4-pentylcyclohexanone

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:

May 14, 1990 - July 17, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EEC Directive 84/449, L 251, B 12, p. 137 - 139 Updating of Toxicological Methods Annex V of the Council Directive 79/831/EEC; Part B

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-pentylcyclohexanone

EC Number:

406-670-4

EC Name:

4-pentylcyclohexanone

Cas Number:

61203-83-6

Molecular formula:

C11H20O

IUPAC Name:

4-pentylcyclohexan-1-one

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Fullinsdorf, CH-4414 Fullinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: approximately 18 hours before treatment with the test article the animals received no food but water ad libitum
- Housing: single, Makrolon Type I, with wire mesh top
- Diet: ad libitum, pelleted standard diet (ALTROMIN)
- Water: tap water, ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 + 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: polyethylene glycol PEG 400
- Justification for choice of solvent/vehicle: On the day of the experiment, the test article was suspended in PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solution were prepared on the day of administration. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

24 h or 48 h

Frequency of treatment:

once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 h sampling time

Doses / concentrations 2

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 h sampling time

Doses / concentrations 3

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 and 48 h sampling time

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally, once
- Doses / concentrations: 20 and 40 mg/kg b.w. (two groups; only 20 mg/kg b.w. dosing is reported)

Examinations

Tissues and cell types examined:

bone marrow/erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available.
The maximum tolerated dose level was determined to be the dose that caused toxic reactions without having major effects on survival within 48 hours.

TREATMENT AND SAMPLING TIMES: Sampling of the bone marrow was done 24 and 48 hours after treatment.

DETAILS OF SLIDE PREPARATION: The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 5 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt, F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w.test substance suspended in PEG 400
All treated animals expressed toxic reactions: reduction of spontaneous activity
Two males and one female: abdominal position
Two males and one female: eyelid closure
Two males and one female: apathy
Two males died within 24 hours after treatment.

In a second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. test item suspended in PEG 400.
All treated animals expressed toxic reactions: reduction of spontaneous activity and apathy.
Two males and one female: eyelid closure
None of the treated animals died. Therefore, 2000 mg/kg b.w. test item was estimated to be a suitable dose.

Any other information on results incl. tables

Table 1: Summary of results

test group	dose mg/kg b.w.	sampling time (h)	PCE with micronuclei	range of micronuclei per 2000 PCE	PCE/NCE
suspending agent	0	24	0.06%	0-3	2000/1518
test article	200	24	0.07%	0-4	2000/1612
test article	670	24	0.06%	0 - 3	2000/1582
test article	2000	24	0.06%	0-4	2000/1907
cyclo- phosphamide	20	24	0.57%	6 -20	2000/1735
suspending agent	0	48	0.05%	0-3	2000/1601
test article	2000	48	0.06%	0-4	2000/1747

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

A study according OECD TG 474 was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was suspended in polyethylene glycol 400. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw . 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCE) per animal were scored.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw.

In a pre-experiment 2000 mg/kg was estimated to be suitable. The animals expressed toxic reactions. After treatment with the highest dose of the test article the number of NCEs was slightly increased as compared to the corresponding negative controls thus indicating that the substance had weak cytotoxic effectiveness.

In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used.

20 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.