4-methylpent-3-en-2-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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1994

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:

4-methylpent-3-en-2-one

EC Number:

205-502-5

EC Name:

4-methylpent-3-en-2-one

Cas Number:

141-79-7

Molecular formula:

C6H10O

IUPAC Name:

4-methylpent-3-en-2-one

Details on test material:

- Name of test material (as cited in study report): mesityl oxide
- Physical state: clear liquid
- Analytical purity: 98.4% (09/19/91); 98.6% (12/5/91)
- Impurities (identity and concentrations): 1.4% as 4-methyl-4-pentene-2-one
- Lot/batch No.: HAEL No_ 91-0033; KAN 900582

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 Inc., St. Constant, Canada
- Age at study initiation: , the males were approximately 65 days old and the females 56 days old at dosing
- Weight at study initiation: the weight range of the mice was 24.2 to 33.8 g.
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: no
- Housing: two or three per cage
- Diet : Agway Prolab® RMII 3000 pellets ad libitum
- Water : tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 72 ± 4
- Humidity (%): 50 ± 20
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To: 11/12/91 to 03/18/92

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: solubility
- Concentration of test material in vehicle:
The test substance was analyzed using gas chromatography/mass spectrosoopy and the structure was confirmed as that of 4-methyl-3-pentene-2-one. The dosing solutions used in the micronucleus studies were analyzed by gas chromatography to confIrm their concentrations. The results are as follows:
Nominal Concentration Analytical Concentration Analytical/Nominal Percent
mg/g mg/ml
17.0 15.69 92.3
34.0 34.56 101.6
68.0 64.09 94.3
The mean ratio of the analytical concentration/nominal concentration was 0.928. Thus, the actual dose levels of mesityl oxide in these studies are lower by about 7 percent than the nominal dose levels given in the tables.
- Amount of vehicle (if gavage or dermal): no data

Duration of treatment / exposure:

Single administration

Post exposure period:

24, 48 and 72 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
170, 340 and 680 mg/kg bw
Basis:

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (40 mg/kg)

Examinations

Tissues and cell types examined:

Polychromatic erythrocytes from the bone marrow

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels used in the micronucleus assay were chosen based upon the LD50 study. The initial study was begun on October 8, 1991. Based upon the reported ip LD50 for mesityl oxide of 354 mg/kg (Krasavage et al., 1982), groups of three males and three females were administered intraperitoneal doses of 500, 250 and 125 mg/kg mesityl oxide (in corn oil). Within 30 minutes after dosing, the males (3/3) receiving the high dose were ataxic and 1/3 high-dose, 1/3 low-dose and 2/3 mid-dose animals were lethargic. At 1.5 hours, none of the animals were ataxic, however, 1/3 of the low-dose and 2/3 mid-dose animals were lethargic. At 7 hours, all clinical signs were normal for the males and all survived the three-day observation period. For the females, those receiving the high dose (3/3) were ataxic at 15 minutes, and 2/3 of the mid-dose and 2/3 of the low-dose animals were lethargic. By 1.5 hours post-dose, 2/3 of the high-dose and one of the mid-dose animals were observed to be lethargic but none were ataxic. At 7 hours, all the females appeared normal and all survived the three-day observation period except one which died as the result of an accident.
A second study was begun on October 9, 1991 in which three males and three females were dosed at 750 mg/kg. All males and females were ataxic and lethargic at approximately 30 minutes after dosing, and one female died during this interval. By seven hours, all survivors were normal except one female that exhibited unkempt hair coat. Some ocular discharge was seen on the second day.
On October 21, 1991, groups of three males and three females were dosed with 1,000 and 875 mg/kg mesityl oxide. All six animals in the groups dosed with 1,000 mg/kg were found dead within 40 minutes. Two of the three males and one of the three females dosed at 875 mg/kg died during the first hour and another of the females was found dead at 48 hours. The remaining animals, 1 male and 1 female, survived the three-day observation period. The survival data were plotted on linear coordinates and the LD50 was estimated to be 850 mg/kg.
Therefore, the high dose in the micronucleus study was set at 680 mg/kg (0.8 times LD50), the mid-dose was set at one half of the high dose, or 340 mg/kg, and the low dose was one fourth of the high dose, or 170 mg/kg.

DETAILS OF SLIDE PREPARATION:
At the appropriate harvest time, the animals were euthanatized with CO2 and the adhering soft tissue and epiphyses of both femurs were removed. The marrow was flushed from the bone and transferred to centrifuge tubes containing 2 ml fetal calf serum (one tube for each animal). Following centrifugation to pellet the tissue, most of the supernatant was drawn off, the cells were resuspended, and the suspension spread on glass microscope slides and air-dried. The slides were then fixed in methanol, stained in Wright's-Giemsa stain, and rinsed in deionized water (Schmid, 1975). After being air-dried, the slides were coverslipped using mounting medium. Each slide was then re-labeled using ccded information generated by a computerized random number generation scheme. Thus, the identity of each slide was not known to those evaluating the slides.

Slides were scored for micronuclei and the relative numbers of polychromatic (PCE) and normochromatic (NCE) cells. One thousand PCEs per animal were routinely scored, except in cases of significant bone marrow depression. The frequency of micronucleated cells was expressed as number of micronucleated cells based on the total number of PCEs present in the microscopie fields. The frequency of PCEs versus NCEs was determined by counting the NCEs observed while scoring the 1,000 PCEs for micronuclei.

Evaluation criteria:

The criteria for determining a positive response involved a statistically significant dose-related increase in micronucleated PCEs, or the detection of a reproducible and statistically significant increase in micronucleated PCEs for at least one dose level. A test article that induced neither a statistically significant dose response nor a statistically significant and reproducible increase at one dose level was considered negative. In either case, the final decision was based on the scientific judgment of the Study Director.

Bone marrow depression was defined as a statistically significant decrease in the percent PCE ([PCE/PCE + NCE] x 100) in treated animals compared to the corresponding untreated control group.

Statistics:

The data were examined using plots, descriptive statistics, rank transformation, analysis of variance, Tukey's HSD Test and Dunnett's t-test. Statistical significance was detected using an alpha risk of <= 0.05.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

mortality and bone marrow depression at the top dose

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Mortality and clinical signs
One female in the 24-hour harvest time group, two females in the 48-hour group and one female in the 72-hour group died within two hours of being dosed at 680 mg/kg. All other animals survived the treatments and provided bone marrow specimens for analysis at the appropriate harvest times. Hypothermia (1/5 females at 680 mg/kg), ocular discharges (all surviving mesityl oxide treated animals) and ptosis (all surviving mesityl oxide treated animals) were observed on the day of dosing_ Ocular discharges and ptosis were observed on the day following dosing for the 680 mg/kg animals. All animals appeared normal on subsequent examinations. Ataxia and lethargy were not observed during the micronucleus study.
- Induction of micronuclei (for Micronucleus assay):
The test article did not indure significant increases in micronucleated polychromatic erythrocytes (MNPCEs) in either the male or female mice, at the 24-, 48- or 72-hour harvest times when compared with the concurrent negative controls. Conversely, the positive control, CPA, induced highly significant increases in MNPCEs in both the male and female mite at 24 hours with concurrent bone marrow depression
- Ratio of PCE/NCE (for Micronucleus assay):
The test article produced statistically significant bone marrow dépression (decrease in the (PCE/NCE+PCE) ratio) at the 72-hour harvest time for the high-dose male mice.

Applicant's summary and conclusion

Conclusions:

Interpretation of results : negative
Mesityl oxide is negative in the in vivo mouse bone marrow micronucleus assay at a dose level which produced deaths in the female animals and statistically significant bone marrow depression in the males.

Executive summary:

The ability of mesityl oxide to induce micronuclei in bone marrow polychromatic erythrocytes was evaluated after a single administration by intraperitoneal injection at 0, 170, 340, or 680 mg/kg to male and female Swiss CD-1 mice. The high dose of 680 mg/kg (80 % of the LD₅₀) was chosen following a three-day study in which the LD₅₀,was determined to be 850 mg/kg. The animals were dosed with the test substance and were sacrificed at 24, 48, and 72 hours after dosing for extraction of the bone marrow. Ten animals (five males and five females) were randomly assigned to each dose harvest time group. A negative control group (corn oil only) was included at each harvest time and a positive control group, dosed with cyclophosphamide (CPA) at 40 mg/kg, was included at the 24-hour sampling time only.

Mesityl oxide did not induce significant increases in micronucleated polychromatic erythrocytes (MNPCEs) in either the male or female mice, at the 24-, 48- or 72-hour harvest times when compared with the concurrent negative controls. Conversely, the positive control, CPA, induced highly significant increases in MNPCEs in both the male and female mice at 24 hours with concurrent bone marrow depression. In the micronucleus test, at the 680 mg/kg dose, one female in the 24-hour harvest time group, two females in the 48-hour harvest time group and one female in the 72-hour harvest time group died. The test article produced statistically significant bone marrow depression (decrease in the (PCE/NCE+PCE) ratio) at the 72-hour harvest time for the high-dose male mice. Therefore, it is concluded that mesityl oxide is negative in the in vivo mouse bone marrow micronucleus assay at a dose level which produced deaths in the female animals and statistically significant bone marrow depression in the males.