Octanoic acid, compound with dicyclohexylamine (1:1)

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19.11.2013 - 14.2.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 483 (Mammalian Spermatogonial Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian germ cell cytogenetic assay

Test material

Test material information

Constituent 1

Reference substance name:

Dicyclohexylamine

EC Number:

202-980-7

EC Name:

Dicyclohexylamine

Cas Number:

101-83-7

Molecular formula:

C12H23N

Test material form:

not specified

Specific details on test material used for the study:

Designation: Dicyclohexylamine
Chemical name: Dodecahydrodiphenylamine
CAS no.: 101-83-7
EC no.: 202-980-7
Batch no.: 89030400
Characteristics: Colourless liquid, strong fishy odour
Content:
99.62% Diclohexamine
0.330% Organic impurities
< 0.050% Water

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: approx. 8 weeks
- Weight at study initiation: 267 - 335 g
- Fasting period before study: approx. 16 hours before administration
- Housing:groups of 2 - 3 in MAKROLON cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 5 adaptation days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 3°C
- Humidity (%): at least 30% but not exceeding 70%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Sesame oil

Details on exposure:

Administration volume: 20 mL/kg b.w.

Duration of treatment / exposure:

24 and 48 hours

Frequency of treatment:

single dose

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7

Control animals:

yes, concurrent vehicle

Positive control(s):

Designation: Mitomycin C4
Dose level: 5 mg/kg b.w.
Route of administration: Intraperitoneal injection
Vehicle: Aqua ad iniectabilia
Administration volume: 20 mL/kg b.w.

Examinations

Tissues and cell types examined:

spermatogonial cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The dose levels should cover a range from the maximum toxicity to little or none.The highest dose is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. The highest dose may also be defined as a dose that produces some indication of toxicity in the spermatogonial cells (e.g. reduction in the ratio of spermatogonial mitoses to first and second meiotic metaphases; this reduction should not exceed 50%).

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Three dose levels are used for the first sampling time (24 h).Only the highest dose is used at the later sampling time (48 h).

DETAILS OF SLIDE PREPARATION:
Seven (7) hours prior to the sampling time, the animals received 4 mg Colchicine6/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.

METHOD OF ANALYSIS:
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.

Evaluation criteria:

Mitotic index
% of cells with gaps
% of cells with aberrations including gaps
% of cells with aberrations excluding gaps

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) 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).

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Dose Range Finding Study:

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.

Main Study:

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).

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 data8 (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.

Applicant's summary and conclusion

Conclusions:

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:

Dicyclohexylamine was assayed in an in vivo mammalian spermatogonial chromosome 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.