2-(3,5-dimethylhex-3-en-2-yloxy)-2-methylpropyl cyclopropanecarboxylate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 17, 2008 To April 02, 2009.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study according to OECD 474 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

Adopted July 21, 1997

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on 2009-10-29 / Signed on 2009-03-30.

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2-{[(2R)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate; 2-{[(2S)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate

EC Number:

700-118-9

Cas Number:

676532-44-8

Molecular formula:

C16H28O3

IUPAC Name:

2-{[(2R)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate; 2-{[(2S)-3,5-dimethylhex-3-en-2-yl]oxy}-2-methylpropyl cyclopropanecarboxylate

Test material form:

liquid

Details on test material:

- Description: Colourless liquid
- Formula: C16H28O3
- Molecular weight: 268,4 g/mol

Specific details on test material used for the study:

- Storage conditions: In the refrigerator at +2 to +8°C, protected from light

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

The mouse is an animal that has been used for many years as a suitable experimental animal in cytogenetic investigations. There are many data available from such investigatios, which my be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experimentations also may be useful for the design and the performance of the micronucleus test.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd.
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: Mutagenicity Experiment: males mean value: 37.0 g (SD+/-2.6 g), females mean value 31.4 g (SD+/-3.0 g). Plasma Sampling: males mean value 39.2 g (SD+/- 2.6 g).
- Assigned to test groups randomly: yes, based on equalization of group mean body weights.
- Housing: Single (Cage type: Makrolon Type I, with wire mesh top).
- Diet: A pelleted standard diet (Harlan Laboratories) was provided ad libitum.
- Water: Animals were allowed free access to tap water (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: no less than 5 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 25 - 85%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle.

IN-LIFE DATES: From: November 17, 2008 To April 02, 2009.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used:vegetable oil; Corn oil
- Justification for choice of solvent/vehicle: the vehicle was chosen to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
- Lot/batch no. (if required): C8267 (Sigma)
- Purity: 100%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
All dose formulations were administered at a dose volume of 10 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, positive control and vehicle: 24 hr
- For high dose: 24 and 48 hr

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Group I (Corn Oil)

Doses / concentrations 2

Dose / conc.:

187.5 mg/kg bw/day

Remarks:

Group II (Low dose)

Doses / concentrations 3

Dose / conc.:

375 mg/kg bw/day

Remarks:

Group III (Mid dose)

Doses / concentrations 4

Dose / conc.:

750 mg/kg bw/day

Remarks:

Group IV (High dose)

No. of animals per sex per dose:

- Pre-experiment: 2 animals/sex/dose (100, 500, 1000 and 750 mg/kg bw/day).
- Main Experiment: 6 animals/sex/dose (0, 187.5, and 375) and 12 animals/sex/dose at 750 mg/kg bw/day.

Control animals:

yes, concurrent vehicle

Positive control(s):

Positive control: cyclophosphamide (Sigma-Aldrich Vertriebs GmbH)
- Justification for choice of positive control(s): according to the OECD 474 guideline
- Route of administration: orally, once
- Doses / concentrations: 10 mL/kg bw
- Lot/batch no: C 0768
- Purity: > 98%

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. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe. The cell suspension was centrifuged at 1500 rpm (390 x G) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The mear was air dried and then stained with May-Grünwald (Merck, 64293 Darmstadt, Germany)/Giemsa (Merck, 64293 Darmstadt, Germany). Cover slips were mounted with EUKITT (Kindler, 79110 Freiburg, Germany). At least one slide was made from each bone marrow sample.
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 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.
- Micronuclei (M)
- The proportion of polychromatic erythrocytes to total erythrocytes was also recorded per 1000 erythrocytes per each animal (PCEs/ECs ratio).

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 or 2000 mg/kg as the upper limit for non-toxic test items. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The administered volume was 10 mL/kg bw.
Three adequately spaced dose levels by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

OTHER:
Blood serum samples from satellite control and high dose animals were taken 1 and 4 hours after oral dosing and the plasma analyzed for the presence of the test item.

Evaluation criteria:

A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group. Statisticals methods (nonparametric Mann-Whitney test will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical methods (nonparametric Mann-Whitney test) will be used as an aid in evaluating the results.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 4 animals (2 males and 2 females)/dose received orally a test substance formulated in corn oil at 100, 500, 1000 and 750 mg/kg bw.
- Solubility: no data
- Clinical signs of toxicity in test animals: No mortality was observed in any treatment group.
* The animals treated with 100 mg/kg bw did not express any toxic reactions.
* The animals treated with 500 mg/kg bw exhibited ruffled fur during the course of the study.
* The animals treated with 1000 mg/kg bw expressed toxic reactions, reduction of spontaneous activity, abdominal position and ruffled fur during the course of the study.
* The animals treated with 750 mg/kg bw exhibited ruffled fur during the course of the study.

Based upon the results of the dose range finding study, a dose of 750 mg/kg was estimated to be the maximum tolerated dose for male and female mice. Therefore, the doses of 187.5, 375 and 750 mg/kg were tested in the micronucleus test.


RESULTS OF DEFINITIVE STUDY
- Clinical Signs: no mortality was observed at all tested doses. The animals expressed clinical signs:
* The animals treated with the vehicle did not express any toxic reactions.
* The animals treated with 187.5 mg/kg bw exhibited ruffled fur during the course of the study.
* The animals treated with 375 mg/kg bw expressed ruffled fur during the course of the study.
* The animals treated with 750 mg/kg bw exhibited ruffled fur and reduction of spontaneous activity during the course of the study.

- 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.
- Ratio of PCE/NCE (for Micronucleus assay): the mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group.
- Appropriateness of dose levels and route: the bioanalysis showed that plasma samples of the animals treated with the high dose did not contain quantifiable amounts of the test item at both tested sampling intervals (1 and 4 h post treatment). The test item concentration in all plasma samples was below LLOQ. The ester compound of the test substance might have been hydrolyzed to the corresponding carboxylic acid by esterase enzymes, that are highly active in rodent plasma. Since no enzyme inhibitor was used during blood sampling, probably the hydrolysis of the test substance took place immediately. However, the bioavailability of the test substance could be confirmed, based on the clinical signs observed at all tested doses, which were taken to indicate that systemic absorption and exposure to the target tissue had been achieved.
- Statistical evaluation: statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test (see Table). In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with the test substance were near to the value of the vehicle control group.

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Executive summary:

In an in vivo micronucleus test, performed according to OECD 474 and GLP, NMRI male and female mice were exposed to the test substance in corn oil by oral gavage. In the dose range-finding study, 4 mice (2 males and 2 females) were exposed to the test substance at 100, 500 or 1000 mg/kg bw. Based upon the clinical signs of toxicity observed in the dose range finding study, a dose of 750 mg/kg bw was estimated to be the maximum tolerated dose for male mice. Therefore, twelve mice (6 males and 6 females) per group were treated with the vehicle, positive control (cyclophosphamide monohydrate) or 187.5, 375, or 750 mg/kg bw of the test substance and were euthanized 24 hours after treatment. In addition, six mice per group were treated with the vehicle or 750 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). 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.. The animals dosed with the test substance at 750 mg/kg bw and three controls (only corn oil) were bled at 1, and 4, hours post-dose (controls only at 1 hour).

 

Plasma was analyzed as a confirmation of systemic exposure to the test substance. All measured concentrations of the test substance in the plasma samples of animals dosed with the test substance were below the lower limit of detection. No mortality was observed in any of the treatment groups, but clinical symptoms were observed in mice of the 187.5, 375, and 750 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 750 mg/kg bw (estimated maximum tolerated dose) did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow of NMRI mice. Therefore, the test substance was concluded to be negative in the in vivo micronucleus assay.

This study is considered as acceptable and satisfies the requirement for chromosome aberration endpoint.