test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. It is concluded that the test item is not clastogenic in the micronucleus test under the experimental conditions described.
Executive summary:

The test item was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect on erythrocytes in bone marrow. The study procedures described in this report were based on the most recent OECD and EEC guidelines. The test substance was dissolved in corn oil. Five male animals were used in each of the six treatment groups, including negative and positive controls. All groups received a single oral intubation. The negative and positive control groups were treated with vehicle and 50 mg/kg body weight (b.w.) of cyclophosphamide (CP), respectively. Animals were dosed with the test item at 1500 (two groups), 750 (one group), and 375 (one group) mg/kg b.w.. Two animals dosed with 1500 mg/kg b.w. died within 21 hours after dosing. All animals dosed with 1500 mg/kg b.w. showed the following toxic signs after dosing: lethargy and hunched posture. Four animals also showed ataxia and five animals had a rough coat. Two animals also had closed eyes. One of these animals had ventral recumbency and the other animal also had tremors and was breathing slow. The animals dosed with 750 and 375 mg/kg b.w. and the control animals showed no abnormalities after dosing. Bone marrow of the groups treated with the test item was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of animals treated with the test item. 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. The groups that were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, which reflects a lack of toxic effects of this compound on the erythropoiesis. The groups that were treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls, demonstrating toxic effects on erythropoiesis. It is concluded that the test item is not clastogenic in the micronucleus test under the experimental conditions described in this report.