Registration Dossier

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: Guideline study (OECD 474); GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
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): Phthalide
- Physical state: Solid, white
- Analytical purity: 100%
- Lot/batch No.: S12233-025
- Stability under test conditions: The stability of the test substance under storage conditions throughout the study period is guaranteed.
- Storage condition of test material: room temperature

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, Germany.
- Age at study initiation: 5 - 8 weeks.
- Weight at study initiation: 27 g (mean).
- Assigned to test groups randomly: yes, under following basis: plan prepared with an appropriate computer program.
- Housing: individually in Makrolon cages.
- Diet: Standardized pelleted feed (Maus/Rafte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 5 days


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


Administration / exposure

Route of administration:
oral: gavage
Vehicle:
DMSO is used up to 4 ml/kg body weight only, the DMSO solution was filled up to 10 ml/kg body weight with olive oil and mixed thoroughly.
Duration of treatment / exposure:
The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment.
Frequency of treatment:
single application.
Post exposure period:
24 hours and 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
187.5, 375.0 and 750.0 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPP) / Vincristine Sulphate (VCR)
- Route of administration: CPP: orally/ VCR: intraperitoneally
- Doses / concentrations: CPP: 20 mg / VCR: 0.15 mg

Examinations

Tissues and cell types examined:
bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Pretests were performed

DETAILS OF SLIDE PREPARATION:
The bone marrow was prepared according to the method described by SCHMID, W. and SALAMONE, M. et al.
• The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
• After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was at 37°C (about 2 mL/femur).
• The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS.
• 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

• The slides were stained in eosin and methylene blue (modified 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 Giemsa solution (15 mL Giemsa, 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:
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 statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
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 were 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/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
poor general state
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes

Any other information on results incl. tables

Summary of Micronucleus Test Results:

Dose: mg/kg bw Total No. PCE´s NCE´s/PCE´s

Micronuclei in PCE´s (‰)

Micronuclei in NCE´s (‰)

 
DMSO (24 h) 20000 7161 1.6 0.8
DMSO (48 h) 20000 10024 1.9 1.6
187.5 (24 h) 20000 7084 2.5* 2.3
375 (48 h) 20000 6441 2.1 1.6
750 (24 h) 20000 6930 1.9 0.7
750 (48 h) 20000 11208 2.2 1.4  
20 mg/kg bw Cyclophosphamide (24 h)  10000 3923 16.9** 0.8  
0.15 mg/kg bw Vincristine (24 h)  10000 5030 58.7** 1.4  

WILCOXON TEST (ONE-SIDED) : *: p <= 0 .05, **: p <= 0.0 1

According to the results of the present study, there are thus no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the vehicle control and the 3 dose groups (187.5 mg/kg, 375.0 mg/kg and 750.0 mg/kg) or between the two sacrifice intervals (24 and 48 hours).

 

The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) or large micronuclei (d > D/4) did not deviate from the vehicle control value at any of the sacrifice intervals and was within the historical control range. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected .

 

The statistically significance (p< 0.05) observed at the lowest dose of 187.5 mg/kg body weight after a sacrifice interval of 24 hours is without any biological relevance; a value of 2.5‰ micronucleated polychromatic erythrocytes is well within the historical control range and there is not any dose-response relationship.

Under the experimental conditions chosen here, the test substance phthalide did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei, thus has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Applicant's summary and conclusion