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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:
2004-10-27 to 2004-12-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

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
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
adopted July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
June 08, 2000
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Version / remarks:
EPA 712-C-98-226, August 1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2-ethylhexyl) carbonate
EC Number:
238-925-9
EC Name:
Bis(2-ethylhexyl) carbonate
Cas Number:
14858-73-2
Molecular formula:
C17H34O3
IUPAC Name:
bis(2-ethylhexyl) carbonate
Test material form:
other: liquid

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: minimum 7 weeks
- Weight at study initiation: males: 30.0 - 36.0 g, females 24.0 - 30.0 g
- Assigned to test groups randomly: yes
- Fasting period before study: no data
- Housing: 5 animals of identical sex per cage, Macrolon Type III (Hereto), granulated soft wood bedding (ALTROMIN)
- Diet: in all probability ad libitum, standard laboratory pelleted mouse diet (ALTROMIN)
- Water: ad libitum, tap water
- Acclimation period: minimum of 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19- 25
- Humidity (%): 55 +/- 10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: cotton seed oil
- Justification for choice of solvent/vehicle: The solvent was chosen according to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: 200 mg/mL
Details on exposure:
The test item was prepared and diluted in cotton seed oil within 1 h before treatment.
At the beginning of the experiment the animals were individually weighed and the administered volume adjusted to the animal's body weight. The animals received the test item once ip.
Duration of treatment / exposure:
The animals received the test item once ip.
Sampling of the bone marrow was carried out on animals 24 and 48 h after treatment.
Frequency of treatment:
single exposure
Post exposure period:
24 and 48 hours
Doses / concentrations
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide, purity: at least 98%
- Justification for choice of positive control(s): not given, however, cyclophosphamide is a standard positive control substance recommended by the guidelines
- Route of administration: single i.p.
- Doses / concentrations: 40 mg/kg bw dissolved in 0.9% NaCl, application volume 10 mL/kg bw

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
The selection of the highest applied dose based on the results of the pre-experiment for toxicity. The main experiment was performed as a limit test
with 2000 mg/kg bw.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
24 h preparation interval: 2000 mg/kg bw, diluted in cotton seed oil, application volume 10 mL/kg bw
48 h preparation interval: 2000 mg/kg bw, diluted in cotton seed oil, application volume 10 mL/kg bw

DETAILS OF SLIDE PREPARATION:
Bone marrow was obtained from the femurs immediately following sacrifice by cervical dislocation of the animals. Cells were removed from the femurs by cutting off the epiphyses and by flushing the marrow out with fetal calf serum using a 5 ml syringe. The cell suspension was centrifuged at 200 x g for 10 minutes and the supernatant was discarded. A drop of the resuspended cell pellet was spread on a slide as a smear. This was air-dried and stained with May-Grünwald/Giemsa. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
All slides, including those of positive and negative controls, were coded before microscopic analysis. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 2000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect an eventually occurring cytotoxic effect of the test item the ratio between immature and mature erythrocytes was determined. At least 200 immature erythrocytes were counted per animal and the result was expressed as relative PCE (reI. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).
Evaluation criteria:
EVALUATION OF RESULTS
There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECD guideline, the biological relevance as well as the statistical significance of the results are the criterion for the interpretation.
A test item is considered to be negative if there is no biological relevant and/or statistical significant increase in the number of micronucleated cells at any dose level. However, both biological relevance and statistical significance will be considered together.

ACCEPTANCE CRITERIA
The data generated are considered acceptable if:
- at the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20% of the mean weight of each sex,
- the background frequency of micronucleated cells is in the normal range as reported in the literature or within the laboratory's historical range,
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.
Statistics:
For the statistics the nonparametric Mann-Whitney test was used.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
The test item was suspended in cotton seed oil. 200 mg/mL of the test item was used as highest dose group corresponding to 2000 mg/kg bw according to the OECD guideline 474. The volume administered was 10 mL/kg bw ip. Three male and three female mice were treated with the test item. No
systemic toxicity was observed in any of the animals evaluated. The animals survived 72 h after the treatment.
The selection of the highest dose was conducted in accordance with the following current international guidelines for assessment of acute toxicity
(OECD 420 and 423), particularly with respect to selection of dose spacing and animal welfare aspects.


RESULTS OF DEFINITIVE STUDY
- Toxicity: 2000 mg/kg bw was tested as the maximum dose in the main experiment. The volume administered was 10 mL/kg bw. No toxicity was noted after administration of the test item in any of the animals evaluated up to 48 h.
- Induction of micronuclei (for Micronucleus assay): The negative controls (24 h and 48 h) evaluated were within the range of the historical control data. The mean values of micronuclei observed for the negative control (24 h) were 0.13 % for male and 0.06 % for female mice. The mean values for the 48 h negative control were 0.09 % for the male and 0.13 % for the female mice. The values noted for the dose groups which were treated with the test item (24 h, 48 h) were within the historical control data range. The values noted for the 24 h treatment group were 0.13 % for the male and 0.09 % for the female mice. The animal groups which were treated with the test item and prepared 48 h after the treatment showed mean values of micronuclei of 0.10 % male mice and 0.09 % female mice. All mean values of micronuclei formation noted after treatment with the test item were within the historical control data of the negative control. No biologically relevant increase of micronuclei was found.
- Ratio of PCE/NCE (for Micronucleus assay): The negative controls (24 h, 48 h) were within the historical control range of the negative control. The values noted for the 24 h negative control were 0.54 for the male and 0.58 for the female mice. The values detected for the 48 h negative control were 0.55 for the male and 0.55 for the female mice. The relative PCE of the animal groups, which were treated with 2000 mg/kg bw test item (24 h, 48 h) were within the historical control data. The values noted for the 24 h treatment were 0.50 for the male and 0.54 for the female mice. The animal groups which were treated with 2000 mg/kg bw test item and prepared 48 h after the treatment showed mean values of relative PCE of 0.53 (male mice) and 0.56 (female mice).
- Appropriateness of dose levels and route: application of the limit dose of 2000 mg/kg bw, as recomended by the guidelines
- Statistical evaluation: The nonparametric Mann-Whitney test was performed to verify the results. No statistically significant enhancement (p<0.05) of cells with micronuclei was noted in any dose group of the test item evaluated.

RESULTS OF POSITIVE CONTROL
Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control which induced a significant increase of induced micronucleus frequency (percentage of cells with micronuclei was 2.60 % for male and 2.05 % for female mice). This demonstrates the validity of the assay.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
It can be stated that during the study described and under the experimental conditions reported, the test item Bis(2-ethylhexyl) carbonate did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, Bis(2-ethylhexyl) carbonate is considered to be non-mutagenic in the Mammalian Erythrocyte Micronucleus Test.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay according to OECD Guideline No. 474, July 21, 1997, EU Method B12, June 08, 2000 and US EPA Guideline OPPTS 870.5395, EPA 712-C-98-226, August 1998, 5 male and 5 female animals/harvest time were treated i.p. with Bis(2-ethylhexyl) carbonate (99-100 % a.i.) at the limit dose of 2000 mg/kg bw. Bone marrow cells were harvested at 24 and 48 hours post-treatment. The vehicle was cotton seed oil.

There were no signs of toxicity during the study. Bis(2-ethylhexyl) carbonate was tested at an adequate dose, as the tested dose is the limit dose recommended by the guidelines. The positive control induced the appropriate response.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time.

This study is classified as acceptable. This study satisfies the requirement of the mentioned guidelines for in vivo cytogenetic mutagenicity data.