N-cyclohexylcyclohexanamine 2-[1-[[[(1R)-1-[3-[(1E)-2-(7-chloroquinolin-2-yl)ethenyl]phenyl]-3-[2-(2-hydroxypropan-2-yl)phenyl]propyl]sulfanyl]methyl]cyclopropyl]acetate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15-09-2009 to 11-10-2009

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Dicyclohexylamine

EC Number:

202-980-7

EC Name:

Dicyclohexylamine

Cas Number:

101-83-7

IUPAC Name:

N-cyclohexylcyclohexanamine

Test material form:

liquid

Test animals

Species:

mouse

Strain:

other: Hsd: ICR (CD-1)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Frederick MD
- Age at study initiation: 6- 8 weeks
- Weight at study initiation:
Dose Range Finding Study: Males: 28.6 - 31.3 g
Females: 24.4 - 28.5 g
Definitive Micronucleus Study: Males: 28.9 - 34.4 g
Toxicokinetic portion of the Study: Males: 28.2 - 35.3 g

- Assigned to test groups randomly: yes, base don equalisation of group mean body weights
- Fasting period before study: no
- 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 (e.g. ad libitum): A certified laboratory rodent chow (Harlan 20 I 8C Certified Global Rodent Diet) was provided ad libitum. The food was analyzed by the manufacturer for the concentrations of specified heavy metals, atlatoxin, chlorinated hydrocarbons, organophosphates and specified nutrient
- Water (e.g. ad libitum): Animals were allowed free access to tap water, which meets U.S. EPA drinking water standards [water source is Washington Suburban Sanitary Commission (WSSC) Potomac Plant]. Drinking water was monitored at least annually for levels of specified microorganisms, pesticides, heavy
metals, alkalinity and halogens.

- Acclimation period: Virus antibody-free (VAF) mice were obtained from a supplier that monitored mice for evidence of ectoparasites, endoparasites, pathogenic bacteria, mycoplasmas, and appropriate murine viruses and were quarantined (acclimatized) for no less than 5 days after receipt. At BioReliance, mice were observed each day for signs of illness and other conditions of poor health. All mice were judged to be healthy prior to utilization in the study

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

IN-LIFE DATES: From:15/09/2009 To: 11/10/2009

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: based on solubility test
- Concentration of test material in vehicle: 2.5, 5, 10, 20 and 30 mg/mL
- Amount of vehicle (if gavage or dermal): 20mL/kg
- Type and concentration of dispersant aid (if powder): N/A
- Lot/batch no. (if required):
- Purity:

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Solubility Test: A solubility test was conducted to demonstrate the solubility of the test article in corn oil at I 00 rng/ml.. The test was conducted by combining I 00 mg of the test article with 1 mL of corn oil. The mixture was vortexed for I minute. A pale yellow clear solution was obtained as a result that can be withdrawn with a 22 G needle. The formulation was examined after 30 minutes of preparation and was observed to be a clear pale yellow solution. Based on this. corn oil was determined to be the appropriate test article vehicle.

Preparation of Test Article Dose Formulations: The test article dose formulations were prepared fresh for each phase of the study prior to dose administration. The amount of test article weighed for each concentration was corrected for test article purity using a correction factor of 1.02. All formulations for the dose range finding study (5, 10, 20 and 30 mg/mL), and all formulations for the definitive micronucleus study (2.5, 5 and I0 mg/mL) were prepared as follows:

I. An appropriate amount of the test article was weighed separately for each concentration into a container of appropriate size.
2. An appropriate volume of vehicle was added to the test article until the final volume was achieved.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

N/A

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

dose range finding

Doses / concentrations 2

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

dose range finding

Doses / concentrations 3

Dose / conc.:

400 mg/kg bw (total dose)

Remarks:

dose range finding

Doses / concentrations 4

Dose / conc.:

600 mg/kg bw (total dose)

Remarks:

dose range finding

Doses / concentrations 5

Dose / conc.:

50 mg/kg bw (total dose)

Remarks:

main study

Doses / concentrations 6

Dose / conc.:

100 mg/kg bw (total dose)

Remarks:

main study

Doses / concentrations 7

Dose / conc.:

200 mg/kg bw (total dose)

Remarks:

main study

No. of animals per sex per dose:

dose range finding: Total of 6 animals per sex were assigned to each treatment group. Six male and 6 female mice each were exposed to either the vehicle control or to Dicyclohexylamine at I 00, 200, 400 and 600 mg/kg. Due to mortality at two highest doses, 3 animals dosed with the vehicle or the test article at
I00 and 200 mg/kg were euthanized at 24 hr post-dose and the remaining 3 animals per sex and treatment groups were euthanized at 48 hr post-dose.

main study: 5 males per dose per timepoint (24 and 48 hrs)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide 50mg/kg- oral gavage 24 hrs sampling

Examinations

Tissues and cell types examined:

Bone marrow: polychromatic erythrocytes, normochromatic erythrocytes

Details of tissue and slide preparation:

IN the DRF study and definitive study, bone marrow was collected at 24 and 48 hr post-dose. ln both studies, animals were euthanized by exposure to C02 followed by 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 1mL fetal bovine serum. The tubes were identified by labels containing the study. group and animal numbers. The bone marrow cells were pelleted by centrifugation at approximately l 00 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 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 successful microscopic evaluation of the first set of slides.

Evaluation criteria:

Two-thousand PCEs per mouse were screened (scored) for the presence of micronuclei resulting in evaluation of a total of I 0,000 PCEs per each treatment group. The number of NCEs and micronucleated NCEs (MNCEs) in the field of 1000 total erythrocytes (ECs) was determined for each animal in order to determine the proportion of polychromatic erythrocytes to total erythrocytes (PCEs/ECs). In addition, the proportion of polychromatic erythrocytes to total of 1000 erythrocytes (PCE/ECs ratio) was determined per each animal and group.
In the definitive study, the incidence of micronucleated polychromatic erythrocytes per 2000 PCEs for each mouse and per l 0,000 PCEs for each treatment group was determined.

Statistics:

Statistical significance was determined using the Kastenbaum-Bowman tables which are based on the binomial distribution (Kastenbaum and Bowman, l970)

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

maximum tolerated dose 200 mg/kg bw

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Dose Rangefinding Study

In Life Evaluation: All mice exposed to vehicle control or I 00 mg/kg of Dicyclohexylamine appeared normal during the course of the study. Lethargy and piloerection were observed at 200 mg/kg in both sexes. 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 to this was one female mouse at 400 mg/kg that was humanely euthanized, approximately 5 hours post-dose, after being lethargic, hunched, with piloerection and irregular breathing and after having convulsions. No appreciable reductions in the mean group body weights were observed in any of the treatment groups.

Bone Marrow Bioavailability/Cytotoxicity: Reductions in the PCE/EC ratio of 2% (males) and 8% (females) at 100 mg/kg and reductions of 12% (males) and 22% (females) were observed at 24 hours post-dose. A reduction of only 4% in the male group at 200 mg/kg and no reduction in the PCE/EC ratio at l 00 mg/kg in male and female groups were observed at 48 hr post-dose. The magnitude of these reductions indicated that the test article was bioavailable but did not markedly inhibit erythropioesis.

Definitive Micronucleus Study:

In Life Evaluation: No mortality was observed in any of the treatment groups during the course of definitive micronucleus study. All mice exposed to the control article (vehicle or positive) or to Dicyclohexylamine at 50 or 100 mg/kg appeared normal during the course of the study. All mice at 200 mg/kg appeared lethargic and had piloerection following dose administration, indicating that animals were exposed to the test article.

Bone Marrow Evaluation:
The incidence of micronucleated polychromatic erythrocytes per I 0,000 polychromatic erythrocytes scored (2000 PCEs/mouse) and the proportion of polychromatic erythrocytes per total erythrocytes· are summarized and presented for each treatment group by sacrifice time in Table 13.6. Individual data are presented in Tables 13.7 and 13.8. (see attachment)
No reductions in the ratio of polychromatic erythrocytes to total erythrocytes in the test article groups relative to the respective vehicle control groups were observed at 24 or 48 hours after dose administration.
The incidence of micronucleated PCEs per 10,000 PCEs in test article groups was not statistically increased relative to their respective vehicle controls regardless of dose level or bone marrow collection time (p > 0.05, Kastenbaum-Bowman Tables).
CP, the positive control, induced a statistically significant increase in micronucleated PCEs in male mice relative to the vehicle control (p < 0.05, Kastenbaum-Bowman Tables).
The incidence of micronucleated PCEs in the vehicle control groups did not exceed the historical vehicle control range.
The vehicle and positive controls were consistent with the historical control data, indicating that all criteria for a valid test were met as described in the protocol and that there was no problem with the test system or the quality of the test.

Any other information on results incl. tables

All criteria for valid test were met because of the following:

The incidence of micronucleated polychromatic erythrocytes in the vehicle control group did not exceed the historical vehicle control range.

The incidence of micronucleated polychromatic erythrocytes in the positive control group was  significantly  increased relative to the respective vehicle  control  groups  (p <=0.05, Kastenbaum-Bowrnan Tables).

Applicant's summary and conclusion

Conclusions:

Under the conditions of the assay described in this 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 rnicronucleated polychromatic erythrocytes in bone marrow. Therefore, Dicyclohexylamine was concluded to be negative in the micronucleus test using male ICR mice.