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
Study period:
May 2001 - November 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study procedures described in this report were based on the following guidelines: - European Economic Community (EEC). Directive 2000/32/EC, Part B: Methods for the Determination of Toxicity; B.12: "Mutagenicity: In Vivo Mammalian Erythrocyte Micronucleus Test" (published June 8, 2000), - Organisation for Economic Co-operation and Development (OECD), OECD Guidelines for the Testing of Chemicals, Guideline No. 474: Mammalian Erythrocyte Micronucleus Test (adopted July 21, 1997). - United States Environmental Protection Agency Title 40 - Protection of environment, - Part 799 - Identification of Specific Chemical substance and Mixture testing requirements -, subpart H - Health Effects Test Guidelines - Sec, 799.9539 TSCA Mammalian Erythrocyte Micronucleus Test (revised July 1, 2001).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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
GLP compliance:
yes (incl. certificate)
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Description: White powder of a crystalline substance consisting of colourless crystals
storage: At room temperature in the dark
stability: Stable
expiry date: 10 April 2008

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS: NMRI BR mice (SPF)
- Source: Charles River, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: The body weights of the mice at the start of the treatment were within 20% of the sex mean.
- Assigned to test groups randomly: The animals were allocated to treatment groups as they came to hand from the delivery boxes
- Fasting period before study:
- Housing: The animals were group housed (5 animals per sex per cage) in labe lied polycarbonate cages (type M II height: 14 cm) containing sterilised sawdust as bedding material (Litalabo; S.P.P.S., Argenteuil, France). Paper bedding was provided as nest material (Enviro-dri, TecniLab-SMI
SV, Someren, The Netherlands). Certificates of analysis of bedding and paper were examined and then retained in the NOTOX archives.
- Diet: The animals were provided with standard pelleted laboratory animal diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: at least 5 days before start of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C (actual range 21.4 - 22.9°C)
- Humidity (%): 30 - 70% (actual range 41 - 78%)
- Air changes (per hr): air-conditioned room, approx. 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours dark per day.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Vecicle: Yes
The test item was dissolved in physiological saline (Ziekenhuis Apotheek Noordoost-Brabant, Den Bosch, The Netherlands).
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The mice received a single intraperitoneal injection of a maximum required (high), an intermediate and a low dose of the test item within 1 hour after preparation of the test item.
The dosing volume was 10 ml/kg body weight.

Duration of treatment / exposure:
Dose range finding study: a single dose with 2000 mg/kg b.w.
main test: a single dose with dose levels of 2000, 1000 and 500 mg/kg b.w.
Frequency of treatment:
Dose range finding study: a single dose
main test: a single dose
Post exposure period:
sampling time: 24 and 48 hours
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
2000 mg/kg b.w.
Basis:
nominal conc.
Dose range finding study
Remarks:
Doses / Concentrations:
2000 mg/kg b.w.
Basis:
nominal conc.
Main Test
Remarks:
Doses / Concentrations:
1000 mg/kg b.w.
Basis:
nominal conc.
Main Test
Remarks:
Doses / Concentrations:
500 mg/kg b.w.
Basis:
nominal conc.
Main Test
No. of animals per sex per dose:
Dose range finding study: 3 male and 3 female animals dosed with 2000 mg/kg b.w.
Main test: 5 male and 5 female animals were used in each treatment group dosed with 2000, 1000 and 500 mg/kg b.w.
Control animals:
yes
Positive control(s):
cyclophosphamide:
-CAS# 50-18-0, Endoxan,
-from: Asta-Werke, Germany
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg/kg body weight.
- dissolved in physiological saline
- route and frequency of administration and the volume administered of the negative and positive control were the same as those of the test article

Examinations

Tissues and cell types examined:
Bone marrow (cell suspension)
Details of tissue and slide preparation:
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Bone marrow of the groups treated with the test item was sampled 24 or 48 hours after dosing.
Bone marrow of the negative control group was isolated 24 hours after dosing.
Bone marrow of the positive control group was isolated 48 hours after dosing.

Both femurs were removed and freed of blood and muscles.
The bone was flushed with approximately 2 ml of fetal calf serum (Invitrogen Corporation, Breda, The Netherlands). The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.

PREPARATION OF THE BONE MARROW SMEARS:
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide, which was previously
cleaned (24 h immersed in a 1: 1 mixture of 96% (v/v) ethanol/ether (Merck, Darmstadt, Germany) and cleaned with a tissue) and marked (with the NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides
at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol (Merck) and air-dried overnight. Two slides were prepared per animal.#

STAINGING OF THE BONE MARROW SMEARS:
The slides were automatically stained using the "Wright-stain-procedure" in an "Am es" HEMAtek slide stainer (Miles, Bayer Nederland B.V.). The dry slides were dipped in xylene (Klinipath, Duiven, The Netherlands) before they were embedded in Pertex (Klinipath) and mounted with a
coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. An adhesive label with NOTOX study identification number and code was stuck over the marked slide. At first the slides werescreened at a magnification of 1 OOx for regions of suitable technical quality, Le. where the cells were weil spread, undamaged and weil stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

OTHER:
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes.
b) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range (mean ± three times the standard deviation): Males: 0.9%0 ± 3.2%0 indicated are means for n=210 (range: 0 - 6).
Females: 1.0%0 ± 3.2%0 indicated are means for n=130 (range: 0 - 4).
Statistics:
Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
It induced a biologically as weil as a statistically significant (Wilcoxon Rank Sum Test, onesided,
p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at
any dose or at any sampling time) in the combined data for both sexes or in the data for
male or female groups separately.
A test substance is considered negative in the micronucleus test if:
None of the tested concentrations or sampling times showed a statistically significant
(Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated
polychromatic erythrocytes either in the combined data for both sexes or in the data for male
or female groups separately. The preceding criteria are not absolute and other modifying factors may enter into the final
evaluation decision.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
positive
Remarks:
statistically significant increase in the mean number of micronucleated polychromatic erythrocytes
Toxicity:
no effects
Remarks:
no decrease in the ratio of polychromatic to normochromatic erythrocytes
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Selection of an adequate dose range for the micronucleus main test was based on a dose range finding study. The test procedure and conditions were similar to those applied in the main test One dose group comprising of 3 males and 3 females received a single dose of the test substance.
The study duration per dosing was 3 days. During this period mortality and physical condition were recorded at least once a day.
- Dose range: 2,000 mg/kg b.w.
- Clinical signs of toxicity in test animals: no abnormalities after dosing.
- Evidence of cytotoxicity in tissue analyzed

RESULTS OF DEFINITIVE STUDY
5 male and 5 female mice were used per sampling time in each treatment group. The animals were dosed once.

Any other information on results incl. tables

Dose range finding study

In the dose range finding test, 3 male and 3 female animals dosed with 2,000 mg substance showed no abnormalities after dosing.

Micronucleus main test

Mortality and systemic toxic signs

All animals showed no abnormalities after dosing.

Micro-nucleated polychromatic erythrocytes

The mean number of micro-nucleated polychromatic erythrocytes, scored in the test item, treated groups were compared with the corresponding solvent control group.

The test item induced a statistically significant increase in the mean number of micro-nucleated polychromatic erythrocytes compared to the corresponding solvent control group in male and female animals dosed with 2,000, 1,000, and 500 mg/kg body weight at the 24 h sampling time. The number of micro-nucleated polychromatic erythrocytes in the bone marrow of all animals treated with the test item and sampled after 24 hours was above the historical control data range. Therefore the increases are biologically relevant and the test item is considered clastogenic.

The test item did not induce a statistically significant increase in the mean number of micro-nucleated polychromatic erythrocytes compared to the corresponding solvent control group in male and female animals dosed with 2,000 mg/kg body weight at the 48 h sampling time. The incidence of micro-nucleated polychromatic erythrocytes in the bone marrow of all negative control animals was within the historical solvent control data range.

Ratio polychromatic to normochromatic erythrocytes:

The animals of the groups, which were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): positive
It is concluded that this test is valid and the substance is clastogenic in the Micronucleus Test under the experimental conditions.
Executive summary:

The test item induced a statistically significant increase in the mean number of micro-nucleated polychromatic erythrocytes compared to the corresponding solvent control group in male and female animals dosed with 2,000, 1,000, and 500 mg/kg at the 24 h sampling time. The number of micro-nucleated polychromatic erythrocytes in the bone marrow of all animals treated with the test item and sampled after 24 h was above the historical control data range. Therefore the increases are biologically relevant and the test item is considered clastogenic.