[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-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:

2009-02-03 to 2009-02-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according 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:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

EC Number:

275-156-8

EC Name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Cas Number:

71048-82-3

Molecular formula:

C13H20O

IUPAC Name:

[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one

Test material form:

liquid

Test animals

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: approximately 7 to 9 weeks old
- Weight at study initiation: Dose Range Finding Study: Males: 29.5 – 32.6 g; Females: 29.4 – 32.9 g. Definitive Micronucleus Study: Males: 30.3 – 34.8 g ; Toxicokinetic Portion of the Study: Males: 29.1 – 33.6 g
- Assigned to test groups randomly: yes, based on equalization of group mean body weights.
- Housing: Mice of the same sex were housed up to five per rodent Micro-Barrier cage. Cages were placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system. The purpose of this system was to supply uninterrupted positive air to each individual rodent Micro-Barrier cage and to capture the effluent air from each cage and re-filter the air (HEPA) prior to introducing the air back into the cage.
- Diet: A certified laboratory rodent chow (Harlan 2018C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, organophosphates and specified nutrients.
- Water: Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards.
- Acclimation period: no less than 5 days after receipt

ENVIRONMENTAL CONDITIONS
Animals were housed in an AAALAC accredited facility with a controlled environment of 50 ± 20% relative humidity and 72 ± 3°F temperature with a 12 hour light/dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Corn oil

Details on exposure:

All dose formulations were administered at a dose volume of 20 mL/kg by a single oral gavage. Oral gavage was performed using appropriate size disposable polypropylene syringes with gastric intubation tubes (needles).

Duration of treatment / exposure:

Single dose

Frequency of treatment:

Single dose

Post exposure period:

For low and mid-dose and positive control: 24 hr
For high dose and vehicle alone: 24 and 48 hr

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

437.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

875 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

5 male mice per dose group per sacrifice time

Control animals:

yes, concurrent vehicle

Positive control(s):

50 mg/kg bw Cyclophosphamide monohydrate

Examinations

Tissues and cell types examined:

Bone marrow erythrocytes

Details of tissue and slide preparation:

Bone Marrow Collection and Slide Preparation: In the micronucleus test, at the scheduled bone marrow collection time, five mice per treatment were euthanized by CO2 asphyxiation and the incision of the diaphragm. Immediately following euthanasia, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labeled centrifuge tube containing approximately 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation at approximately 100 x g for five minutes and the supernatant was drawn off, leaving a small amount of serum with the remaining cell pellet. The cells were resuspended in the remaining serum and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol, and one set of slides was stained with Acridine orange (a nucleic acid specific stain) and used in microscopic evaluation.
Scoring for Micronuclei: To control for bias, bone marrow slides were coded using a random number table by an individual not involved with the scoring process. Using a fluorescent microscope and medium magnification (400X; blue excitation filter in the range of 440-490 nm and barrier filter combination at 520 nm), an area of acceptable quality was selected such that the cells were well spread and stained. Using oil immersion (1000X), the following cell populations and cellular components were evaluated and enumerated:
- Polychromatic erythrocytes (PCEs): Two-thousand PCEs per each animal were screened (scored) for the presence of micronuclei resulting in evaluation of a total of 10,000 PCEs per each treatment group.
- Normochromatic erythrocytes (NCEs): The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) is determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs ratio). The incidence of MNCEs per 2000 PCEs was enumerated for each animal, but the results were not presented in this report or used in analysis of the test article clastogenic response since the primary target cells are the PCEs.
- The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (PCEs/ECs ratio).

Evaluation criteria:

All conclusions were based on sound scientific judgment taking into account biological relevance. However, as a guide to interpretation of the data, the test substance would have been considered to induce a positive response if a dose-responsive increase in the incidence of micronucleated polychromatic erythrocytes was observed and one or more doses were statistically elevated relative to the vehicle control (p ≤ 0.05, Dunnett’s t-test) at any sampling time.
The study was considered to be valid based on the following: (1) Animals dosed with the test substance exhibited clinical signs of toxicity and loss in body weights indicating that animals were exposed to the test substance. (2) Measurable concentrations of the test substance were detected in the plasma samples of animals dosed with the test substance indicating systemic exposure. (3) The incidence of micronucleated polychromatic erythrocytes in the vehicle (negative) control group did not exceed the historical vehicle control range. (4) The incidence of micronucleated polychromatic erthrocytes in the positive control group was significantly increased relative to the vehicle (negative) control group (p ≤0.05, Dunnett’s t-test).

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using one way analysis of variance (ANOVA, p≤ 0.050) and Dunnett’s t-test (post hoc) for group mean comparison and regression analysis (p ≤ 0.001) for detecting any trend using square-root transformed data (square root of MPCEs/2000 PCEs +1). All analyses were performed separately for each sampling time.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Dose Range-Finding Study:
In the dose range-finding study, one male at 1500 mg/kg and one female at 2000 mg/kg was found dead during the course of the study. Male mice exhibited lethargy, piloerection, hunched position and diarrhea at all dose levels (1100, 1250, or 1500 mg/kg) during the course of the study. In addition, palpebral closure was observed at 1250 and 1500 mg/kg. Female mice exhibited lethargy, piloerection and diarrhea at all dose levels (1500, 1750, or 2000 mg/kg) during the course of the study. In addition, hunched position was observed at 1750 mg/kg and palpebral closure was seen at 1750 and 2000 mg/kg during the course of the study. Reductions in mean (group) body weights up to 10.8% and 11.6% were observed in male and female groups, respectively. Based upon the results of the dose range finding study, a dose of 1250 mg/kg was estimated to be the maximum tolerated dose for male mice. Therefore, the doses of 435.7, 875 and 1250 mg/kg were tested in the micronucleus test. Furthermore, since no apparent differences (two fold difference in MTD) in the toxicity between male and female mice were observed, the micronucleus test was conducted using male mice only.
Definitive Micronucleus Study:
- Clinical Signs: No mortality was observed in any treatment group. All mice in the control groups and in test substance group at 437.5 mg/kg appeared normal during the course of the study. Piloerection and diarrhea was observed at 875 mg/kg, while lethargy, piloerection, diarrhea, hunched position, and palpebral closure were observed in mice at 1250 mg/kg. Reductions in mean (group) body weights of up to 8.4% and 9.6% were observed in the 24 and 48 hours treatment groups, respectively.
- Bone Marrow Evaluation: No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the 24 or 48 hr test substance groups relative to the respective vehicle control groups were observed. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in test substance groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration (p > 0.050, ANOVA, Dunnett’s t-test). CP, the positive control, induced a statistically significant increase in the incidence of micronucleated PCEs relative to the vehicle control (p ≤ 0.050, Dunnett’s t-test). The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met as specified in the protocol.
Systemic Exposure: The results indicate that the animals were exposed to the test substance and that systemic exposure increased with increasing dose of the test substance.

Applicant's summary and conclusion

Conclusions:

A single oral administration of the test substance at doses up to and including 1250 mg/kg (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male ICR mice. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay (OECD 474).

Executive summary:

In an in vivo micronucleus test, performed according to OECD 474 and in compliance with GLP, ICR mice were exposed to the test substance in corn oil by oral gavage. In the dose range-finding study, three male mice were exposed to the test substance at 1100, 1250 or 1500 mg/kg bw and three female mice were exposed to 1500, 1750 or 2000 mg/kg bw. Based upon the results of the dose range finding study, a dose of 1250 mg/kg bw was estimated to be the maximum tolerated dose for male mice. Furthermore, since a two fold difference in the MTD dose between male and female mice was not observed, the micronucleus test was conducted using male mice only. In the micronucleus test, five male mice per group were treated with the vehicle, positive control (cyclophosphamide monohydrate) or 437.5, 875 or 1250 mg/kg bw of the test substance and were euthanized 24 hours after treatment. In addition, five male mice per group were treated with the vehicle or 1250 mg/kg bw of the test substance and were euthanized 48 hours after treatment. At the time of euthanasia, femoral bone marrow was collected and bone marrow smears (slides) were prepared and stained with Acridine orange stain (a nucleic acid specific stain). Bone marrow cells (polychromatic erythrocytes (PCEs); 2000 PCEs/animal) were examined microscopically for the presence of micronuclei (micronucleated PCEs; MPCEs). A statistical analysis of data was performed using one way analysis of variance (ANOVA, significant level of p≤0.050) and Dunnett’s t-test (post hoc). In addition, the ratio of polychromatic erythrocytes to total of 1000 erythrocytes (PCEs/ECs ratio) was determined as an indicator of bone marrow exposure to the test substance and subsequently as a measure of test substance cytotoxicity. Additional 6 satellite groups per each test substance dose (each composed of 3 mice) were assigned for blood collection. These animals were dosed with the test substance at 437.5, 875 or 1250 mg/kg bw and were bled at 15 minutes, 30 minutes, 1, 2, 4, and 8 hours post-dose. Blood from the 3 main study animals, dosed with the vehicle or test substance, was also collected at 24 hours post-dose just prior to bone marrow collection. Plasma was analyzed as a confirmation of systemic exposure to the test substance.

Measurable concentrations of the test substance were detected in the plasma samples of animals dosed with the test substance indicating systemic exposure. No mortality was observed in any of the treatment groups, but clinical symptoms and weight loss (about 6 -10% relative to pretreatment weight) were observed in mice of the 875 and 1250 mg/kg bw dose groups. No appreciable reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the bone marrow were observed in the test substance groups relative to the respective vehicle control groups suggesting that the test substance did not inhibit erythropoiesis. No statistically significant increase in the incidence of micronucleated polychromatic erythrocytes in the test substance groups relative to the respective vehicle control groups was observed at 24 or 48 hours after dose administration. The positive control induced a statistically significant increase in the incidence of micronucleated polychromatic erythrocytes relative to the vehicle control. The number of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range. Based upon this, all criteria for a valid test were met.

Under the conditions of this test, a single oral administration of the test substance at doses up to and including 1250 mg/kg bw (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of male ICR mice. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.