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:
15. Nov 2005 - 16. Feb 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform GLP study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Directive 2000/32/EC, Annex 4C, May 19, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan WInkelmann GmbH, Borchen, Germany
- Age at study initiation: 5-8 weeks (males), 7-11 weeks (females)
- Weight at study initiation: 36 males (for NMT) 37.86 +/- 3.04 g; 9 males (for blood plasma analysis) 36.62 +/- 1.59 g; 36 females (for MNT) 28.3 +/- 2.00 g
- Assigned to test groups randomly: yes
- Housing: singly in Makrolon Type I cages with wire mesh top
- Diet (e.g. ad libitum): pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: min. 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 24-70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: Nov 15 To: Dec 15 2005

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

On the day of the experiment, the test item was formulated in corn oil. The vehicle was chosen to its relative non-toxcity for the animals. All animals received a single standard volume of 10 mL/kg bw.
Duration of treatment / exposure:
single oral application
Frequency of treatment:
once
Post exposure period:
24 and 48 h
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA, 40 mg/kg bw

Examinations

Tissues and cell types examined:
In order to quantify the concentration of the test item in blood plasma 3 additional males were treated with the test item orally. The highest dose (and one untreated group) to be investigated in the micronucleus assay was applied. 1 and 4 hours respectively, after the treatment, the blood of the animals was collected in heparinised tubes, and following the animals were sacrificed. The blood was centifuged, the plasma was isolated and stored at -80°C for one year after the finalisation of the report.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reporducibly or 2000 mg/kg bw as the upper limti for non-toxic test items. Zhe maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 h. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose levels spaced by factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animals (including the controls) were weighed and the individual volume to be administered was adjusted to the animals body weight. The animals received the test item, the vehicle ot the positive control substance once. Twelve animals, six males and six females, were treated per dose group and sampling time. The animals of all dose groups were examined for acute toxic symptoms at intervals of around 1h, 2-4h, 6h and 24 h after administration of the test item. Sampling of the bone marrow was done 24 and 48 h after treatment, respectively.

DETAILS OF SLIDE PREPARATION:
The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm for 10 min. and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald/Giemsa. Cover slips
were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animals for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrycytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.
Ten animals per test group were evaluated as described.
Evaluation criteria:
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single does group. Statistical methods were used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
nonparametric Mann-Whtney test

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
During the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.
Executive summary:

The test item was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test item was formulated in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 and 48 h after a signle administration of the test item the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males and 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 PCE per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erytthrycytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

The following dose levels of the test item were investigated:

24 h preparation interval: 500, 1000 and 2000 mg/kg bw

48 h preparation interval: 2000 mg/kg bw

As estimated by a pre-experiment 2000 mg/kg bw was suitable.

The mean number of PCE was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicatingf that the test item did not have any cytotoxic properties in the bone marrow.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after treatment with the test item. The mean values of micronuclei observed after treatment were below or near to the value of the vehicle control group.

40 mg/kg be cyclophosphmide administered orally was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimetal conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.