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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Remarks:
source of read-across
Adequacy of study:
key study
Study period:
From December 09th, 1997 to January 07th, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
read-across source

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted May 26, 1983
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Reference substance name:
Similar Substance 02
IUPAC Name:
Similar Substance 02

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, CH-4414 Füllinsdorf.
- Age at the acclimatization: 8-12 weeks.
- Weight at study initiation: males mean value 28.8 g (SD ± 2.6 g); females mean value 25.9 g (SD ± 1.8 g).
- Assigned to test groups randomly: yes.
- Fasting period before study: approximately 18 hours before treatment the animals received no food but water ad libitum.
- Housing: single, in Makrolon Type I, with wire mesh top.
- Diet: pelleted standard diet, ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: minimum 5 days.
- Quarantine: the animals were under quarantine in the animal house of testing facility for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 24 - 70 %
- Photoperiod: artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Solvent: on the day of the experiment, the test article was formulated in PEG 400.
- Justification for choice of solvent: the vehicle was chosen due to its relative non-toxicity for the animals.
Details on exposure:
All animals received a single standard volume of 10 ml/kg body weight orally.

Pre-experiment for toxicity
A preliminary study on acute toxicity was performed with a small group of animals 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 article and examined for acute toxic symptoms at intervals of 1 h, 6 h, 24 h, and 48 h after administration of the test article.
Frequency of treatment:
Once and twice at an interval of 24 hours
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
at 24 hours
Dose / conc.:
670 mg/kg bw/day (actual dose received)
Remarks:
at 24 hours
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
at 24 and48 hours
No. of animals per sex per dose:
Six males and six females were assigned to each test group.
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control: cyclophosphamide.

Examinations

Tissues and cell types examined:
Polychromatic erythrocytes (PCE) in the bone marrow.
Details of tissue and slide preparation:
Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.

Preparation of the animals
The animals were sacrificed by cervical dislocation. 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-Griinwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

Analysis of cells
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 normochromatic erythrocytes per 2000 the PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described.
Evaluation criteria:
A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test.
However, both biological and statistical significance should be considered together.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight toxic reactions
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The animals expressed slight toxic reactions.
The mean number of normochromatic erythrocytes was increased after treatment with the test article as compared to the mean value of NCEs of the vehicle controls, indicating that test item had 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 administration of the test article. The mean values of micronuclei observed after treatment with test item were close to the vehicle control value.

POSITIVE CONTROL
40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
The substance is considered to be non-mutagenic in the micronucleus assay.
Executive summary:

The study was performed to investigate the potential of test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in polyethylene glycol 400 (PEG 400). PEG 400 was used as vehicle control. The volume administered orally (gavage) was 10 ml/kg b.w.. 24 h and 48 h after a single administration of the test article the bone marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE.

The following dose levels of the test article were investigated: at 24 h preparation interval 200, 670 and 2000 mg/kg b.w.; at 48 h preparation interval 2000 mg/kg b.w..

The highest guideline-recommended dose (2000 mg/kg) was estimated by a pre-experiment to be suitable. The animals expressed slight toxic reactions. After treatment with the test article the mean number of NCEs was increased as compared to the corresponding vehicle controls thus indicating that test item had cytotoxic effectiveness.

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 after administration of the test article and with any dose level used.

40 mg/kg b.w. cyclophosphamide administered per os was used as positive control which showed a statistically significant increase of induced micronucleus frequency.

Conclusion

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

Therefore, the substance is considered to be non-mutagenic in the micronucleus assay.