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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): DIMETHYLBENZYLIDENECYCLOPENTANONE 95%
- Physical state: Solid (powder), white
- Analytical purity: 96.6%
- Lot/batch No.: COD-000462
- Storage condition of test material: Room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Germany GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: ca. 29 g
- Assigned to test groups randomly: yes, under following basis: according to a randomization plan prepared with an appropriate computer program.
- Housing: Makrolon cages, type Ml
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): Drinking water from bottles
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
In comparison to other commonly used vehicles (e.g. water, DMSO, corn oil, etc.) an aqueous CMC formulation is the most suitable. Therefore, an aqueous CMC formulation (0.5%) was selected as the vehicle.
Details on exposure:
The substance to be administered per kg body weight was suspended in a 0.5% CMC formulation. To achieve homogeneity of the test substance in the vehicle, the test substance preparation was stirred with an ultraturrax. All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
two adminstrations with a 24-hour interval between administrations
Frequency of treatment:
twice
Post exposure period:
24 h after the 2nd administration
Doses / concentrations
Remarks:
Doses / Concentrations:
500, 1000 and 2000 mg/kg bw
Basis:
analytical conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
1 x 20 mg cyclophosphamide (CPP, for clastogenic effects) and 1 x 0. 15 mg vincristine (VCR, for aneugenic effects); both in 10 mL test solution

Examinations

Tissues and cell types examined:
bone marrow: polychromatic erythrocytes
Details of tissue and slide preparation:
Preparation of the bone marrow
The bone marrow was prepared according to the method described by SCHMID, W. (1976, 1977) and SALAMONE, M. et al. (1980).

Stainling of the slides
- The slides were stained in eosin and methylene blue (modifled May-Gruenwald solution or Wrights solution) for about 5 minutes.
- After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
- Subsequently, the slides were stained in Giemnsa solution (15 mL Giemnsa, 185 mL purified water) for about 15 minutes.
- After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.
Evaluation criteria:
Acceptance criteria
The mouse micronucleus test is considered valid if the following criteria are met:
- The quality of the slides must allow the identification and evaluation of a sufficient number of analyzable cells, i.e. >= 2000 PCEs and a clear differentiation between PCEs and NCEs.
- The ratio of PCEs/NCEs in the untreated animals (negative control) has to be within the normal range for the animal strain selected.
- The number of cells containing micronuclei in negative control animals has to be within the range of the historical control data both for PCEs and for NCEs.
- The two positive control substances have to induce a significant increase in the number of PCEs containing small and large micronuclei within the range of the historical control data or above.

Assessment criteria
A finding is considered positive if the following criteria are met:
- Significant and dose-related increase in the number of PCEs containing micronuclei.
- The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met:
- The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal was used as a criterion for the rank determination for the U test. Significances were identified as follows:
* p <= 0.05
** p <= 0.01

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
CLINICAL EXAMINATIONS
The two oral administrations of the vehicle in a volume of 20 mL/kg body weight were tolerated by all animais without any signs or symptoms.
The administration of the test substance did not led to any clinical signs of toxicity.
Neither the single administration of the positive control substance, cyclophosphamide, in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0. 15 mg/kg body weight caused any evident signs of toxicity.

Any other information on results incl. tables

Result summary table

Substance Dose (µg/mL) harvest (h) MN in PCEs (%) MN in PCEs (%) Cells with MN <D/4 (%) Cells with MN >D/4 (%)
vehicle 0.5% CMC 24 0.9 1.3 0.9 0.0
test substance 500 24 1.9* 0.3 1.9* 0.0
  1000 24 1.0 0.8 1.0 0.0
  2000 24 1.3 2.0 1.3 0.0
positive ctrl CPP: 20 24 14.8** 0.4 14.8** 0.0
  VCR: 0.15 24 51.1** 1.3 41.5** 9.6**

* p <= 0.05

** p <= 0.01

Applicant's summary and conclusion