Diisopentyl ether

Genetic toxicity: in vivo

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: GLP compliant, guideline study, no restrictions, fully adequate for assessment.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Diisopentyl ether

EC Number:

208-857-4

EC Name:

Diisopentyl ether

Cas Number:

544-01-4

Molecular formula:

C10H22O

IUPAC Name:

3-methyl-1-(3-methylbutoxy)butane

Details on test material:

- Name of test material (as cited in study report): Diisopentyl ether
- Analytical purity: 99.2%
- Lot/batch No.: TAP 645
- Storage condition of test material: 2-8 °C

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks old
- Weight at study initiation: 34.3 ± 2.2 g
- Housing: animals were group housed (5 animals per sex per cage) in labelled Macrolon cages (MIII type, height 15 cm)
- Diet (e.g. ad libitum): free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)
- Water (e.g. ad libitum): free access to tap water.
- Acclimation period: at least 6 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.1-21.4°C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12h/12h
- Air changes (per hr): approx. 15 changes per hour

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

- Dose selection rationale: In order to set the dose levels for the main study, a dose range finding study was performed. One dose group of 3 females and 3 males received a single dose of 2000 mg/kg bw by oral gavage. The animals showed no treatment related clinical signs or mortality after dosing. Based on the results of this range finding study, dose levels for the main study were: 500, 1000 and 2000 mg/kg body weight. Since there were no differences between sexes in toxicity only male animals were used in the main study.
- Preparation of dosing solutions: The total dosages (500, 1000 and 2000 mg/kg bw) were adminstered as single dose by oral gavage. Feed was withheld 3-4 h prior to dosing. The dosing volume was 10 ml/kg body weight. Diisopentyl ether concentrations were prepared on the day of administration.

Duration of treatment / exposure:

single dose

Frequency of treatment:

once

Post exposure period:

24-48 hr

Doses / concentrations

Remarks:

Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

5 male animals

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (40 mg/kg bw) was administered once by oral gavage as a solution in physiological saline (dose volume 10 mL/kg bw).

Examinations

Tissues and cell types examined:

Bone marrow of the groups treated with diisopentyl ether was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. 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 216 g for 5 min.

Details of tissue and slide preparation:

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 remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed in a 1:1 mixture of 96% (v/v)
ethanol (Merck, Darmstadt, Germany)/ether (Merck) and cleaned with a tissue. 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. The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer (Miles, Bayer Nederland B.V.).

Evaluation criteria:

The number of micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

A micronucleus test is considered acceptable if it meets the following criteria:
a) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range.
b) 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.
c) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range

Statistics:

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) or and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

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 and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Mortalities
The animals of the groups treated with 2000, 1000 and 500 mg diisopentyl ether/kg body weight and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.

Micronucleus counts
- No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Diisopentyl ether treated animals compared to the vehicle treated animals.
- The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.
- Cyclophosphamide induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes

Normochromatic to polychromatic erythrocyte ratios
The groups that were treated with Diisopentyl ether showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
It is concluded that this test is valid and that diisopentyl ether is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.

Executive summary:

In a GLP-compliant erythrocyte micronucleus test, according OECD guideline 474, male animals were dosed via oral gavage with vehicle or with 2000, 1000 and 500 mg Diisopentyl ether per kg body weight. A positive control group was dosed via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals. No treatment related clinical signs or mortality were noted in any animal.

Bone marrow of the groups treated with Diisopentyl ether was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively. No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with Diisopentyl ether. The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range. Cyclophosphamide induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Therfore, the study was considered valid.

The groups that were treated with diisopentyl ether showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is therefore concluded that, under the conditions used in this study, Diisopentyl ether is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg (the maximum recommended dose in accordance with current regulatory guidelines).