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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity testing was conducted on the two ionic constituents of the registered substance. DCHA showed no mutagenic activity in standard Ames tests with and without metabolic activation system but induced chromosomal aberrations in Chinese hamster (CHL) cells at high concentrations and short exposure time (exposure time: 6 hours (a) with S9-mix at 0,6 mg/ml; (b) without S9-mix at 0,8 and 1.0 mg/ml).

Sodium montelukast did not induce any mutagenic or genotoxic effects in a battery of in vitro genotoxicity assays: microbial reverse mutation, CHO chromosome aberration and V79 HPT locus mammalian cell gene mutagenicity.

Link to relevant study records

Reference

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

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

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)

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.

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

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

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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Given the positive in vitro genotoxicity assay for DCHA, subsequent in vivo testing was conducted; In an in vivo study, using the In Vivo Mammalian Erythrocyte Micronucleus Test, DCHA induced the production of the micronuclei, which is the result of chromosomal damage to the mitotic apparatus in the erythroblasts of Wistar rats. Conversely, in the Mouse Bone Marrow Erythrocyte Micronucleus Test, 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. In order to resolve the in vivo clastogenicity of the substance, Dicyclohexylamine was tested in In Vivo Mammalian Spermatogonial Chromosome Aberration Test in Rats by Oral Administration) which resulted in negative results.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

18-11-2013 to 12-3-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

yes

Remarks:

 The supplier of colcinine was SIGMA-ALDRICH CHEMIE and not Fluka, Neu-Ulm, Germany as stated in the Study Plan.  The vehicle was sesame oil and not water for injection.

Qualifier:

according to guideline

Guideline:

EU Method B.23 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

yes

Remarks:

 Colchicine (4 mg/kg b.w., i.p.) was administered to the animals seven hours and not three hours prior to sampling. These minor changes did not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Specific details on test material used for the study:

Batch no. 89030400
Content 99.62% Diclohexamine
0.330% Organic impurities
< 0.050% Water

Species:

rat

Strain:

other:

Details on species / strain selection:

Rat (Rattus norvegicus) / CD / Crl: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

Number of animals 49 animals
i.e. 7 animals per group
Age (at start of administration) approx. 8 weeks
Body weight 267 - 335 g
Identification of animals By coloured marks and cage label
Acclimatisation period At least 5 adaptation days
ommercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest,
Germany; composition of the diet: see Appendix 2) served as food. Feeding was
discontinued approx. 16 hours before administration; only tap water was then
available ad libitum.
Periodic analysis of the food for contaminants based on EPA/USA

1
is conducted at
least twice a year by LUFA-ITL
2
(see Appendix 2: Limitation for contaminants in the
diet). Certificates of analysis of the composition and for contaminants are provided by
the manufacturer and are QAU archived.

Housing
Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt,
Germany) was used as bedding material for the cages. The cages were changed and
cleaned twice a week.
Periodic analysis of the bedding material for contaminants based on EPA/USA is
conducted at least once a year by LUFA-ITL (see Appendix 2: Limitation for
contaminants in the bedding material).
The animals were kept in groups of 2 - 3 in MAKROLON cages (type III plus) at a
room temperature of 22°C  3°C (maximum range) and the relative humidity was at
least 30% but not exceeding 70%, ideally 50-60%. Deviations from the maximum
range caused for example during cleaning procedures were dealt with in SOPs.
The rooms were lit (about 150 lux at approx. 1.50 m room height) and darkened for
periods of 12 hours each.

Drinking water
Drinking water in drinking bottles was offered ad libitum.
Drinking water is examined periodically according to the 'Deutsche Trinkwasserverordnung
2001',
[German
Regulations
on drinking water, 2001] by the Hamburger
Wasserwerke, 20539 Hamburg, Germany, at least four times a year (see Appendix 2:
Limitation for contaminants in the drinking water). In addition, drinking water samples taken at LPT are analysed by LUFA-ITL once a
year for means of bacteriological investigations according to the 'Deutsche
Trinkwasserverordnung 2001, Anlage 1' [German Regulations on drinking water
2001, Addendum 1].

Route of administration:

oral: gavage

Vehicle:

sesame oil

Details on exposure:

Route of administration Oral by gavage

Frequency of administration Single dose

Vehicle Sesame oil

Administration volume 20 mL/kg b.w.

Selection of
route of administration The oral route is the most likely route of exposure.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

once

Post exposure period:

N/A

Dose / conc.:

62.5 mg/kg bw (total dose)

Dose / conc.:

125 mg/kg bw (total dose)

Dose / conc.:

250 mg/kg bw (total dose)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

4
Designation Mitomycin C

Dose level 5 mg/kg b.w.

Route of administration Intraperitoneal injection

Vehicle Aqua ad iniectabilia

Administration volume 20 mL/kg b.w.

Tissues and cell types examined:

testes - spermatogonial cells

Details of tissue and slide preparation:

Seven (7) hours prior to the sampling time, the animals received 4 mg Colchicine
/kg
b.w. i.p.. The animals were sacrificed by ether. After having removed the tunica
albuginea, the seminiferous tubules of both testicles were exposed to hypotonic
1% sodium citrate solution for 20 minutes. Afterwards, still in toto, the seminiferous
tubules were fixed in freshly prepared methanol/glacial acetic acid (3 + 1) fixative
and the samples were left overnight (about 16 hours) at low temperature (0°C -
4°C). In order to prepare the slides the samples were centrifuged and the fixative was
completely removed with a Pasteur pipette.
The samples were gently resuspended by adding 60% acetic acid. Approx. 50 µL of
the cell suspension were dropped onto a slide prewarmed to 48C and spread. The
air-dried cells were stained in 10% Giemsa (in buffered phosphate solution, pH 7.2)
for 45 minutes. Afterwards the slides were mounted. At least two slides were
prepared per animal.

Evaluation criteria:

Slides for evaluation were coded before microscopic analysis and examined at a
magnification of 1000 x (Planapochromat 100/1.25). The mitotic index was
determined by counting the number of metaphases per 1000 cells in each cell
preparation. The mean mitotic index of the animals per group was compared with the
mean mitotic index of the negative control (mitotic index: 1.0).
The analysis of structural aberrations (chromosome- and chromatid type) was carried
out in 200 cells per animal. Cells with an incomplete number of centromeres or
insufficient spreading were not used for analysis.
Morphological observations were scored as follows and are listed in this report:

Gap:
Chromatid-type aberration:
Chromosome-type aberration: S
Numerical aberration:
Polyploidy:
Fragment:

Metaphases were assigned to one of the following 5 categories:

1: normal metaphases
2: 1 - 2 breaks and/or inter- or intrachanges
and/or fragments
3: metaphases with multiple aberrations
4:pulverized metaphases (more than 50% of
the chromatin present in form of point-like
fragments)
5: polyploidy / endoreduplication

In addition, the total number of gaps in 200 metaphases was recorded.
Metaphases which differed from the normal diploid complement were excluded from
evaluation, however, any test item-related variations in the normal chromosome
number were noted (polyploidy).

The following criteria determine the acceptability of a test:
a) The negative control is consistent with published norms for historical negative
control data.
b) The positive control fulfills the positivity criteria.
c) An adequate number of cells and doses have been analyzed.
d) The selection of the top dose is consistent with the described MTD.

Statistics:

The assessment was carried out by a comparison of the samples with the positive
and the vehicle references, using a chi-square test corrected for continuity according
to YATES (COLQUHOUN, 1971)
7
as recommended by the UKEMS guidelines (The
United Kingdom Branch of the European Environmental Mutagen Society: Report of
the UKEMS subcommittee on guidelines for mutagenicity testing, part III, 1989:
Statistical evaluation of mutagenicity data).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

see below

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

In a range-finding study two male animals per dose were employed. Three dose levels

of 125, 250 and 500 mg Dicyclohexylamine/kg b.w. were tested. A dose level of

125 mg/kg b.w. revealed slight signs of toxicity 60 minutes after administration. 250

mg Dicyclohexylamine/kg b.w. caused slight to moderate signs of toxicity 15 minutes

to 3 hours after administration. The high dose level of 500 mg/kg b.w. revealed slight

to severe signs of toxicity 15 to 60 minutes and death within 3 hours after

administration (see Appendix 3).  

For the main study three ascending doses of 62.5, 125 or 250 mg

Dicyclohexylamine/kg b.w., p.o. were administered. Further groups received the

vehicle (sesame oil) and one further group the positive reference item Mitomycin C

(5 mg/kg b.w., i.p.). Each group consisted of 7 male rats.  

No signs of systemic toxicity were noted at 62.5 mg/kg b.w.. A dose level of

125 mg/kg b.w. revealed slightly reduced motility, slight ataxia and slightly reduced

muscle tone 60 minutes after administration. The high dose level of 250 mg/kg b.w.

revealed slightly to moderately reduced motility, slight to moderate ataxia, slightly to

moderately reduced muscle tone and slight to moderate dyspnoea 15 minutes to 6

hours as well as slight to severe tonic convulsions 30 minutes to 6 hours and slight

pilo-erection 30 minutes to 3 hours after administration (sacrifice after 24 or 48

hours).

In each group, the chromosome preparations from only five animals were used for

evaluation. Two (2) sampling times were employed in this study: 24 hours after

administration, samples were prepared from the negative reference item, positive

reference item and all 3 doses of test item-treated animals; 48 hours after

administration, samples were prepared only from vehicle and high dose-treated

animals.  

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated

with Dicyclohexylamine ranged from 0.8% to 1.9%. These results were within the

normal range as no significant difference was observed compared to negative control

(0.7% or 1.1%). These results were within the range of published historical negative

control data (fragments: 1.2±0.44%, exchanges: 0.8±0.44%, damaged

chromosomes: 0.8±0.44%).

The percentage of cells with gaps was also within the range of the negative control

(treated groups: 2.1% to 3.3%; control group: 1.8% to 3.9%).  

No reduction of the mean mitotic index of the animals per group compared with the

mean mitotic index of the negative control was noted.

The positive control, Mitomycin C, induced significant levels of chromosomal

aberrations.  

Summarised data on structural aberrations in mammalian spermatogonial cells are

presented in Table 1, the individual values in Table 2, results on clinical signs in

Table 3 and body weight in Table 4.

Conclusions:

In conclusion, under the present test conditions, Dicyclohexylamine tested up to the
maximum tolerated dose of 250 mg Dicyclohexylamine/kg b.w. by oral administration
showed no mutagenic properties in the mammalian spermatogonial chromosome
aberration test of the rat at the two tested sampling times of 24 hours and 48 hours.
In the same system, Mitomycin C (positive reference item) induced significant
damage.

Executive summary:

aberration test in the rat for the detection of damage to the chromosomes employing

three dose levels.

The dose levels had been selected based on a range-finding study employing two

male animals per dose. Three dose levels of 125, 250 and 500 mg

Dicyclohexylamine/kg b.w. were tested. A dose level of 125 mg/kg b.w. revealed

slight signs of toxicity 60 minutes after administration. 250 mg Dicyclohexylamine/kg

b.w. caused slight to moderate signs of toxicity 15 minutes to 3 hours after

administration. The high dose level of 500 mg/kg b.w. revealed slight to severe signs

of toxicity 15 to 60 minutes and death within 3 hours after administration. The

administration volume was 20 mL/kg b.w..  

For the main study three ascending doses of 62.5, 125 or 250 mg

Dicyclohexylamine/kg b.w., p.o. were administered. Further groups received the

vehicle (sesame oil) and one further group the positive reference item Mitomycin C

(5 mg/kg b.w., i.p.). Each group consisted of 7 male rats.  

No signs of systemic toxicity were noted at 62.5 mg/kg b.w.. A dose level of

125 mg/kg b.w. revealed slightly reduced motility, slight ataxia and slightly reduced

muscle tone 60 minutes after administration. The high dose level of 250 mg/kg b.w.

revealed slightly to moderately reduced motility, slight to moderate ataxia, slightly to

moderately reduced muscle tone and slight to moderate dyspnoea 15 minutes to 6

hours as well as slight to severe tonic convulsions 30 minutes to 6 hours and slight

pilo-erection 30 minutes to 3 hours after administration (sacrifice after 24 or 48

hours).

In each group, the chromosome preparations from only five animals were used for

evaluation. Two (2) sampling times were employed in this study: 24 hours after

administration, samples were prepared from the negative reference item, positive

reference item and all 3 doses of test item-treated animals; 48 hours after

administration, samples were prepared only from vehicle and high dose-treated

animals.  

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated

with Dicyclohexylamine ranged from 0.8% to 1.9%. These results were within the

normal range, and no significant difference was observed compared to negative

control (0.7% or 1.1%).  

The percentage of cells with gaps was also within the range of the negative control

(treated groups: 2.1% to 3.3%; control group: 1.8% to 3.9%).  

No reduction of the mean mitotic index of the test item-treated animals per group

compared with the mean mitotic index of the negative control was noted.

The positive control, Mitomycin C, induced significant levels of chromosomal

aberrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based upon a weight of evidence approach, the registered substance does not fulfil the criteria for classification as a germ cell mutagen:

Both sodium Montelukast and DCHA individually are Ames negative and neither substances have a structural alert. Sodium montelukast is negative in the CHO chromosome aberration study, with apparent cytotoxicity at low concentrations (50% MI at ~30 µg/mL); DCHA only induces a positive effect in CHL cells at concentrations >=600µg/mL (3.3mMol). It can be predicted that a chromosome aberration test conducted on the registered substance would result in no significant levels of genotoxicity, based on the relative molecular weights of the constituents compared to the registered substance, and the cytotoxicity induced by sodium Montelukast in the CHO test system that would limit the maximum tested concentration of the registered substance.

Given the positive in vitro genotoxicity assay for DCHA, subsequent in vivo testing was conducted; In an in vivo study, using the In Vivo Mammalian Erythrocyte Micronucleus Test, DCHA induced the production of the micronuclei, which is the result of chromosomal damage to the mitotic apparatus in the erythroblasts of Wistar rats. Conversely, in the Mouse Bone Marrow Erythrocyte Micronucleus Test, 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. In order to resolve the in vivo clastogenicity of the substance, Dicyclohexylamine was tested in In Vivo Mammalian Spermatogonial Chromosome Aberration Test in Rats by Oral Administration) which resulted in negative results.

The rat MN assay has several deficiencies compared to the mouse MN study including a much lower number of erythrocytes counted for PCE and mPCE enumeration and a lack of historical control data. Of these the lack of historical control data is most crucial as the results of the assay should be compared for significance to both the historical and concurrent negative control means and standard deviation. This reduces the weight that should be afforded to the rat MN study.

The conclusion is that the weight of evidence shows that montelukast DCHA should be considered non-genotoxic based on the evidence presented.