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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
None

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: solid
Specific details on test material used for the study:
None

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
None
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation:
- Weight at study initiation: minimum 10 weeks
- Assigned to test groups randomly: yes
- Fasting period before study:
- Housing: placed singly in Makrolon Type I cage, with wire mesh top(ALTROMIN, D-4937 Lage/Lippe, F.R.G.)
- Diet (e.g. ad libitum): pelleted standar diet
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-3
- Humidity (%): relative humidity not regulated

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 4 % carboxynethylcellulose
- Justification for choice of solvent/vehicle: The vehicle was chosen to its nontoxicity for the animals
- Concentration of test material in vehicle: 20 ml/kg b.w.
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test article was suspended in 4 % CMC.
Duration of treatment / exposure:
24, 48 and 72 hours
Frequency of treatment:
once
Post exposure period:
approximately 18 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
no data
No. of animals per sex per dose:
5 males / test
5 females / test
Positive control(s):
CPA; Cyclophaosphamide
- Justification for choice of positive control(s): The stability of CPA at room temperature is good. At 20 °C only 1 % of CPA is hydrolysed per day in aqueous solution.
- Route of administration: orally
- Doses / concentrations: 10 ml/kg bw

Examinations

Tissues and cell types examined:
polychromatic erythrocytes (PCE) per animal were scored for micronuclei.
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 reproducibly. The volume to be administered should be compatible with physiological space available.
The maximum tolearated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 72 hrs.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hrs after treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with 2.0 ml fetal calf serum, using a 5 ml syringe, into 1 ml fetal calf serum. The cell suspension was centrifuged at 1000 rpm for 5 minutes and the supernatent 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-Griiwald/Giemsa. Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G). 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. 1000 polychromatic erythrocytes(PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normocrochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.
Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:
A test article is classified as mutagenic if it induces either a statistically significant dose related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article neither a statistically significant dose related increse in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the non parametric Mann-Whitney test.
Statistics:
Statistical significance at the five percent level (p<0.05) was eveluated by means of the non parametric Mann-Whitney test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
slight toxic reactions
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
None

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:
FAT 40'007/B was considered to be non-clastogenic in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of FAT 40'007/B to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was suspended in 4% carboxymethylcellulose (CMC). This suspending agent was used as negative control. The volume administered orally was 20 ml/kg bw. 24 h, 48 h and 72 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. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

To describe a cytotoxic effect due to the treatement 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 1000 PCE.

The following dose level of the test article was invetigated:

24 h, 48 h and 72 h preparation interval: 5000 mg/kg bw.

In a pre-experiment this dose level was estimated to be the maximum attainable dose. The animals expressed slight toxic reactions. After treatment with the test article the ration between PCEs and NCEs was not affected as compared to the corresponding negative controls thus indicating no cytotoxic effects. In comparison with the corresponding negative controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. An appropriate reference mutagen was used as positive control which showed a distinct increase of induced micronucleus frequency.

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 in the bone marrow cells of the mouse. Therefore, FAT 40'007/B was considered to be non-clastogenic in this micronucleus assay.