Cyclopentane, methoxy-

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

Study period:

21 November 2001 - 08 January 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD, EC and US EPA test guidelines, and in compliance with GLP, so the data is considered reliable without restrictions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-090-6

EC Name:

-

Cas Number:

5614-37-9

Molecular formula:

C6 H12 O

IUPAC Name:

Cyclopentyl methyl ether

Test material form:

liquid

Specific details on test material used for the study:

- Analytical purity: 99.96%
- Lot/batch No.: 011107

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: 28 - 32 g (males) and 22 - 26 g (females)
- Assigned to test groups randomly: Yes
- Diet (e.g. ad libitum): Free access
- Water (e.g. ad libitum): Free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2°C
- Humidity (%): 55±15%
- Photoperiod (hrs dark / hrs light): 12 hours light per day

IN-LIFE DATES: From: 21 November 2001 To: 08 January 2002

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Corn oil
- Concentration of test material in vehicle: 100 or 50 mg/mL (preliminary study); 25, 50, or 100 mg/kg (Micronucleus test)
- Amount of vehicle (if gavage or dermal): 20 mL/kg

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solutions of the test substance in corn oil were prepared freshly on the day of use.

Duration of treatment / exposure:

A single oral dose was administered. Observation or sacrifice times were as follows:
48 hours (preliminary study)
24 hours (micronucleus test); in addition, additional animals in the negative control group and highest test group were sacrificed 48 hours after exposure

Frequency of treatment:

N/A (A single dose was administered)

Post exposure period:

Refer above for details.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Preliminary test - 2 males and 2 females per group (2 groups)
Micronucleus test - 7 males per dose in the 500 and 1000 mg/kg dose groups; 14 males in the control and 2000 mg/kg dose groups.

Control animals:

yes, concurrent vehicle

Positive control(s):

mitomycin C
- Route of administration: Oral gavage (solution in purified water)
- Doses / concentrations: 12 mg/kg (solution concentration = 0.6 mg/mL)

Examinations

Tissues and cell types examined:

Bone marrow from femurs

Details of tissue and slide preparation:

The bone marrow of both femurs from each animal was flushed out and pooled in a total volume of 2 ml of pre-filtered foetal calf serum. The cells were sedimented by
centrifugation, the supernatant was discarded and the cells were resuspended in a small volume of fresh serum. A small drop of the cell suspension was transferred to a glass microscope slide and a smear was prepared in the conventional manner (Schmid 1976). Three smears were made from each animal. The prepared smears were fixed in methanol (> 10 minutes). After air-drying the smears were stained for 10 minutes in 10% Giemsa (prepared by 1 : 9 dilution of Giemsa with purified water). Following rinsing in purified water and differentiation in buffered purified water, the smears were rinsed in purified water, air-dried and mounted with coverslips using DPX.

Evaluation criteria:

The stained smears were examined (under code) by light microscopy to determine the incidence of micronucleated cells per 2000 polychromatic erythrocytes per animal. Usually only one smear per animal was examined. The remaining smears were held temporarily in reserve in case of technical problems with the first smear.

Micronuclei were identified by the following criteria:
• Large enough to discern morphological characteristics
• Should possess a generally rounded shape with a clearly defined outline
• Should be deeply stained and similar in colour to the nuclei of other cells - not black
• Should lie in the same focal plane as the cell
• Lack internal structure, ie they are pyknotic
• There should be no micronucleus-like debris in the area surrounding the cell.

The proportion of immature erythrocytes for each animal was assessed by examination of at least 1000 erythrocytes. A record of the number of micronucleated mature erythrocytes observed during assessment of this proportion was also kept as recommended by Schmid (1976).

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

A number of clinical signs were noted following administration at 1000 and 2000 mg/kg, but all animals in all groups survived to the scheduled sacrifice times.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Doses producing toxicity:
In the preliminary test, two groups of animals were dosed with the test substance at concentrations of 1000 and 2000 mg/kg. At 2000 mg/kg, clinical signs observed included underactivity, overactivity, flattened posture, abnormal gait, fast and irregular respiration, reduced righting reflexes. At 1000 mg/kg, clinical signs of underactivity, abnormal gait and fast respiration were observed. All animals survived to scheduled termination.

Results showed that a dose level of 2000 mg/kg was tolerated. This dose was therefore selected as an appropriate maximum for use in this test.

Observations: No statistically significant increases in the frequency of micronucleated immature erythrocytes and no substantial decreases in the proportion of immature erythrocytes were observed in mice treated with the test substance and killed 24 or 48 hours later, compared with the vehicle control values.

Applicant's summary and conclusion

Conclusions:

It is concluded that the substance did not show any evidence of causing chromosome damage or bone marrow cell toxicity when administered orally by gavage in this in vivo test procedure.