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 vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration 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

Specific details on test material used for the study:

Purity: 99.63% (September 18, 1996 analysis)
(Impurities: Dicyclohexyl imine: 0.119%)
Solubility: Water: slightly soluble 0.16 g/m100 ml
Alcohol, ether, benzene, acetone: soluble

Stability: Stable (The purity was 99.75% (analyzed April 9, 1997) in an analysis
of the residual test substance performed by the study requesting party after
the experiment was completed.)

Storage conditions: Cold, dark location (4°C), airtight stopper (nitrogen gas seal)

Method

Species / strain

Species / strain / cell type:

other: Chinese Hamster Lung (CHL)

Details on mammalian cell type (if applicable):

Study cell line
Chinese hamster lung-derived fibroblasts (CHL) (purchased on January 13, 1985 from National Institute of Health Sciences, Mutated Gene Department (previously: Tokyo Institute of Hygienic Sciences, Mutagenic Property Department)) were used. For the test cells, DMSO was added to a planktonic cell liquid at a rate of 10% and once it had been stored in liquid nitrogen, it was returned to a medium and was used up to a passage number of four times after thawing.

5. Culture Medium
Eagle-MEM powder medium (Gibco Laboratories, lot number 73K2362) was prepared in accordance with the conventional procedure and inactivated (56°C, 30 minute heat processing) and calf serum (Gibco Laboratories, lot number 36N2761) was added to this at a rate of 10%.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 fraction: phenobarbital (PB), 5,6-benzoflavone (BF) induced.

Test concentrations with justification for top dose:

We performed the cell growth inhibition test described below at concentrations of 100, 200, 300, 400, 500, and 600 µg/ml for 24-hour processing and at concentrations of 100, 150, 200, 250, 300, and 350 µg/ml for 48-hour processing using the continuous processing method and at concentrations of 400, 600, 800, 1000, 1200, and 1400 µg/ml with and without the S9 mix using the short-time processing method in order to investigate the appropriate concentration of the test substance for a chromosomal aberration study. Two petri dishes were used for each concentration in the study.
From the results of the cell growth inhibition test, the test substance concentrations included concentrations above and below the 50% cell growth inhibition concentration and considering the need to obtain data for concentrations above the three pertinent concentrations, the six concentrations of 100, 200, 250, 300, 400, and 500 µg/ml were established for the continuous processing method and the six concentrations of 100, 200, 400, 600, 800, and 1000 µg/ml were established for the short-time processing method.

Vehicle / solvent:

The test substance is very insoluble in water and according to the introductory investigation, it is insoluble in dimethyl sulfoxide (DMSO) but is soluble in acetone, so acetone was used as the solvent.
DMSO (Wako Pure Chemical Industries, Ltd., lot number WDG4420) was used as the positive control substance MNNG and B[a]P solvents.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: N-methyl-N’-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

Preparation of the test substance solution
The test substance solution was prepared by dissolving the test substance in acetone at the time of use to make the maximum concentration of the test solution (stock solution). More specifically, the undiluted solution was 99.9 mg/ml in the continuous processing method and 139.9 mg/ml in the short-time processing method. Next, one part of the undiluted solution was consecutively diluted with acetone to make the specified concentrations of the test solution. The amount of the test substance solution added was set to 1.0% (v/v) of the medium amount of each petri dish.

Cell processing
With the continuous processing method, a 2 ml medium containing 6 × 103 cells/ml was added to a round plastic petri dish (Becton Dickinson) with a diameter of 3.5 cm, and then three days after incubation was started, 0.02 ml of acetone (negative control) or the test substance solution was added and it was incubated for 24 and 48 hours. At the same time, with the short-time processing method, cells were incubated using the same method as in the continuous processing method and 0.02 ml of acetone of the test substance solution was added to the petri dish without replacing the culture medium for cases without the S9 mix. In cases with the S9 mix, the medium in the petri dish was removed, 2 ml of the S9 mix diluting solution (S9 diluted six times in the medium) was added, and then 0.02 ml of acetone or the test substance solution was added to the petri dish. Next, the medium was removed after six hours of incubation, the cell surfaces were washed three times with fresh culture medium, 2 ml of the new culture medium was added, and it was incubated for 18 hours. The culture medium was removed after incubation was completed, the cell surfaces were washed twice with physiological saline, 10% formalin aqueous solution was added, and it was fixed for approximately 10 minutes. After fixing, it was washed with water and then stained for approximately 10 minutes in a 0.1% crystal violet aqueous solution. After washing with water, it was left to dry naturally overnight at room temperature.

Cell proliferation rate measurement
The cells that had been fixed and stained in 8-2) above had the cellular density measured from the contrasting density of the staining using a monolayer culture densitometer (Olympus Corporation, Monocellator) and the cell proliferation rate of each concentration group was obtained when the cell proliferation rate of the negative control group was 100%.



As shown in the table below, cell growth inhibition exceeding 50% was observed in 24-
hour continuous processing at 400 µg/ml and higher and in 48-hour continuous processing at 250 µg/ml and higher and the 50% cell growth inhibition concentration was determined to be between 300-400 µg/ml and between 200-250 µg/ml, respectively.
In the short-time processing method, cell growth inhibition exceeding 50% was observed without the S9 mix at 600 µg/ml and with the S9 mix at 1000 µg/ml and the 50% cell growth inhibition concentration was determined to be between 400-600 µg/ml and between 800-1000 µg/ml, respectively. At concentrations of 800 µg/ml or higher, when the test substance solution was added to the culture medium in the petri dish, oil droplet-like products were observed temporarily on the medium surface, but they quickly disappeared.

Chromosomal aberration test

1) Test substance and positive control substance concentrations
From the results of the cell growth inhibition test, the test substance concentrations included concentrations above and below the 50% cell growth inhibition concentration and considering the need to obtain data for concentrations above the three pertinent concentrations, the six concentrations of 100, 200, 250, 300, 400, and 500 µg/ml were established for the continuous processing method and the six concentrations of 100, 200, 400, 600, 800, and 1000 µg/ml were established for the short-time processing method. The positive control substance MNNG was tested at a concentration of 2.5 µg/ml and B[a]P was tested at a concentration of 10 µg/ml.

2) Test substance and positive control substance solution preparation
The test substance solution was prepared by being dissolved in acetone at time of use to prepare the maximum concentration of the test solution (stock solution). More specifically, the concentration of the stock solution was set at 50.0 mg/ml for the continuous processing method and set at 100.1 mg/ml in the short-time processing method. Next, following the same operations as in the method described in 8-1) above, one part of the stock solution was sequentially diluted with acetone to prepare the specified concentrations of the test solution. For the positive control substances, a 0.5 mg/ml MNNG test solution was prepared and a 2.0 mg/ml B[a]P test solution was prepared.

3) Cell processing
Five ml of a medium containing 4 × 103 cells/ml was added to a plastic round petri dish (Becton Dickinson) with a diameter of 6 cm and after 3 days of incubation, it was processed using the following method. For incubation, two petri dishes were used for each concentration.

(1) Continuous processing method
In this method, 0.05 ml of acetone and each test substance solution and 0.025 ml of the MNNG test solution were added to each petri dish and they were incubated for 24 and 48 hours.

(2) Short-time processing method
In the method without the S9 mix, 2 ml of the culture medium was taken from each petri dish, 0.03 ml of acetone and each test substance solution and 0.015 ml of the B[a]P test solution were added to each petri dish and incubated. In the method with the S9 mix, 0.5 ml was added to each petri dish after removing 2.5 ml of the culture medium from each petri dish and additionally, 0.03 ml of acetone and each test substance solution and 0.015 ml of the B[a]P test solution were added to each petri dish and incubated. Whether with or without the S9 mix, the culture medium was removed after six hours of incubation and the cell surface was washed three times with fresh culture medium, 5 ml of new culture medium was added, and it was incubated for another 18 hours.

Chromosomal sample preparation
Two hours before sample preparation, colcemid was added to each petri dish during incubation such that the final concentration became 0.2 µg/ml. After incubation was completed, the culture medium was removed, and the cells separated from the petri dish after processing with 0.2% trypsin aqueous solution 2 ml, transferred to a centrifuge tube into which 5 ml of the fresh medium had been input, and centrifuged at 1000 rpm for five minutes. The supernatant was thrown out, 4 ml of the hypotonic solution 75 mM potassium chloride aqueous solution was added to the cell sedimentation and suspended, then hypotonic treatment was performed at 37°C for 15 minutes. After the hypotonic treatment, fixng was performed by adding 1 ml of a cooled methanol/acetic acid (3:1) mixture (v/v). It was centrifuged at 1000 rpm for five minutes, the supernatant was thrown out, and the cell sediment suspended and fixed in 4 ml of fixing solution. After this procedure was performed three times, an appropriate density of cells was suspended in a small amount of fixing solution, single drops were dropped in two locations on the slide glass, and it was left to dry overnight at room temperature. After drying, it was stained for 15 minutes with diluted 1.4% giemsa solution using a Sorensen buffer solution (pH 6.8).

After washing, it was dried and the chromosome specimen was prepared. Three specimens were prepared per petri dish.

6) Chromosomal observation
The chromosomes were observed at a total magnification of 600× using a no cover field lens. The samples were all coded and it was performed as a blind trial. The number of chromosomes in which the chromosome could be clearly identified along with each test concentration was 100 per petri dish for 25±2 metaphase images, more specifically, 200 chromosomes per two petri dishes for one concentration were observed.

7) Chromosomal aberration classification and count
Chromosomal aberration classification of structural abnormalities included gaps (chromatid type and chromosome type), chromatid type breaks and exchanges, chromosome type breaks and exchanges (e.g. dicentric, circularized chromosomes), and other (fragmentation). Numerical aberrations were recorded only for polyploid cells.
Gaps targeted were sites in which chromosome staining could not be observed at all and such sites were chromatid-width or larger and were on the longitudinal line of a chromatid. However, if the non-stained site was on the longitudinal line of a chromatid, but was markedly separated, it was considered a break.
For the counting of chromatid aberrations, a cell that has even one of the abnormalities classified above was recorded as an abnormal cell and a tally of the types of abnormalities was kept. For the total number of structural abnormalities, the number of abnormal cells observed in the 200 cells observed was divided into cases of cells that included only gaps and cases that did not include only gaps.

8) Determination of results
In determining the study results, quantification of the significant difference (Considering redundancy, 5% or 1% divided by the number of the processing group was used for the significance level) between the negative control group and each concentration group was performed using Fisher’s exact test when a significant difference (significance level of 5% or less) was observed with the multi-sample chi-square test for structural abnormalities that include gaps and the rate of occurrence of polyploidy cells. As a result, there was a significant increase in the occurrence rate of chromatid aberration cells due to the test substance in two or more concentrations when compared with the negative control group and concentration dependence and reproducibility were observed, so a positive determination was made.

Evaluation criteria:

As a result, there was a significant increase in the occurrence rate of chromatid aberration cells due to the test substance in two or more concentrations when compared with the negative control group and concentration dependence and reproducibility were observed, so a positive determination was made

Statistics:

see above

Results and discussion

Test results

Key result

Species / strain:

mammalian cell line, other: Chinese Hamster Lung (CHL)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

1. Chromosomal aberration test (continuous processing method)

 The results are shown in Table 1. The occurrence rate of cells with chromatid structural

abnormalities was 0.5% in both the 24 hour and 48 hour processing of the negative control group. Occurrence rates in the ranges of 0 – 1.5% and 0.5 – 1.0% were observed in the test substance group for 24-hour processing and 48-hour processing, respectively, but no statistically significant difference was observed when compared with the negative control group. However, the rates of occurrence of chromosomal structural abnormalities due to MNNG at 24-hour processing and 48-hour processing in the positive control group were 91.5% and 82.0%, respectively, and pronounced clastogenesis was confirmed.

           Polyploid cells indicating numerical abnormalities were not observed in the negative control group or the test substance group. In the positive control group, a low rate of occurrence of 0.5% was observed only at 48-hour processing.

           No observable metaphase images were seen at the 400 and 500 µg/ml concentrations in 48-hour processing due to cytotoxicity of the test substance.

Chromosomal aberration test (short-time processing method)

           The results are shown in Table 2. The occurrence rate of cells with chromosome structural abnormalities in the negative control group without the S9 mix and with the S9 mix were 1.5% and 0.5%, respectively. On the other hand, occurrence rates of 1.0, 1.0, 2.5, and 10.0% were observed in the test substance group without the S9 mix at the 100, 200, 400, and 600 µg/ml concentrations, respectively, and the increase in the occurrence rate at the 600 µg/ml concentration was statistically significant when compared with the negative control group. In addition, occurrence rates of 0.5, 1.5, 0, 0.5, 13.5, and 31.0% were observed with the S9 mix at the 100, 200, 400, 600, 800, and 1000 µg/ml concentrations, respectively, and the occurrence rates at 800 and 1000 µg/ml were high values, statistically significant when compared with the negative control group. Moreover, concentration-dependence was observed between the 600 to 1000 µg/ml concentrations.

           At the same time, the occurrence rate of cells with abnormal chromosomal structure due to B[a]P in the positive control group was 1.0% without the S9 mix and 56.5% with the S9 mix, and we confirmed that B[a]P was metabolically activated and pronounced chromosomal aberrations were induced.

           Polyploid cells were not observed in the negative control and positive control groups. In the test substance group without the S9 mix and with the S9 mix, occurrences rates in the ranges of 0 – 1.5% and 0 – 1.0% were observed, respectively, but both were low values, so no significant difference was observed when compared with the negative control group.

           Observable metaphase images were not seen at 800 and 1000 µg/ml concentrations without the S9 mix due to test substance cell toxicity. In addition, when the test substance solution was added to the medium in the petri dish at the 800 and 1000 µg/ml concentrations, oil droplet-like formations were observed temporarily on the medium surface, but they quickly disappeared.

D₂₀value⁴⁾

           An increase in chromosomally-aberrant cells was observed in the short-time processing method, so the D₂₀value (the necessary test substance concentration for inducing aberrations in 20% of metaphase images, mg/ml) was calculated.

           The results are shown in the table below, and the 0.96 mg/ml, which is the smallest S value (an indicator based on the concept that, of the D₂₀values referenced, the correlation coefficient r is large and the higher the number of groups, including the negative control group, the more applicable it is) was considered the D₂₀value of this test substance.

 

Short-time processing method	D20value (mg/ml)	S value
Without S9 mix	1.50	70.94
With S9 mix	0.96	24.01
	1.94	61.42

 

Applicant's summary and conclusion

Conclusions:

No increase in cells with chromosomal aberrations was observed in the continuous processing method in the dicyclohexylamine in vitro chromosomal aberration study using Chinese hamster lung-derived fibroblasts. However, in the short-time processing method, a significant increase in cells with aberrant chromosomal structure was observed without the S9 mix at 600 µg/ml, which was the maximum concentration of the 100 – 600 µg/ml concentrations that could be tested. In addition, a significant concentration-dependent increase in cells with aberrations in chromosomal structure was observed with the S9 mix.
As a result, a positive determination of dicyclohexylamine clastogenic properties in CHL cells was made under the conditions of this study. In addition, the D20 value of this test substance was 0.96 mg/ml in the short-time processing method. These study results indicate an obvious positive result in CHL cells even looking at it from biological determination criteria 5) in which an occurrence rate of cells with chromosomal aberrations of 10% or higher is considered a positive result.
Dicyclohexylamine mutagenicity has already been reported to be positive in a chromosomal aberration study 6) using human lymphocytes, but it has been reported to be negative in a reverse mutation study 7) using Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and a transformation study 8) using Syrian hamster-derived BHK21cl13 cells.