[No public or meaningful name is available]

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 10 June 2005; Experiment start date - 14 June 2005; Experiment end date - 01 August 2005; Study completion date - 06 October 2005.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

FAT40819/A TE

IUPAC Name:

FAT40819/A TE

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): FAT 40819/A
- Purity: Approx. 77%
- Lot/batch No.: Red ROE 420 BOP 01/04
- Expiration date: 2 November 2009
- Stability: At room temperature 7 days
- Stability in solvent: In saline, polyethylene glycol, carboxymethylcellulose at room temperature 7 days and in vaseline and FCA at room temperature 1 day
- Storage conditions: At room temperature, in the desiccator

Specific details on test material used for the study:

Identity: FAT 40819/A
Description: Red brown powder
Batch number: Red ROE 420 BOP 01/04
Purity: approx. 77 %
Stability of test item: Stable under storage condition
Expiry date: 02 November 2009
Stability of test item dilution: Stable in PEG 300 for at least 7 days at room temperature
Storage conditions: At room temperature.

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, D-33178, Borchen
- Age at start acclimatization: 8-10 weeks
- Weight at start of treatment: Males 40.0 g (SD ± 3.4 g), Females: 31.5 g (SD ± 1.6 g)
- Assigned to test groups randomly: yes
- Housing: Individually, in Makrolon Type I cages, with wire mesh top (EHRET GmbH, D-79302 Emmendingen) with granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen).
- Diet: Pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen).
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Roßdorf)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Deionised water. The vehicle was chosen to its relative non-toxicity for the animals.
Volume: 20 mL/kg bw

Details on exposure:

On the day of the experiment, the test item was formulated.

Frequency of treatment:

Single treatment

Post exposure period:

24 hours for all doses, 48 hours for the 2000 mg/kg bw dose group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Negative control

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Mid dose

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide;
- Route of administration: orally
- Doses: 40 mg/kg bw
- Volume administrated: 10 mL/kg bw

Examinations

Tissues and cell types examined:

Normochromatic and polychromatic erythrocytes

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 around 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.
- The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
- Three adequate spaced dose levels spaced by a 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.

DETAILS OF SLIDE PREPARATION:
- The animals were sacrificed using CO2 following by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal 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 resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-64293 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-79110 Freiburg). 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 animal for micronuclei. To describe 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. Ten animals (5 males, 5 females) per test group were evaluated as described.

Evaluation criteria:

The study was considered valid as the following criteria are met: the negative controls are in the range of our historical control data; the positive controls are in the range of our historical control data; at least 4 animals per group and sex can be evaluated; PCE to erythrocyte ratio should not be less than 20 % of the negative control.

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. 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:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

The test substance did not induce micronuclei in bone marrow cells of the mouse.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test, tested according to OECD guideline 474, 6 NMRI mice per sex were treated once by oral gavage with the test substance (500, 1000, 2000 mg/kg bw) dissolved in water followed by a 24 or 48 hours post exposure period. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei and at least 2000 polychromatic erythrocytes (PCEs) per animal were scored. The mean number of polychromatic erythrocytes was not decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that the test substance had no cytotoxic properties in the bone marrow. The analysis of the plasma samples of the animals treated with 2000 mg/kg b.w., showed, that significant levels (38.8 - 67.7 µg/mL) of the test item could be detected in the plasma 1 hour after treatment. The level dropped to 2.1 - 4.1 µg/mL at 4 hours sampling interval. Thus, the bioaviability of the test item could be confirmed. 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 with the test substance were below or near to the value of the vehicle control group. In conclusion, it can be stated that 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.