9-Decenamide, N,N-dimethyl-

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:

23 April 2014 to 12 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study conducted according to OECD 474 test guideline.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N-dimethyldec-9-enamide

EC Number:

806-919-0

Cas Number:

1356964-77-6

Molecular formula:

C12H23NO

IUPAC Name:

N,N-dimethyldec-9-enamide

Test material form:

other: colourless clear liquid

Details on test material:

- Name of test material (as cited in study report): Steposol MET-10U
- Physical state: clear colourless liquid
- Analytical purity: 98.5%
- Lot/batch No.: 5104-14-006
- Storage condition of test material: room temperature protected from light

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., Portage, MI
- Age at study initiation: 52 days
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Individually in stainless steel caging
- Diet (e.g. ad libitum): Rodent lab diet ad libitum
- Water (e.g. ad libitum): Reverse osmosis drinking water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 50 +/- 20
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 24 April 2014 To: 12 June 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: none

Duration of treatment / exposure:

Once daily for 3 consecutive days

Frequency of treatment:

3 doses

Post exposure period:

1 day

Doses / concentrations

Remarks:

Doses / Concentrations:
75, 150, 300 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

6

Control animals:

yes

Positive control(s):

- cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Gavage
- Doses / concentrations: 60 mg/kg formulated in distilled water

Examinations

Tissues and cell types examined:

Bone marrow polychromatic erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Maximum tolerated dose level

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Treated daily for 3 days and sample 1 day after final dose

DETAILS OF SLIDE PREPARATION: Bone marrow smears were prepared byplacing approximately 1 drop of cell suspension onto a minimum of 2 appropriately labeled, clean microscope slides. Each slide was coded so that the treatment group would not be revealed during subsequent analysis. The slides were air dried, fixed in 100% methanol for approximately20 minutes, and allowed to air dry a second time. Prior to analysis, the coded slides were stained with acridine orange (A/O) staining.
solution

METHOD OF ANALYSIS: Two separate evaluations were made for each slide: 1) a total of 1000 erythrocytes (both polychromatic erythrocytes [PCEs] and normochromatic erythrocytes [NCEs]) per animal were counted and the PCE: total erythrocytes [TE] ratio was determined; and 2) the
number of micronucleated PCEs from a total of 2000 PCEs was scored per animal.

OTHER:

Evaluation criteria:

Criteria for Positive Response:
The test substance may be considered positive for induction of micronuclei if a dose-related increase in the percentage of PCEs with micronuclei is observed and a statistically significant increase relative to the negative control is seen at one or more dose levels. Statistical significance will not be the only factor in determining a positive response. Other criteria, such as historical control values, that take into account the biological relevance of the results will be considered in the final evaluation.
Criteria for Negative Response:
The test substance is considered to be negative for inducing the formation of micronuclei in PCEs if there is no statistically significant increase in the
percentage of PCEs with micronuclei when compared to the concurrent negative control at any dose level.
Criteria for Equivocal Response:
Cases which do not clearly fit into the positive or negative criteria may be judged equivocal. In these cases the Principal Investigator, based on sound
scientific judgment, may take additional factors into consideration in evaluating the test results. As a general rule the biological relevance of any result will be considered first.

Statistics:

The percentages of micronucleated cells in PCEs and in the ratio of PCEs to TEs for the test substance-treated and vehicle control group (Group 1) were subjected to a parametric one-wayANOVA (Snedecor and Cochran, 1980) to determine intergroup differences. If the ANOVA revealed statisticallysignificant (p≤0.05) intergroup variance, Dunnett's test (Dunnett, 1964) was used to compare the test substance-treated groups to the vehicle control group (Group 1). In addition, the positive control and vehicle control groups were compared using a separate parametric one-wayANOVA. Statistical significance was assessed at a 95% confidence level (p≤0.05).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 300, 400, 500 mg/kg
- Clinical signs of toxicity in test animals: Clinical observations were noted.
- Rationale for exposure: LD50 is 550 mg/kg

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The test substance did not produce an increase in the mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) compared to the negative control group.
- Ratio of PCE/NCE (for Micronucleus assay): No bone marrow cytotoxicity(decreases in the ratio of polychromatic to total erythrocytes,
PCE:TE ratio) was noted in anytest substance-treated group.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
Based on the results of the definitive phase of this study, oral administration of the test substance once daily to Crl:CD-1 mice for 3 consecutive days resulted in a negative response for induction of bone marrow micronuclei at dosage levels up to 300 mg/kg/day.

Executive summary:

OBJECTIVE

The objective of this studywas to assess the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCEs) in mouse bone marrow following 3 consecutive days of treatment administered byoral gavage. This assayevaluated compounds for in vivo clastogenic activity and/or disruption of the mitotic apparatus.

STUDY DESIGN

RANGE-FINDING PHASE

The test substance was administered orally bygavage once daily for 3 consecutive days to 3 groups (Groups 2-4) of Crl:CD1(ICR) mice. Dosage levels were 300, 400, and 500 mg/kg/day for Groups 2, 3, and 4, respectively. A concurrent control group (Group 1) received deionized water on a comparable regimen. The dose volumes based upon specific gravity were 0.57, 0.34, 0.46, and 0.57 mL/kg for all Groups 1-4, respectively. Each group (Groups 1-4) consisted of 3 animals/sex. All surviving animals were euthanized on study day 3 and discarded.

All animals were observed twice daily for mortalityand moribundity. Clinical examinations were performed at the time of dose administration and approximately 1 to 2 hours following dosing. Detailed physical examinations were performed and individual bodyweights were collected at least once weekly during acclimation, 1 week (± 2 days) prior to randomization, at the time of animal selection for randomization, on study day 0, and prior to the scheduled euthanasia. Individual food weights were recorded for 1 week (± 2 days) prior to randomization and from study day 0 until just prior to the scheduled euthanasia.

DEFINITIVE PHASE

The test substance was administered orally by gavage once daily for 3 consecutive days to 3 groups (Groups 2-4) of Crl:CD1(ICR) mice. Dosage levels were 75, 150, and 300 mg/kg/dayfor Groups 2, 3, and 4, respectively. A concurrent negative control group (Group 1) received deionized water on a comparable regimen. A positive control group (Group 5) received a single oral dose of 60 mg/kg cyclophosphamide monohydrate (CPS) at a dose volume of 10 mL/kg on studyday2, the day prior to the scheduled euthanasia. The dose volumes based upon specific gravity were 0.34, 0.09, 0.17, and 0.34 mL/kg for Groups 1-4, respectively. Each group (Groups 1-5) consisted of 6 animals/sex. All animals were euthanized on studyday3 and discarded following bone marrow collection.

All animals were observed twice daily for mortalityand moribundity. Clinical examinations were performed at the time of dose administration and approximately 1 to 2 hours following dosing. Detailed physical examinations were performed and individual bodyweights were collected at least once weekly during acclimation, 1 week (± 2 days) prior to randomization, at the time of animal selection for randomization, on study day 0, and prior to the scheduled euthanasia. Individual food weights were recorded for 1 week (± 2 days) prior to randomization and from study day 0 until just prior to the scheduled euthanasia. Bone marrow collection for micronucleus evaluation was performed for 5 of 6 animals/sex/group at the scheduled euthanasia (study day 3). All animals were discarded without necropsy. Bone marrow smears were prepared and the coded slides were counted for polychromatic, normochromatic, and micronucleated polychromatic erythrocytes following the final bone marrow sample collection on study day 3.

RESULTS

RANGE-FINDING PHASE

A single 300 mg/kg/day group female (no. 15542) was euthanized in extremis and a single 500 mg/kg/daygroup male (no. 15472) was in moribund condition and was scheduled to be euthanized in extremis on study day 2; however the animal died prior to euthanasia. Clinical observations noted for these animals on the dayof death consisted of prostration, dermal atonia, pale or cool extremities, cool body, labored respiration, gasping, decreased respiration rate, and/or yellow material on selected bodysurfaces (around mouth, urogenital area, anogenital area, and/or ventral trunk). Although no macroscopic findings were noted for either of the 2 early death animals and a microscopic evaluation was not conducted, the early death of the 500 mg/kg/day group male was considered potentially test substance-related. Test substance-related clinical observations in animals surviving to the scheduled euthanasia were limited to single incidences of yellow material around the urogenital area and/or dermal atonia in the 400 and 500 mg/kg/day group males Test substance-related mean bodyweight losses were noted in all test substance-treated male and female groups and correlated with lower mean food consumption in all male groups and in the 300 and 500 mg/kg/daygroup females.

DEFINITIVE PHASE

There were no test substance-related effects on survival. Test substance-related clinical observations of rales and yellow material on selected body surfaces (around the mouth and/or urogenital area) were noted in all test substance-treated male and female groups as earlyas study day1, at time of or 1-2 hours post-dosing. Additional test substance-related clinical observations on study days 2 and 3 included dermal atonia, decreased defecation, small feces, labored respiration, gasping, pale extremities, swollen abdominal area, and/or yellow material on the ventral trunk. Test substance-related mean bodyweight losses were noted in all test substance-treated male and female which resulted in 10.4% to 16.3% lower mean bodyweights in male groups and 10.2% to 12.1% lower mean bodyweights in the female groups at the end of the study when compared to the control group. The bodyweight losses correlated with lower food consumption in all test substance-treated male and female groups. The test substance did not produce an increase in the mean number of micronucleated polychromatic erythrocytes (%MN-PCEs) compared to the negative control group. No bone marrow cytotoxicity(decreases in the ratio of polychromatic to total erythrocytes, PCE:TE ratio) was noted in any test substance-treated group. Therefore, the test substance met the criteria for a negative response for bone marrow cytotoxicity and clastogenicity and/or disruption of the mitotic apparatus under the conditions of this assay.

CONCLUSIONS

Based on the results of the definitive phase of this study, oral administration of the test substance once daily to Crl:CD-1 mice for 3 consecutive days resulted in a negative response for induction of bone marrow micronuclei at dosage levels up to 300 mg/kg/day.