Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1990-05-30 to 1990-10-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report date:
1990

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.11 (Mutagenicity - In Vivo Mammalian Bone-Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
4-(trans-4-propylcyclohexyl)acetophenone
EC Number:
406-700-6
EC Name:
4-(trans-4-propylcyclohexyl)acetophenone
Cas Number:
78531-61-0
Molecular formula:
Hill formula: C17H24O CAS formula: C17H24O
IUPAC Name:
1-[4-(4-propylcyclohexyl)phenyl]ethan-1-one

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 10 weeks
- Weight at study initiation: approx. 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: yes, approx. 18 hrs before treatment
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +/- 3 °C
- Humidity: 30-70 %
- Air changes: not specified
- Photoperiod: 12 / 12 hrs dark / hrs light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
Polyethylenglycol 400
Duration of treatment / exposure:
single gavage application
Frequency of treatment:
once
Post exposure period:
24 or 48 hours
Doses / concentrationsopen allclose all
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
24 hours post-exposure period
Dose / conc.:
670 mg/kg bw/day (actual dose received)
Remarks:
24 hours post-exposure period
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
Remarks:
24 and 48 hours post-exposure period
No. of animals per sex per dose:
5 per sex per dose per post exposure period
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Justification for choice of positive control: as recommended by the guideline
- Route of administration: oral (gavage)
- Doses / concentrations: 20 and 40 mg/kg bw (two groups)

Examinations

Tissues and cell types examined:
bone marrow of femora
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: according to guideline the MTD was applied as high dose

TREATMENT AND SAMPLING TIMES: single treatment followed by either 24 or 48 hours post-treatment period

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 fetal calf serum, using a 5 mL syringe. The cell suspension was centrifuged at 1500 rpm for 5 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-6100 Darmstadt, F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). 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. 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 same sample and expressed in normochromatic erythrocytes per 2000 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 producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the non-parametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Doses producing toxicity: at 2000 mg/kg bw unspecific signs of toxicity were observed but no mortality occurred; slight cytotoxicity (PCE/NCE) was seen at this dose level at 48 h post-treatment evaluation

Observations:
No increase in the micronucleus rate in animals treated with the test item.

Any other information on results incl. tables

Please refer to background material attached.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study the test item was found to be not clastogenic.
Executive summary:

This study was performed to investigate the potential of the test item to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was suspended in polyethylene glycol 400. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw. 24 h and 48 h after a single application of the test article the one marrow cells were collected for micronuclei analysis. Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCE) per animal were scored. 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:

24 h preparation interval: 200, 670 and 2000 mg/kg bw
48 h preparation interval: 2000 mg/kg bw

In a pre-experiment 2000 mg/kg bw was estimated to be suitable. The animals expressed toxic reactions. After treatment with the highest dose of the test article the number of NCEs was slightly increased as compared to the corresponding negative controls thus indicating that the test item had weak cytotoxic effectiveness. In comparison to the corresponding negative controls there was no enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used. 20 mg/kg bw cyclophosphamide administered per gavage 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 with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

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