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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
Study period:
From March 28 to November 20, 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline No. 474 (1997 version).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

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:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
GLP principles of the German "Chemikaliengesetz" (inspected on May 15 and June 26, 2001/signed on September 24, 2001)
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Clear liquid / colourless - yellowish
- Homogeneity: Homogeneous
- Storage condition of test material: Stored in refrigerator, protected from light.
- Stability: Stability under storage conditions was confirmed by reanalysis

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland GmbH
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 29 g (mean weight)
- Assigned to test groups randomly: yes, using an appropriate computer program.
- Housing: Animals were housed individually in Makrolon cages, type Ml.
- Diet: Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: At least 5 days

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

IN-LIFE DATES: From: March 28, 2003 To: November 20, 2003

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, olive oil was selected as the vehicle, which had been demonstrated to be suitable in the in vivo micronucleus test and for which historical data are available.
- Concentration of test material in vehicle: 25, 50 and 75 mg/mL (main study).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test material was dissolved in olive oil. All test substance formulations were prepared immediately before administration. The stability of the test substance at 4°C in the vehicle over a period of 4 days was verified analytically.

ANALYSIS OF FORMULATION: For the determination of the test substance concentrations in the vehicle, 3 samples of each dose were taken from the test substance preparations, kept at room temperature until the treatment of the last animal (approximately 1 h) and then deep-frozen until they were determined analytically . The determination of the concentrations in the vehicle was carried out by HPLC.

DOSE VOLUME: 10 mL/kg bw
Duration of treatment / exposure:
Single intraperitoneal administration
Frequency of treatment:
Single intraperitoneal administration
Post exposure period:
250 and 500 mg/kg bw: 24 h
750 mg/kg bw: 24 and 48 h
Doses / concentrationsopen allclose all
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Remarks:
actual administered by intraperitoneal injection
Dose / conc.:
500 mg/kg bw/day (actual dose received)
Remarks:
actual administered by intraperitoneal injection
Dose / conc.:
750 mg/kg bw/day (actual dose received)
Remarks:
actual administered by intraperitoneal injection
No. of animals per sex per dose:
Main study: 5 males/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide and vincristine sulphate.
- Justification for choice of positive control(s): Cyclophosphamide and vincristine sulphate are well established reference clastogens and aneugens, respectively.
- Route of administration: Intraperitoneal
- Doses / concentrations: Cyclophosphamide: 20 mg/kg bw; vincristine sulphate: 0.15 mg/kg bw.

Examinations

Tissues and cell types examined:
- 2000 polychromatic erythrocytes (PCEs) were evaluated per animal and investigated for micronuclei (MN).
- The normochromatic erythrocytes (NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded.
- Ratio of PCEs/NCEs.
- Number of small MN (diameter of MN < cell diameter/4) and of large MN (diameter of MN ≥ cell diameter/4).
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
- Dose selection was based on the range-finding test conducted in male and female animals at 500, 750, 1000 and 2000 mg/kg bw. Mortality was observed at 2000 mg/kg bw; evident signs of toxicity observed and some animals were sacrificed moribund at 750 and 1000 mg/kg bw; all animals survived but evident signs of toxicity were observed at 500 mg/kg bw. Therefore, a dose of 750 mg/kg bw was selected as the highest dose and 500 and 250 mg/kg bw were administered as further doses in the main study.

TREATMENT AND SAMPLING TIMES:
- The animals were sacrificed and the bone marrow of the femurs was prepared at 24 and 48 h after administration in the highest dose group of 750 mg/kg bw and in the vehicle controls.
- In the test groups of 500 and 250 mg/kg bw and in the positive control groups, only 24 h sacrifice interval was investigated.

DETAILS OF SLIDE PREPARATION:
- The bone marrow was prepared according to the method described by Schmid (1976, 1977) and Salamone et al (1980). Animals were sacrificed by cervical dislocation and femurs were prepared by dissection and removing all soft tissues. Femurs of the mice were removed and the bone marrow cells were extracted with fetal calf serum. After centrifugation, the supernatant was removed and a drop of the cell suspension was placed and spread on a slide.
- Slides were air-dried and stained in eosin and methylene blue (modified May-Grunwald solution or Wrights solution) for about 5 minutes, then followed by rinsing in purified water, stained in Giemsa solution for about 15 minutes and rinsed twice in purified water. Slides were clarified in xylene and then mounted in Corbit-Balsam.

METHOD OF ANALYSIS:
- Slides were examined under microscope to determine the frequency of micronuclei in 2000 polychromatic erythrocytes (PCEs) per animal. In addition, normochromatic erythrocytes (NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. Ratio of PCEs to NCEs calculated. Number of small MN (diameter of MN < cell diameter/4) and of large MN (diameter of MN ≥ cell diameter/4) recorded.
Evaluation criteria:
- A test substance is considered positive if the following criteria are met:
Significant and dose-related increase in the number of PCEs containing micronuclei observed; 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 number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.
Statistics:
- 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
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
squatting posture, irregular respiration and poor general state
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 500, 750, 1000 and 2000 mg/kg bw
- Rationale for exposure: Determination of the acute intraperitoneal toxicity of test material.
- Clinical signs of toxicity in test animals: Mortality was observed at 2000 mg/kg bw; evident signs of toxicity observed and some animals were sacrificed moribund at 750 and 1000 mg/kg bw; all animals survived but evident signs of toxicity were observed at 500 mg/kg bw.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): Test material did not lead to any increase in the rate of micronuclei. 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.

- Ratio of PCE/NCE (for Micronucleus assay): No dose-dependent inhibition of erythropoiesis induced by the treatment of mice with test material was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Any other information on results incl. tables

Table 7.6.2/1: Micronucleus data

 

 

Vehicle Control (0 mg/kg bw)

250 mg/kg bw

500 mg/kg bw

750 mg/kg bw

CCP

20 mg/kg bw

VCR

0.15 mg/kg bw

Interval

24 h

48 h

24 h

24 h

24 h

48 h

24 h

24 h

Total No. of PCEs

10000

10000

10000

10000

10000

10000

10000

10000

Total No. of NCEs

4594

3439

3755

4646

2476

2804

3920

5374

MN in PCEs (%)

1.4

0.7

1.6

1.8

1.2

1

11.1**

47.2**

MN in NCEs (%)

0.7

1.5

0.5

1.1

0.8

0.4

2

0.9

 

PCEs: Polychromatic erythrocytes

NCEs: Normochromatic erythrocytes

MN: Micronuclei

CCP: Cyclophosphamide

VCR: Vincristine

* : Statistically significant (Wilcoxon test, one-sided) vs. Vehicle control group, p<= 0.05

**: Statistically significant (Wilcoxon test, one-sided) vs. Vehicle control group, p<= 0.01

- The administration of the test substance at 250, 500 and 750 mg/kg bw led to squatting posture, poor general state and irregular respiration.

Applicant's summary and conclusion

Conclusions:
Under the test conditions, test material is not classified as clastogenic or aneugenic according to the criteria of the Annex VI of the of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vivo bone marrow micronucleus test, performed according to OECD guideline 474 (1997 version) and in compliance with GLP, Crl:NMRI mice (5 males/dose) were administered once intraperitoneally with test material, dissolved in olive oil, at the dose levels of 250, 500 and 750 mg/kg bw in a volume of 10 mL/kg bw. Vehicle control group was administered with olive oil and positive control groups were given cyclophosphamide (20 mg/kg bw) and vincristine (0.15 mg/kg bw) by the same route. Animals were sacrificed and the bone marrow of the femurs was prepared 24 and 48 h after administration in the 750 mg/kg bw dose group and in the vehicle controls. In the test groups of 250 and 500 mg/kg bw and in the positive control groups, only 24 h sacrifice interval was investigated. After staining of the preparations, 2000 polychromatic erythrocytes (PCEs) were evaluated per animal and investigated for micronuclei. The normocytes (NCEs) with and without micronuclei occurring per 2000 polychromatic erythrocytes were also recorded. Range-finding test was conducted to determine the dose levels for main study.

Test material did not lead to any increase in the number of PCEs containing either small or large micronuclei. The rate of micronuclei was always close to the range as that of the concurrent vehicle controls in all dose groups and at all sacrifice intervals and within the range of the historical control data. No inhibition of erythropoiesis determined from the ratio of PCE/NCE was detected. Both of the positive control chemicals, i.e. cyclophosphamide for clastogenicity and vincristine for spindle poison effects, led to the expected increase in the rate of polychromatic erythrocytes containing small or large micronuclei indicating the validity of the study.

Under the test conditions, test material is not classified as clastogenic or aneugenic according to the criteria of the Annex VI of the of the Regulation (EC) No.1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for mammalian erythrocyte micronucleus test.