4-propylcyclohexanone

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:

1990-06-28 to 1990-09-03

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

4-propylcyclohexanone

EC Number:

406-810-4

EC Name:

4-propylcyclohexanone

Cas Number:

40649-36-3

Molecular formula:

C9H16O

IUPAC Name:

4-propylcyclohexan-1-one

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Portage, Michigan, USA
- Age at study initiation: 35 - 37 days
- Weight at study initiation: 17-27 g (males); 17-24 g (females)
- Fasting period before study: animals were starved overnight prior to dosing
- Housing: They were housed in groups of no more than 3 animals of the same sex in polypropylene cages with wire mesh lids and solid floors containing wood shavings to a depth of approximately 1 cm.
- Diet: Special Diets Services Ltd, RM1. (E). SQC. pellets; ad libitum
- Water: Tapwater; ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24 °C
- Humidity (%): 55-60%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing preparations were made by suspending the test chemical in 0.5% (w/v) carboxymethyl cellulose (CMC) immediately prior to assay to give the top concentrations. Dilutions were made using CMC and the test chemical preparations used as shown in table 1 in the section "Any other information on materials and methods incl. tables".

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

375 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

750 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (CPA)
- Doses / concentrations: CPA was freshly dissolved in distilled water at 0.8 and 3.2 mg/mL and administered at 20 and 80 mg/kg.

Examinations

Tissues and cell types examined:

bone marrow/erythrocytes

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES: Test chemical and vehicle treated mice were killed in groups of 5 males and 5 females after 24 and 48 hours; CPA-treated mice were killed after 24 hours.

DETAILS OF SLIDE PREPARATION: Both femurs from each animal were exposed, removed and cleaned of adherent muscle and connective tissue so that the shank could be removed from the ends by using fine clippers or scissors. Disposable centrifuge tubes containing 1 mL of foetal bovine serum (FBS) were labelled with individual animal numbers. Some of the FBS was drawn into a syringe with a fresh needle, and this was used to aspirate the contents of the femur into the centrifuge tube. The tubes were centrifuged at 1250 x 'g' for 2-3 minutes, the serum was carefully aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of 3 slides labelled with study number, sampling time, sex, date of preparation and tag number. The latter served as the code so analysis could be conducted 'blind'. The end of a clean slide was placed across the drop, and a smear made by drawing the clean slide along the labelled slide. Slides were allowed to air-dry before being stained according to the modification of Gollapudi and Kamra. Slides were fixed for 5 minutes in absolute methanol. One slide from each set of 3 was taken (the others were kept in reserve unless required) and after rinsing several times in water, slides were stained for 10 minutes in filtered Gurr's Giemsa R66 stain diluted 1:6 (v/v) in distilled water. Slides were rinsed, and allowed to dry thoroughly before clearing in xylene for 3 minutes. When dry, the slides were mounted with coverslips.

METHOD OF ANALYSIS: Slides from the mice treated with 80 mg/kg CPA were initially checked as described below to ensure the system was operating satisfactorily. The slides from all dose groups sacrificed after 24 hours, and the slides from the top dose and control groups sacrificed after 48 hours were grouped according to sampling time and sex and handed to a person not connected with the dosing phase of the study to be analysed. Starting at one corner of the slide, and following a zig-zag pattern to avoid crossing the same area more than once, a series of random fields were observed under high power. Initially the relative proportions of polychromatic erythrocytes (PCE), seen as pale blue or blue/grey enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller yellow/orange-stained enucleate cells, were determined using tally counters until a total of at least 1000 (PCE plus NCE) cells had been analysed. Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these 2 phases of counting were recorded.

Evaluation criteria:

Evaluation criteria
The test chemical was to be considered as clearly positive in this assay if:
i) a statistically significant increase in the frequency of micronucleated PCE occurred for at least one dose at one kill time.
ii) the frequency of micronucleated PCE at such a point exceeded the historical vehicle control range.
iii) corroborating evidence was obtained e.g. increased but insignificant frequencies of micronucleated PCE at the other doses or kill time, and dose response profiles.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Two males receiving 1500 mg/kg died

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
An initial range finding study was conducted using small groups of male and female mice (3/sex/dose). Doses of 892.5, 1373, 2113, 3250 and 5000 mg/kg test item were tested. An LD50 of approximately 2300 mg/kg was determined in the rang finding study.
A dose equivalent to 50-80% of the LD50 is considered acceptable as a maximum dose level, so 1500 mg/kg (approx 65%) was chosen as an appropriate upper dose level for the micronucleus study.

Any other information on results incl. tables

Validity of study
The data confirm that:
i) the heterogeneity chi-square test provided evidence of acceptable variability between animals within each group.
ii) the incidence of micronucleated PCE in vehicle control groups fell within or close to the historical vehicle control range.
iii) at least 8 animals (males and females together) out of each group at each kill time were available for analysis. Two males receiving 1500 mg/kg died before sampling was possible. This observation indicated that it would not have been practicable to administer the test agent at a higher dose. The reduction in the number of animals available for sampling did not affect the validity of the assay because extra animals were dosed.
iv) the positive control chemical (CPA) induced a clear and statistically significant increase in the frequency of micronucleated PCE at both dose levels. In all of the CPA-treated mice, numbers of micronucleated PCE clearly exceeded those seen in vehicle controls.

Table 3: Summary of group mean data for test chemical, vehicle and positive controls

Treatment Group	Kill Time (hours)	Sex	Mean Ratio PCE/NCE	Group mean ffrequency of micronucleated PCE (per 1000)
				Per sex	Per treatment group
Vehicle control	24	M	0.82	0.90	0.75
		F	1.05	0.60
	48	M	1.10	0.50	0.45
		F	1.09	0.40
Test item 375 mg/kg	24	M	1.02	0.80	0.75
		F	1.14	0.70
Test item 750 mg/kg	24	M	1.20	0.60	0.70
		F	0.80	0.79
Test item 1500 mg/kg	24	M	1.23	0.40	0.60
		F	0.97	0.79
	48	M	1.09	0.30	0.30
		F	1.18	0.30
Positive control CPA 20 mg/kg	24	M	0.87	5.36	4.43
		F	0.99	3.49
Positive control CPA 80 mg/kg	24	M	0.70	43.54	44.87
		F	0.74	46.20

Applicant's summary and conclusion

Conclusions:

It is concluded that the test item was not able to induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 1500 mg/kg, a dose equivalent to approximately 65% of the 3 day LD50.

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 choice of dose levels was based on an initial toxicity range-finder study in which the test item made up in 0.5% (w/v) carboxymethyl cellulose (CMC) was administered to mice orally. Three male and 3 female mice each received the test article at a dose of 892.5, 1373, 2113, 3250 and 5000 mg/kg. The LD50 calculated from the pattern of mortality was found to be approximately 2300 mg/kg. For the micronucleus test the test item was made up as described and administered at 375, 750 and 1500 mg/kg to groups of 5 male and 5 female mice killed 24 and 48 hours after treatment. A small number of animals receiving the highest dose of the test item died prior to sampling indicating that it would not have been possible to administer the test chemical at a higher dose. The negative (vehicle) control in the study was 0.5% CMC also administered orally. Groups of 5 male and 5 female mice treated with this were killed and sampled after 24 and 48 hours. Cyclophosphamide (CPA), the positive control, was dissolved in water and administered orally at 20 and 60 mg/kg to groups of 5 male and 5 female mice which were killed after 24 hours. Positive control animals exhibited increased numbers of micronucleated polychromatic erythrocytes (PCE) such that the micronucleus frequency in both groups was significantly greater than in controls. Negative (vehicle) control mice exhibited normal ratios of PCE to NCE (normochromatic erythrocytes) and normal frequencies of micronucleated PCE within historical negative control ranges. Slides from all dose groups sacrificed after 24 hours, and slides from top dose and control groups sacrificed after 48 hours were analysed. Mice treated with the test item at all doses and sampling times exhibited ratios of PCE to NCE and frequencies of micronucleated PCE which were similar to vehicle controls. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test chemical at any sampling time. It is concluded that the item was not able to induce micronuclei in the polychromatic erythrocytes of the bone marrow of mice treated up to 1500 mg/kg, a dose equivalent to approximately 65% of the 3 day LD50.