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Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 MAR 2006 - 17 MAY 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted in compliance with the OECD Principles of Good Laboratory Practice and was performed according to Commission Directive 2000/32/EC, the ICH Guidelines, the OECD Guideline for Testing of Chemicals No.474 and the OECD Principles of Good Laboratory Practice.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
21 Jul 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
425-560-7
EC Name:
-
Cas Number:
174063-87-7
Molecular formula:
C33H32O10
IUPAC Name:
2-methylbenzene-1,4-diyl bis{4-[3-(acryloyloxy)propoxy]benzoate}
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 6 to 10 weeks
- Weight at study initiation: 218 - 235 g
- Assigned to test groups randomly: yes, under following basis: The animals were assigned to the cages according to a random list which had been provided by a computer program developed in house. The rats were identified by metal earmarks.
- Housing: The animals were kept individually in Makrolon cages type 3 (floor area: 37.5 x 21.5 cm, height: 13 cm) on softwood chippings
- Diet (e.g. ad libitum): ad libitum (Standard diet from Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum (tap water from Makrolon drinking bottles)
- Acclimation period: The animals were received from the breeding center and were adapted to the laboratory conditions for a period of at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 21
- Humidity (%): 45 - 55
- Photoperiod (hrs dark / hrs light): 12/12
- Atmospheric pressure: 751 - 759 mm Hg

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Miglyol 812 neutral oil (20 mL/kg body weight) Batch: ZDP K34243307
Details on exposure:
As proposed in the EEC Directive and in the OECD Guideline, the animals were treated with the test compounds once. To achieve maximal sensitivity of the test system, the test material was administered intraperitoneally in this investigation. Dosing of the animals of different groups was staggered over one to three day intervals to allow for sufficient sample preparation time.
Duration of treatment / exposure:
single administration
Frequency of treatment:
1
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
316 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
5 per dose and 5 per control
Control animals:
yes, concurrent vehicle
yes, historical
Positive control(s):
Cyclophosphamide (CPA) = Endoxan® (Baxter Oncology, Frankfurt, Germany) provided as ampouls containing:
Cyclophosphamide x H2O 106.9 mg
Cyclophosphamide anhydrous form 100.0 mg
NaCl 45.0 mg
Art. No.: E 432-1
Batch: 4GH156D
Stable until: August, 2007

25 mg Endoxan® (= 16.5 mg CPA) were dissolved in 10 mL Aqua pro injectione directly before use.

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The high dose given in the present study was selected to produce signs of toxicity but no mortality. In preliminary dose-finding experiments, 5 male rats treated intraperitoneally with 1000 mg/kg body weight of the test item (in 20 mL/kg body weight of Miglyol 812 neutral oil) showed clear toxic effects (abdominal position, ptosis and body weight loss) but no mortality.
For these reasons, the dose of 1000 mg/kg body weight was selected as the high dose for male rats in the main study of this investigation. The mid and low dose is obtained by dilution in half-log ranges, i.e. by the divisor of √10 = 3.16.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were treated with the test compounds once. To achieve maximal sensitivity of the test system, the test material was administered intraperitoneally in this investigation. Dosing of the animals of different groups was staggered over one to three day intervals to allow for sufficient sample preparation time.

DETAILS OF SLIDE PREPARATION: Immediately after the rats had been killed by CO2, one femur of each animal was dissected and cleaned from adherent muscles. The epiphyses were cut off and bone marrow cells were flushed out with fetal calf serum (Biochrom, Berlin, Germany) with the aid of a syringe, and suspended in the serum. This suspension was filtered through cellulose and centrifuged for 5 min at 150 x g. The sediment was then resuspended in fetal calf serum and bone marrow smears were prepared from the resulting cell suspension.
After 3 hours of drying, the slides were stained according to a modified Giemsa-staining method described by Gollapudi and Kamra (1979) using Giemsa's solution with Weise buffer solution and mounted in Entellan.

METHOD OF ANALYSIS: A total of 2000 polychromatic erythrocytes per animal were scored for micronuclei using Zeiss light microscopes with plane optics (magnification: 1250 x).
Evaluation criteria:
A positive effect in this test system is defined by the occurrence of mean MN-PCE values of a treatment group which are statistically significantly higher than those of the actual negative control.

A test material is defined as mutagenic in this system if dose-related and/or single, reproducible (in independent experiments) positive effects occur. Establishment of dose-dependent effects of the test material is preferable. For this reason, if a positive effect occurs in a study in which a single, limit dose of 2000 mg/kg has been applicated, 3 different test material doses have to be administered in the supplementary experiment. The above mentioned criteria for a negative or positive test result apply for this experimental design likewise.
Statistics:
Descriptive statistics
For all groups, mean values were calculated of the following parameters:
NCE/PCE - number of normochromatic erythrocytes (NCE) /
number of polychromatic erythrocytes (PCE) / animal
MN-NCE - number of micronuclei-containing cells / 1000 NCE /
animal
MN-PCE - number of micronuclei-containing cells / 1000 PCE/
animal
For the parameter body weight the mean values and the relative body weight gains to the preceding mean values were calculated.

Statistical tests
For further statistical analysis, the numbers of micronuclei-containing polychromatic erythrocytes (MN-PCE), normochromatic erythrocytes (MN-NCE) and the quotient of NCE/PCE per animal were used.

Pairwise comparison
Each treatment group was compared to the negative control.

For comparisons of micronuclei-containing polychromatic and normochromatic erythrocytes, the exact Mann-Whitney-test was used against one-sided alternatives. The p-values (exact significance one-sided) of these comparisons are presented.
For comparisons of the quotient of NCE/PCE, the Dunnett’s t-test was used against two-sided alternatives. The p-values (significance) of these comparisons are presented for those dose groups that showed a higher mean value than the negative control.

Software
The numerical calculation was performed using the computer-aided micronucleus test program (Version 2.3c) and for the exact Mann Whitney test and Dunnett’s t-test the SPSS-System (Version 9.0), running under Windows NT was used.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
some clinical signs at highest dose
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: The high dose given in the present study was selected to produce signs of toxicity but no mortality. In preliminary dose-finding experiments, 5 male rats treated intraperitoneally with 1000 mg/kg body weight of the test item (in 20 mL/kg body weight of Miglyol 812 neutral oil) showed clear toxic effects (abdominal position, ptosis and body weight loss) but no mortality.
For these reasons, the dose of 1000 mg/kg body weight was selected as the high dose for male rats in the main study of this investigation. The mid and low dose is obtained by dilution in half-log ranges, i.e. by the divisor of √10 = 3.16.
- Clinical signs of toxicity in test animals: at 1000 mg/kg bw clinical symptoms were bevelling (4/10 animals), ptosis (1/10 animals), abdominal position (6/10 animals)
- Rationale for exposure: As proposed in the OECD Guideline, the animals were treated with the test compounds once.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): 2000 polychromatic erythrocytes per animal, using 5 males per group, were evaluated. The negative control (solvent) values were all in the expected range predetermined as historical controls of the laboratory. For the positive control, a statistically significant increase in polychromatic erythrocytes with micronuclei was established (p < 0.01). No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei (MN-PCE) was observed for the test item. For the number of normochromatic cells with micronuclei, calculated per 1000 normochromatic erythrocytes by means of the above mentioned quotient, no increase was observed.
- Ratio of PCE/NCE (for Micronucleus assay): No relevant treatment-related variation was observed.
- Appropriateness of dose levels and route: The highest dose revealed toxicity but no mortality.
- Statistical evaluation: No statistically significant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item treated groups (p> 0.05). For statistical methods, please refer to methods section.

Any other information on results incl. tables

Table 1: Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE in different dose groups


Test material Dose Preparation MN-PCEa
[mg/kg] time [h] males
Solvent - 24
1.60
test material 100 24 1.90ns
test material 316 24 1.10ns
test material 1000 24 2.10ns
test material 1000 48 1.00ns
Cyclophosphamide 16.5 24 21.2**


a:     Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE
Statistical significance:         ns: not significant (p > 0.05)    *:  0.01  <  p  ≤  0.05    **:  p  ≤  0.01

Applicant's summary and conclusion

Conclusions:
The test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.
Executive summary:

The registered substance was tested for cytogenicity according to OECD Guideline 474 following GLP.

Purpose
The purpose of the in vivo micronucleus test is to identify agents that cause chromosomal damage or damage to the mitotic apparatus thus providing information on possible mutagenic effects of  the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in human.

Study Design
The micronucleus test was performed according to Commission Directive 2000/32/EC, the ICH Guidelines, the OECD Guideline for Testing of Chemicals No.474 and the OECD Principles of Good Laboratory Practice.
In the current experiment, the test chemical was given once intraperitoneally to male rats at doses of 100, 316 or 1000 mg/kg body weight. Rats of the negative control group received the solvent alone, i.e. an intraperitoneal dose of 20 mL/kg body weight Miglyol 812 Neutral oil. The animals of the positive control group were treated with an oral dose of 16.5 mg/kg body weight cyclophosphamide.
Bone marrow smears were prepared from one femur of each animal and stained with Giemsa's solution. For the high dose groups (1000 mg/kg bw), preparation took place at two different times, i.e. 24 and 48 hours after administration of the test material. For the low and mid dose groups, as well as for the positive and negative control groups, the preparation time was 24 hours after start of the treatment. A total of 30 animals (5 male rats per dose group) were used.
For microscopic investigation, one slide from each animal preparation was coded. The number of polychromatic erythrocytes with micronuclei per 2000 polychromatic erythrocytes per animal was determined.
The quotient of normochromatic to polychromatic erythrocytes was calculated based on the analysis of 1000 erythrocytes per animal. The micronucleated normochromatic erythrocytes were registered when scoring the polychromatic erythrocytes. The number of micronucleated normochromatic erythrocytes per 1000 erythrocytes was then calculated with the aid of the quotient.

Results
Under the conditions of the present study the following results were obtained:

Test material Dose Preparation MN-PCEa
[mg/kg] time [h] males
Solvent - 24
1.60
test material 100 24 1.90ns
test material 316 24 1.10ns
test material 1000 24 2.10ns
test material 1000 48 1.00ns
Cyclophosphamide 16.5 24 21.2**


a:     Mean numbers of polychromatic erythrocytes with micronuclei per 1000 PCE
Statistical significance:         ns: not significant (p > 0.05)    *:  0.01  <  p  ≤  0.05    **:  p  ≤  0.01

The highest test material dose induced some clinical signs of toxicity. A relevant treatment-related variation was observed for the quotient of normochromatic : polychromatic erythrocytes.
The mean numbers of polychromatic erythrocytes with micronuclei for the negative control (solvent) were all in or very close to the expected range predetermined by historical controls of the laboratory. The positive control group (cyclophosphamide) showed the expected significant increase in the number of polychromatic erythrocytes with micronuclei.
No statistically significant or biologically relevant increase in the number of polychromatic erythrocytes with micronuclei was observed in any of the test item-treated groups. The number of normochromatic cells with micronuclei was not increased.

Conclusion
The test material was not mutagenic in the micronucleus test in rats under conditions where the positive control exerted potent mutagenic effects.