A mixture of cis- and trans-cyclohexadec-8-en-1-one

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986-06-17 to 1986-11-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1986

Report date:

1986

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

A mixture of cis- and trans-cyclohexadec-8-en-1-one

EC Number:

401-700-2

EC Name:

A mixture of cis- and trans-cyclohexadec-8-en-1-one

Cas Number:

3100-36-5

Molecular formula:

C16H28O

IUPAC Name:

(8E)-cyclohexadec-8-en-1-one; (8Z)-cyclohexadec-8-en-1-one

Test animals

Species:

mouse

Strain:

Swiss

Remarks:

CD-1 strain, SPF

Details on species / strain selection:

The mouse is a commonly used species for in vivo genotoxicity testing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: Not specified
- Weight at study initiation: 33.1-35 g (males), 27.9-29.9 g (females)
- Assigned to test groups randomly: Yes
- Fasting period before study: 2 hours
- Housing: In Makrolon sterilized cages with a grid cover of stainless steel and with a bedding of sterilized softwood chips. Males were housed individually and females five per cage.
- Diet: Ad libitum (except for the 2-hours period before treatment when food was withheld)
- Water: Ad libitum
- Acclimation period: 28 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40-60
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 0 to day 3

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: None (undiluted test substance), water (negative control), saline (positive control)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was given undiluted by gavage in one single dose of 3 mL/kg bw (corresponding to 2800 mg/kg bw) to males and 5 mL/kg bw (corresponding to 4600 mg/kg bw) to females

Duration of treatment / exposure:

24, 48 and 72 hours

Frequency of treatment:

Single oral dose

Post exposure period:

None

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

4 600 mg/kg bw (total dose)

Remarks:

Used for females

Doses / concentrations 2

Dose / conc.:

2 800 mg/kg bw (total dose)

Remarks:

Used for males

No. of animals per sex per dose:

5 males treated with a single dose of 2800 mg/kg bw and 5 females were treated with a single dose of 4600 mg/kg bw. 5 male negative control animals were treated with 3 mL water and 5 female negative control animals were treated with 5 mL water. 2 animals per sex per dose were treated with the positive conrol (1500 mg/kg bw)

Control animals:

yes

Positive control(s):

Mitomycin C
- Justification for choice of positive control: Well-known clastogen, frequently used for in vivo genotoxicity testing
- Route of administration: Intraperitoneally
- Doses / concentrations: 1.5 mg/ kg bw

Examinations

Tissues and cell types examined:

Bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: From the results of an acute oral toxicity study, it appeared that the acute oral LD50 of the test substance in mice exceeded 5 mL/kg bw, both in males and in females. Nevertheless the test material induced clear signs of intoxication in male mice. Males treated with 5 mL/kg bw showed tremors, paralysis and piloerection an the first post-treatment day, and 2 out of 5 males died. The female mice showed no signs of intoxication and, deaths did not occur. Therefore, in the present study a lower dose was administered to males than to females, i.e. males were given 3 mL/kg bw and females 5 mL/kg bw.

TREATMENT AND SAMPLING TIMES: At 24 (day 1), 48 (day 2) and 72 (day 3) hours after treatment, 5 males and 5 females of the test group and the negative control group, and 2 males
and 2 females of the positive control group were anaesthetized with ether and killed by decapitation. The femurs were dissected free, the bone marrow was flushed from the femurs into a centiifuge tube containing foetal calf Serum and mixed gently. The cell suspension was centrifuged and the excess serum removed.

DETAILS OF SLIDE PREPARATION: A drop of the Suspension was spread on a slide with a ground glass microscope slide. Ten slides were prepared from each animal. The smears were air dried and fixed in methanol for 5 minutes. The smears were stained with acridine orange for approx. 3 minutes at room temperature and rinsed in the buffer for about 2 minutes. The preparations were mounted with the same buffer and examined on the day of staining with a fluorescence microscope.

METHOD OF ANALYSIS: The incidence of MNC (= micronucleated polychromatic erythrocytes) and the ratio P:N ratio of polychromatic to normochromatic erythrocytes) were scored "blindly" in slides that had been coded, before staining, by a qualified person not involved in the microscopic evaluation of the smears. The incidence of MNC was determined in a total of 1000 polychromatic erythrocytes per animal, by examining 200 polychromatic erythrocytes in each of 5 slides of each animal, for the presence of yellowish-green fluorescing micronuclei. The P:N ratio is used as a measure of cytotoxicity. This ratio was determined by counting the numbers of P and N in a total of 1000 erythrocytes per animal (200 erythrocytes per smear).

Evaluation criteria:

Not described.

Statistics:

The numbers of MNC per 1000 polychromatic erythrocytes and the numbers of polychromatic erythrocytes per 1000 erythrocytes (normo- plus polychromatic erythrocytes) were each analysed by 2 linear logistic regression models with factors: time (24, 48 and 72 hours), sex (male, female) and group, and their mutual interactions. The levels of the factor group were successively: 1) water; test substance and 2) water; mitomycin C.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: No dose range-finding study was conducted. Doses were based on the results of an acute oral toxicity study.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: MNC counts per 1000 polychromatic erythrocytes in water control and test animals were fully comparable (see attached background material).
- Ratio of PCE/NCE: There were no signs of cytotoxic effects of the test compound an the bone marrow cells: the ratios of poly- and normochromatic erythrocytes in control and test animals of the same sex and sacrifice time did not show any indication of treatment related cytotoxic effects attributable to treatment with the test compound.
- Appropriateness of dose levels and route: The substance was tested in concentrations which induced slight toxicity but no severe suffering or deaths. The oral route was chosen based on the most probable route of exposure.
- Clinical signs: Piloerection occured 4 hours after treatment in all animals. Three animals (2 males and 1 female) showed paralysis of the hind legs. 24 hours after treatment, one male animal died and some other animals showed rough coats. Males appeared more affected by treatment than females. No specific signs of toxicity were observed at 48 and 72 hours after treatment.

Applicant's summary and conclusion

Conclusions:

In a mammalian erythrocyte micronucleus test according to OECD guideline 474, the results did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated with 2800 (males) or 4600 mg/kg bw (females) of the test substance.

Executive summary:

In an in vivo mammalian erythrocyte micronucleus test according to OECD guideline 474 and GLP, the test item was examined using mice as experimental animals. Two groups of 15 males and 15 females each were treated once orally with, respectively, 3 mL (2.8 g) and 5 mL (4.6 g) of the undiluted test substance per kg body weight. Negative control animals were treated in a comparable way with water only. A positive control group of 6 males and 6 females was treated once intraperitoneally with 1.5 mg of the mutagen Mitomycin C per kg body weight. At 24, 48 and 72 hours after treatment, ten negative controls (5/sex), ten test animals (5/sex) and four positive controls (2/sex) were killed. Samples of bone marrow were collected and bone marrow smears were prepared for microscopic examination. Treatment with- the test material induced overt signs of intoxication and one male died. Mean body weights were not affected. The incidence of micronucleated erythrocytes and the ratios of poly- to normochromatic erythrocytes in test animals were fully comparable to those found in the concomitant negative controls killed at the same exposure time. Animals treated with the mutagen mitomycin C showed an increased incidence of micronucleated polychromatic erythrocytes and a decrease in the ratio of poly— to normochromatic erythrocytes. It was concluded that the results of the micronucleus test did not provide any indication of chromosomal damage and/or damage to the mitotic apparatus in bone marrow cells of mice treated with a high dose of the test substance.