Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 15, 2013 - February 15, 2013

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
GLP compliance:
yes (incl. certificate)
Remarks:
Harlan Cytotest Cell Research GmbH (Harlan CCR), In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid/colorless, clear
- Storage condition of test material: ambient (room temperature)/ under light exclusion

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: pre-experiment: 8 – 9 weeks, main experiment: 9 - 10 weeks
- Weight at study initiation: mean value 36.7 g
- Assigned to test groups randomly: yes
- Housing: single in Makrolon Type II/III cages with wire mesh top(EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: pelleted standard diet, ad libitum, (Harlan Laboratories B.V.; Postbus 6174; 5960 AD Horst; The Netherlands)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 45 - 65 %
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
- Amount of vehicle: 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was dissolved in corn oil.
Duration of treatment / exposure:
single treatment
Frequency of treatment:
once
Post exposure period:
24 h preparation interval: 500, 1000, and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw
Doses / concentrationsopen allclose all
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Seven males were assigned to each test group (except the vehicle and positive control groups with five animals each).
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA; cyclophosphamide
- Route of administration: orally, once
- Volume administered: 10 mL/kg bw
- Doses / concentrations: 40 mg/kg bw

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration. The animals were treated orally with the test item and examined for acute toxic symptoms at intervals of approximately 0-1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three adequately spaced dose levels spaced by a factor of 2 were administered (500, 1000 and 2000 mg/kg), and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment. Since no gender specific differences were observed in the pre-experiment, the main study was performed using males only in accordance with the test guidelines.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed using CO2 followed by bleeding. 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 (390 x g) for 10 minutes 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 (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). 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. Per animal 2000 polychromatic erythrocytes (PCE) were analysed for micronuclei. To investigate a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

OTHER:
The animals of all dose groups, except the positive control were examined for clinical signs at intervals of around 0-1 h, 2 - 4 h, 6 h, 24 h, and/or 48 h after administration of the test item and vehicles.
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 dose group. Statistical methods are used as an aid in evaluating the results, if necessary. 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-Whitney test

Results and discussion

Test results
Sex:
male
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 mg/kg
- Clinical signs of toxicity in test animals: Reduction of spontaneous activity, Eyelid closure, Ruffled fur, Tumbling

RESULTS OF DEFINITIVE STUDY
In the main experiment clinical signs after treatment with the test item comprised ruffled fur (4/7 mice at 1000 mg/kg bw, 14/14 at 2000 mg/kg bw), reduction of spontaneous activity (3/7 mice at 1000 mg/kg bw and 11/14 at 2000 mg/kg bw) as well as tumbling (5/14 mice at 2000 mg/kg bw) No clinical symptoms were observed at 500 mg/kg bw.
After treatment with the test item at the 24 h and 48h preparation interval the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test article did not induce cytotoxic effects 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 administration of the test item. The mean values of micronuclei observed after treatment were below to the value of the vehicle control group and well within the historical control data range. The positive control showed a statistically significant increase of induced micronucleus frequency.

Any other information on results incl. tables

Summary of Micronucleus Test Results

Test group Dose(mg/kg bw) Sampling time (h) PCEs with micronuclei (%) Range PCE per 2000 erythrocytes
Vehicle control 0 24 0.24 2 - 9 1212
Test item 500 24 0.143 1 - 5 1286
Test item 1000 24 0.121 0 - 8 1243
Test item 2000 24 0.214 1 - 9 1210
Positive control 40 24 2.7 33 - 86 1200
Vehicle control 0 48 0.15 2 - 4 1212
Test item 2000 48 0.114 0 - 5 1202

Biometry

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Vehicle control versus test group Significance p
500  mg/kg bw; 24 h n.t. -
1000 mg/kg bw; 24 h n.t. -
2000 mg/kg bw; 24 h n.t. -
40 mg CPA/kg bw; 24 h + 0.004
2000 mg/kg bw; 48 h n.t. -

+ = significant;

n.t = not tested, as the mean micronucleus frequency was not above the vehicle control value

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse and is therefore considered to be non-mutagenic in this micronucleus assay.
Executive summary:

A GLP-compliant study following OECD guideline 474 was performed to investigate the potential of the test article to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test item was dissolved in corn oil, which was also used as vehicle control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.

Seven males per test group (except the vehicle and positive control groups with 5 males only) were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To investigate a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes 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, based on results of a pre-experiment:

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

48 h preparation interval: 2000 mg/kg bw

In the main experiment clinical signs after treatment with the test item comprised ruffled fur (4/7 mice at 1000 mg/kg bw, 14/14 at 2000 mg/kg bw), reduction of spontaneous activity (3/7 mice at 1000 mg/kg bw and 11/14 at 2000 mg/kg bw) as well as tumbling (5/14 mice at 2000 mg/kg bw) No clinical symptoms were observed at 500 mg/kg bw. After treatment with the test item the number of PCEs per 2000 erythrocytes was not substantially decreased as compared to the mean value of PCEs per 2000 erythrocytes of the vehicle control thus indicating that the test substance did not exert a cytotoxic effect in the bone marrow. In comparison to the corresponding vehicle controls there was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test item and with any dose level used. The mean values of micronuclei observed after treatment were below to the value of the vehicle control group and well within the historical control data range. 40 mg/kg bw cyclophosphamide administered orally was used as positive control which showed a substantial increase of induced micronucleus frequency. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test article is considered to be non-mutagenic in this micronucleus assay.