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Toxicological information

Genetic toxicity: in vivo

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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:
15.9.2009-11.10.2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study was carried out in accordance with internationally valid GLP principles.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010
Reference Type:
publication
Title:
Tables for determining the statistical significance of mutation frequencies.
Author:
Kastenbaum, M.A., Bowman, K.O.
Year:
1970
Bibliographic source:
Mutation Res. 1970, 9: 527-549

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2A (1996)
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH S2B (1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dicyclohexylamine
- Physical state: clear colourless liquid
- Analytical purity: 99.7% (GC Area %, Certificate of Analysis), 98.5% (% Area, Analysis Results)
- Purity test date: 31.8.2009
- Lot/batch No.: 1369464 (Certificate of Analysis), 0001369464 (Analysis results, Chemir Analytical Services, Inc.)
- Stability under test conditions: stable
- Storage condition of test material: Ambient (15 to 30°C), in the dark under inert gas

Test animals

Species:
mouse
Strain:
ICR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 6-8 week
- Weight at study initiation: 28.9-34.4 g (Definitive Micronucleus Study)
- Assigned to test groups randomly: Yes. Randomization procedure based on equalization of group mean body weight.
- Housing: Rodent Micro-Barrier cage placed on the racks equipped with an automatic watering system and Micro-VENT full ventilation, HEPA filtered system.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no les than 5 days after receipt

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72 +/- 3 °F
- Humidity (%): 50 +/- 20 %
- Air changes (per hr): at least 10 changes of fresh HEPA-filtered air per every hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of vehicle: The corn oil was selected based on good solubility of the test article in the vehicle and compability of the vehicle with the test system and route administration.
- Concentration of test material in vehicle: 2.5, 5, 10 mg/mL
- Lot/batch no. (if required): 9539J, M.P. Biomedicals, LLC
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test article dose formulations were prepared for each phase of the study prior to dose administration. The amount of the test article weighted for each concentration was corrected for test article purity using a correction factor of 1.02. An appropriate amount of the test article was weighted. An appropriate volume of vehicle was added to the test article untiil the final volume was achieved. Each formulation was votexed for 2 minutes.
Duration of treatment / exposure:
24 and 48 hr
Frequency of treatment:
single oral administration
Post exposure period:
24 and 48 hr
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Test article:
- 50 mg/kg: 5 males
- 100 mg/kg: 5 males
- 200 m/kg: 15 males (Including 5 replacement mice to ensure the availability of five mice for micronucleus analysis)
Control animals:
yes, concurrent vehicle
Positive control(s):
- cyclophosphamide monohydrate
- Route of administration: no data
- Doses / concentrations: 50 mg/kg

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Based primarily on the mortality in DRF study, a dose of 200 mg/kg was determined to be maximum tolerated dose and was tested as the highest dose in the definitive micronucleus study. Mice were exposed to two lower doses - 100 mg/kg and 50 mg/kg.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Twenty four hour post-dose femoral bone marrow was collected from animals dosed with negative or positive control article and from animals dosed with the test article at 50, 100 and 200 mg/kg. At 48 hr post-dose, bone marrow was collected from animals dosed with the vehicle control article and the test article at 200 mg/kg.

DETAILS OF SLIDE PREPARATION: The bone marrow was aspirated into a syringe containing fetal bovine serum. The bone marrow cells were transferred to a labeled centrifuge tube containing approx. 1 mL fetal bovine serum. The bone marrow cells were pelleted by centrifugation and the supernatant was drawn off, leaving a small amount of serum with remaining cell pellet. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse and fixed in methanol. One set of slides was stained with May-Gruenwald-Giemsa stain, permanently mounted, and used in microscopic evaluation. The second set was discarded following succesful microscopic evaluation of the first set of slides.

METHOD OF ANALYSIS: Using a light microscope and a medium magnification (400X) an area of acceptable quality was selected. Using oil immerion (1000X), the evaluation was performad. Polychromatic erythrocytes (PCEs), Normochromatic erythrocytes (NCEs) and Micronuclei (M) were evaluated and enumerated. In addition, the proportion of polychromatic erythrocytes to total of 1000 erythrocytes (PCE/ECs ration) was determined per each animal and group.
Evaluation criteria:
Evaluation of Bioavailability/Cytotoxicity - The proportion of PCEs to total of 1000 erythrocytes was determined for each mouse and treatment group (PCEs/ECs ratio).

Evaluation of Genotoxocity - The incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per 10000 PCEs for each treatment group was determined.
Statistics:
Statistical significance was determinated using the Kasten-Bowman tables which are based on the binomial distribution (Kasten and Bowman, 1970).

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: 100, 200, 400 and 600 mg/kg
- Solubility: good solubility of the test article in the vehicle
- Clinical signs of toxicity in test animals: Lethargy and piloerection were observed at 200 mg/kg in male and female mice. All male and female mice at 400 mg/kg, and 600 mg/kg were convulsing immediately after dose administration and were found dead within 2 hours post-dose. The exception of this was one female mouse at 400 mg/kg that was humanely euthanized, approx. 5 hours post-dose, after being lethargic, hunched, with piloerection and irregular breathing and after having convulsions.
- Evidence of cytotoxicity in tissue analyzed: The magnitude of PCE/ECs ratio reductions indicated that the test article was bioavailable but that did not markedly inhibit erythropoiesis.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no increase in the incidence
- Ratio of PCE/NCE (for Micronucleus assay): no reduction was observed
- Statistical evaluation: no statistically significant changes

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Dicyclohexylamine was concluded to be negative in the micronucleus test using male ICR mice.
Executive summary:

Under the conditions ofthe assay described in report, a single oral administration of Dicyclohexylamine at doses up to and including 200 mg/kg did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow. Therefore, Dicyclohexylamine was concluded to be negative in the micronucleus test using male ICR mice.