3-methyl-4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-3-buten-2-one

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2000

Report date:

2000

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

July, 1997

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (1996 and 1997)

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Ionone, methyl-

EC Number:

215-635-0

EC Name:

Ionone, methyl-

Cas Number:

1335-46-2

Molecular formula:

C14H22O

IUPAC Name:

(1E)-1-(2,6,6-trimethylcyclohex-2-en-1-yl)pent-1-en-3-one

Test animals

Species:

mouse

Strain:

ICR

Details on species / strain selection:

ICR mice for the study were obtained from Harlan Sprague Dawley, Inc., Frederick, MD.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague Dawley, Inc., Frederick, MD
- Age at study initiation: 6 to 8 weeks old
- Weight at study initiation: Male: 25.0 - 29.6 g; Female: 25.2 - 29.7 g
- Housing: AAALAC-accredited facility: five mice per cage in polycarbonate cages which were maintained on stainless steel racks equipped with automatic watering manifolds and which were covered with filter material. Heat-treated hardwood chips were used for bedding.
- Diet: certified laboratory rodent chow ad libitum
- Water: tab water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 72 ± 3°F
- Humidity: 50 ± 20% relative humidity
- Photoperiod: 12 hour light/dark cycle

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Solvent used: corn oil
- Justification for choice of vehicle: compatibility of the vehicle with the test system animals
- Concentration of test material in vehicle: 20 mg/mL

Details on exposure:

The test article-vehicle mixture, the vehicle alone, or the positive control was administered by intraperitoneal injection at a constant volume of 20mL/kg body weight. Intraperitoneal injection was selected to maximize delivery of the test article to the test system. All mice in the experimental and control groups were weighed immediately prior to dose administration, and the dose volume was based on individual body weights. Mice were observed after dose administration for clinical signs of chemical effect.

Frequency of treatment:

Once

Post exposure period:

24 h (462.5 and 925 mg/kg bw); 24 and 48h (1850 mg/kg bw)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

462.5 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

925 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 850 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (462.5 and 925 mg/kg bw); 15 (1850 mg/kg bw)

Control animals:

yes

Positive control(s):

Positive Control Cyclophosphamide (CP), 50 mg/kg, 5 mice per sex, post exposure period 24 h

Examinations

Tissues and cell types examined:

Using oil immersion, 2000 polychromatic erythrocytes were scored for the presence of micronuclei which are defined as round, darkly staining nuclear fragments, having a sharp contour with diameters usually from 1/20 to 1/5 of the erythrocyte. The number of micronucleated normochromatic erythrocytes in the field of 2000 polychromatic erythrocytes was enumerated. The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes.

Details of tissue and slide preparation:

Immediately following sacrifice, 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 capped 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 by aspiration with a capillary pipet 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, stained with May-Gruenwald-Giemsa and permanently mounted.

Evaluation criteria:

In order to quantify the proliferation state of the bone marrow as an indicator of bone marrow toxicity, the proportion of polychromatic erythrocytes to total erythrocytes was determined for each animal and treatment group.

Statistics:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes was determined for each mouse and treatment group. Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, 1970). All analyses were performed separately for each sex and sampling time.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The incidence of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes scored and the proportion of polychromatic erythrocytes per total erythrocytes are summarized and presented for each treatment group by sacrifice time in Table 1. Individual animal data are presented in Tables 2 and 3. Slight to moderate reductions of 6% to 35% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to their respective vehicle controls. Reductions were observed in male and female dose groups 24 hours after treatment with 462.5, 925 and 1850 mg/kg and in female mice 48 hours after treatment with 1850 mg/kg. The reduction in the frequency of polychromatic erythrocytes in the bone marrow suggest that there was bioavailability of the test article to the bone marrow target tissue. A statistically significant increase in micronucleated polychromatic erythrocytes (8 MNPCEV10,000 PCE) in the test article treated group relative to the respective vehicle control group was observed in male mice at 24 hours after treatment with 925 mg/kg (p<0.05, Kastenbaum-Bowman Tables). However, this response is not considered biologically relevant since each of the five animals had no more then 3 MNPCE that are within the range of the historical solvent control (0-7 MN/2000 PCE/animal). No significant increase and no dose-responsive increase was observed in any other test article treated group regardless of dose level, sex, or bone marrow collection time. CP induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p<0.05, Kastenbaum-Bowman Tables).

Any other information on results incl. tables

Table 1:Summary of Bone Marrow Micronucleus Study Using Methyl ionone

 

Treatment	Sex	Time (hr)	Number of Mice	PCE/Total Erythrocytes (Mean ± SD)	Change from Control (%)	Micronucleated Polychromatic Number per 1000 PCEs (Mean ± SD)	Erythrocytes Number per PCEs Scored^1
Corn oil 20 mL/kg	M F	24 24	5 5	0.463 ± 0.04 0.489 ± 0.07	--- ---	0.1 ± 0.22 0.4 ± 0.42	1 / 10000 4 / 10000
Methyl ionone 462.5 mg/kg	M F	24 24	5 5	0.363 ± 0.06 0.377 ± 0.04	-22 -23	0.2 ± 0.27 0.2 ± 0.27	2 / 10000 2 / 10000
925 mg/kg	M F	24 24	5 5	0.344 ± 0.06 0.339 ± 0.04	-26 -31	0.8 ± 0.45 0.5 ± 0.50	*8 / 10000 5 / 10000
1850 mg/kg	M F	24 24	5 5	0.434 ± 0.07 0.319 ± 0.09	-6 -35	0.3 ± 0.27 0.7 ± 0.45	3 / 10000 7 / 10000
CP 50 mg/kg	M F	24 24	5 5	0.334 ± 0.01 0.329 ± 0.07	-28 -33	27.8 ± 4.19 40.4 ± 9.90	*278 / 10000 *404 / 10000
Corn oil 20 mL/kg	M F	48 48	5 5	0.451 ± 0.07 0.485 ± 0.03	--- ---	0.1 ± 0.22 0.2 ± 0.27	1 / 10000 2 / 10000
Methyl ionone 1850 mg/kg	M F	48 48	5 5	0.455 ± 0.03 0.385 ± 0.07	1 -21	0.1 ± 0.22 0.2 ± 0.27	1 / 10000 2 / 10000

 ¹* p≤0.05 (Kastenbaum-Bowman Tables)

 

 

Table 2: Induction of Micronucleated Polychromatic Erythroxcytes in Bone Marrow Cells collected 24 hours after a single dose of Methyl ionone

 

Treatment	Sex	Animal Number	PCE / Total Erythrocytes	Micronucleated PCE (Number / PCE scored)
Corn oil   20 ml/kg	M	101 102 103 104 105	0.413 0.531 0.435 0.475 0.461	0 / 2000 0 / 2000 0 / 2000 0 / 2000 1 / 2000
	F	106 107 108 109 110	0.486 0.379 0.510 0.556 0.512	0 / 2000 1 / 2000 2 / 2000 0 / 2000 1 / 2000
Methyl ionone   462.5 mg/kg	M	111 112 113 114 115	0.334 0.442 0.360 0.400 0.277	1 / 2000 0 / 2000 0 / 2000 1 / 2000 0 / 2000
	F	116 117 118 119 120	0.382 0.387 0.310 0.418 0.387	1 / 2000 1 / 2000 0 / 2000 0 / 2000 0 / 2000
925 mg/kg	M	121 122 123 124 125	0.259 0.330 0.411 0.406 0.312	1 / 2000 3 / 2000 2 / 2000 1 / 2000 1 / 2000
	F	126 127 128 129 130	0.336 0.389 0.306 0.372 0.293	1 / 2000 2 / 2000 0 / 2000 2 / 2000 0 / 2000
1850 mg/kg	M	131 132 133 134 135	0.500 0.494 0.426 0.329 0.421	0 / 2000 1 / 2000 0 / 2000 1 / 2000 1 / 2000
	F	136 137 138 139 140	0.205 0.325 0.327 0.289 0.449	2 / 2000 0 / 2000 2 / 2000 1 / 2000 2 / 2000
CP   50 mg/kg	M	141 142 143 144 145	0.346 0.342 0.318 0.345 0.320	66 / 2000 59 / 2000 44 / 2000 51 / 2000 58 / 2000
	F	146 147 148 149 150	0.334 0.384 0.219 0.374 0.332	73 / 2000 50 / 2000 99 / 2000 88 / 2000 94 / 2000

 

Table 3: Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells collected 48 hours after a single dose of Methyl ionone

 

Treatment	Sex	Animal Number	PCE / Total Erythrocytes	Micronucleated PCE (Number / PCE scored)
Corn oil   20 mL/kg	M	151 152 153 154 155	0.377 0.555 0.468 0.435 0.419	0 / 2000 1 / 2000 0 / 2000 0 / 2000 0 / 2000
	F	156 157 158 159 160	0.499 0.459 0.472 0.459 0.536	1 / 2000 0 / 2000 0 / 2000 1 / 2000 0 / 2000
Methyl ionone   1850 mg/kg	M	*171 162 163 164 165	0.492 0.426 0.478 0.443 0.438	0 / 2000 0 / 2000 1 / 2000 0 / 2000 0 / 2000
	F	166 167 168 169 170	0.400 0.327 0.461 0.431 0.307	0 / 2000 1 / 2000 0 / 2000 0 / 2000 1 / 2000

* = Replacement animal

Applicant's summary and conclusion

Conclusions:

The test item was negative in the mouse micronucleus assay (OECD 474).

Executive summary:

The test item was tested in the mouse micronucleus assay according to OECD 474. The assay was performed in two phases. The first phase, designed to set dose levels for the definitive study, consisted of a pilot toxicity study followed by a toxicity study. The second phase, the micronucleus study, evaluated the potential of the test article to increase the incidence of micronucleated polychromatic erythrocytes in bone marrow of male and female mice. In both phases of the study, test and control articles were administered in a constant volume of 20 mL/kg body weight by a single intraperitoneal injection. Corn oil was determined to be the solvent of choice based on the compatibility of the vehicle with the test system animals. The test article was soluble in corn oil at 20 mg/mL, the maximum concentration tested in the study. Dosing concentrations were delivered to the test system as clear light yellow solutions. In the pilot toxicity study, male mice were dosed with 1, 10, 100, or 1000 mg test article/kg body weight and male and female mice were dosed with 2000 mg/kg. Mortality was observed in 1/5 male mice and 2/5 female mice at 2000 mg/kg. Clinical signs following dose administration included lethargy and piloerection in males at 1000 mg/kg and in males and females at 2000 mg/kg. Convulsions were observed in males and females at 2000 mg/kg and prostration and crusty eyes were observed in female mice at 2000 mg/kg. In the toxicity assay, male and female mice were dosed with 1500 and 1750 mg test article/kg body weight. No mortality was observed in any male or female mice during the course of the study. Clinical signs following dose administration included lethargy and piloerection in males and females at 1500 and 1750 mg/kg. Due to the mortality at 2000 mg/kg in the pilot toxicity study, the high dose for the micronucleus test was set at 1850 mg/kg, which was estimated to be the maximum tolerated dose. In the micronucleus assay, male and female mice were dosed with 462.5, 925 or 1850 mg/kg body weight. Mortality was observed only in 1/15 male mice receiving 1850 mg/kg. This animal was replaced at the time of bone marrow collection with a replacement animal that also received 1850 mg/kg. All treatment groups had five animals for bone marrow analysis. Clinical signs following dose administration included: lethargy and piloerection in males and females at 925 and 1850 mg/kg and crusty eyes in male and female mice at 1850 mg/kg. Bone marrow cells, collected 24 and 48 hours after treatment, were examined microscopically for micronucleated polychromatic erythrocytes. Slight to moderate reduction (up to 35%) in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the test article-treated groups relative to the respective vehicle controls. These reductions suggest bioavailability of the test article to the bone marrow target. A statistically significant increase in micronucleated polychromatic erythrocytes (8 MNPCE/10000 PCE) in test article-treated group relative to the respective vehicle control group was observed in male mice 24 after treatment with 925 mg/kg (p<0.05, Kastenbaum-Bowman). However, this response is not considered biologically relevant, since each of the five animals had no more than 3 MPCE. These numbers of MNPCE are within the range of historical solvent control (0-7 MN/2000 PCE/animal). No significant increase and no dose responsive increase was observed in any other test article treated group regardless of dose level, sex, or bone marrow collection time. The results of the assay indicate that under the conditions described in this report, the test item did not induce a significant increase in micronucleated polychromatic erythrocytes in either male or female mice. The test item was concluded to be negative in the mouse micronucleus assay.