Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-05-23 - 2011-09-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline Study (GLP)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Adopted on July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
Adopted on Aug, 1998
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
4-methylcyclohexane-1,3-diamine
EC Number:
237-666-9
EC Name:
4-methylcyclohexane-1,3-diamine
Cas Number:
13897-55-7
Molecular formula:
C7H16N2
IUPAC Name:
4-methylcyclohexane-1,3-diamine
Constituent 2
Chemical structure
Reference substance name:
2-methylcyclohexane-1,3-diamine
EC Number:
237-667-4
EC Name:
2-methylcyclohexane-1,3-diamine
Cas Number:
13897-56-8
Molecular formula:
C7H16N2
IUPAC Name:
2-methylcyclohexane-1,3-diamine
Details on test material:
- Name of test material (as cited in study report): Methyl-Diamino-Cyclohexan; Lab test item number: 10/0117-1
- Physical state: liquid, colourless to yellowish, clear
- Analytical purity: 98.5 mol% (NMR-spectroscopy)
- Lot/batch No.: 84407247G0
- Stability under test conditions: the stability under storage conditions over the study period was guaranteed.
- Storage condition of test material: room temperature (N2 conditions)
- Other: the homogeneity of the test substance was ensured by mixing before preparation of the test substance preparations.

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10 000 IU / 10 000 μg/mL), 1% (v/v) amphotericin B (250 μg/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
The cell cycle of the untreated V79 cells lasted for about 12 - 14 hours (measurement based on the BrdU method) under the selected culture conditions.
Metabolic activation:
with and without
Metabolic activation system:
1 part S9 (liver of 5 male Wistar rats pre-treated with 80 mg/kg b.w. phenobarbital i.p. and β-naphthoflavone orally) fraction was mixed with 9 parts S9 supplement (cofactors)
Test concentrations with justification for top dose:
- First experiment: 31.3, 62.5, 125.0, 250.0, 500.0 and 750.0 µg/mL (without S9 mix; 4 h exposure and 18 h sampling time); 250.0, 500.0, 750.0, 1000.0 and 1300.0 µg/mL (with S9 mix; 4 h exposure and 18 h sampling time);
- Second experiment: 40.6, 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix; 4 h exposure and 18 h sampling time); 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (with S9 mix; 4 h exposure and 18 h sampling time);
- Third experiment: 40.6, 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix; 18 h exposure and 18 h sampling time); 162.5, 325.0, 650.0 and 1300.0 µg/mL (without S9 mix; 18 h exposure and 28 h sampling time); 81.3, 162.5, 325.0, 650.0 and 1300.0 µg/mL (with S9 mix; 4 h exposure and 28 h sampling time).

- The slides were not scored for mitotic indices or cytogenetic damage because the experiment did not fulfil the recommendations of the current guideline due to technical errors.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: due to the limited solubility of the test substance in water, and in comparison to other commonly used vehicles (e.g. DMSO, ethanol etc.) acetone was selected as the most suitable vehicle, which has been demonstrated to be suitable in the V79 in vitro cytogenetic assay and for which historical control data are available. The final concentration of the vehicle acetone in culture medium was 1% (v/v).
Controls
Untreated negative controls:
yes
Remarks:
MEM medium with or without FCS
Negative solvent / vehicle controls:
yes
Remarks:
medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9 mix: 500 μg/mL ethyl methanesulfonate (EMS); with S9 mix: 0.5 μg/mL cyclophosphamide (CPP); both dissolved in MEM without FCS (2.5 mg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: cells (run through max. 15 routine passages) visually checked for attachement (20-30 h incubation at 37°C, 5% (v/v) CO2 and ≥ 90% humidity in MEM incl. 10% (v/v) FCS, were used for treatment
- Exposure duration: see above (first to fourth experiment)
- Expression time (cells in growth medium): see above; samples were taken at 18 hours (about 1.5-fold of the normal cell cycle time) and 28 hours (more than 1.5-fold of the normal cell cycle time).
- Fixation time (start of exposure up to fixation or harvest of cells): cells were then fixed with methanol : glacial acetic acid (ratio 3 : 1; +4°C) for a total of 30 min.

SPINDLE INHIBITOR (cytogenetic assays): 2 - 3 hours prior to cell harvest, 100 μL colcemide (stock: 10 μg/mL colcemide dissolved in PBS [Phosphate Buffered Saline]) was added to each chamber in order to arrest mitosis in the metaphase.
STAIN (for cytogenetic assays): after drying, the slides were stained with 7.5% (v/v) Giemsa/Titrisol solution pH 7.2 for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the slides were mounted in Corbit-Balsam.

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 100 metaphases per culture were evaluated for structural chromosome aberrations, whereby aneuploid and polyploid cells were recorded separately. Due to clearly positive findings (> 10% aberrant cells exclusive gaps) in all positive control cultures, the analysis of these test groups was restricted to 50 metaphases per culture.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (in general, 1 000 cells, incl. mitotic cells, were counted per culture); cloning efficiency (cells were seeded in cell culture flasks (2.5x10E5 cells per 25 cm2 flask) about 24 - 30 hours prior to exposure. The cells were treated similar to the slides using the same media and test substance concentrations. At the end of the recovery period or at the end of continuous treatment single cell suspensions were prepared from each flask and the cells were counted using a cell counter); at the end of the treatment period, the test cultures of all test groups were checked microscopically for cell morphology, which is an indication of attachment of the cells to the slides.
Evaluation criteria:
The test substance is considered as “positive” if the following criteria are met: (1) a statistically significant, dose-related and reproducible increase in the number of cells with structural chromosome aberrations (excl. gaps); (2) the number of aberrant cells (excl. gaps) exceeds both the concurrent negative/vehicle control value and the historical negative control data range. A test substance generally is considered as “negative” if the following criteria are met: the number of cells with structural aberrations (excl. gaps) in the dose groups is not statistically significant increased above the concurrent negative/vehicle control value and is within the historical negative control data range.
Statistics:
The proportion of metaphases with structural aberrations was calculated for each group. A comparison of each dose group with the negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided. If the results of this test are statistically significant compared with the respective vehicle control, labels (* p ≤0.05, ** p ≤0.01) are printed in the tables.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
under the experimental conditions chosen here, the test substance is not a chromosome-damaging (clastogenic) substance nor did it unduce changes in the number of chromosomes under in vitro conditions using V79 cells.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
no clear mitotic activity suppression indicated by mitotic rates below 50% of control was observed; a dose-dependent growth inhibition was observed in all experiments under any of the experimental conditions.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: pH was not influenced by test substance treatment.
- Effects of osmolality: osmolarity was not influenced by test substance treatment.
- Precipitation: no test substance precipitation in medium was observed.
- Other confounding effects: a dose-related increase in the aberration rates (1.5%, 2.0% and 3.5% aberrant metaphases, excl. gaps) was observed at the concentrations scored for cytogenetic damage (162.5 to 650 μg/mL) in the 2nd Experiment in the absence of S9 mix after 4 hours exposure at 18 hours sampling time. However, all values were equal or below the respective negative control value (3.5% aberrant metaphases, excl. gaps) and clearly within the range of our historical negative control data (0.0% - 5.5% aberrant metaphases, excl. gaps). Thus, this observation has to be regarded as biologically irrelevant.

RANGE-FINDING/SCREENING STUDIES:
In the initial range-finding cytotoxicity test for the determination of the experimental doses, the test substance did not exhibit any pronounced toxicity up to the highest required concentration, 5100 μg/mL (approx. 5000 μg/mL of the active ingredient), at which distinct test substance precipitation was observed.

COMPARISON WITH HISTORICAL CONTROL DATA:
The structural chromosome aberration rates of the vehicle control groups were within the historical negative control data range and, thus, fulfilled the acceptance criteria of this study. The increase in the frequencies of structural chromosome aberrations induced by the positive control substances EMS and CPP clearly demonstrated the sensitivity of the test system and of the metabolic activity of the S9 mix employed. The values were also within the range of the historical positive control data and, thus, fulfilled the acceptance criteria of this study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Strongly reduced cell numbers were observed from 1250 μg/mL onward in the absence of S9 mix and from 5000 μg/mL onward in the presence of S9 mix in the 1st experiment after 4 hours treatment. The cell numbers were also strongly reduced at 650 μg/mL (37.4% of control) and above in the absence of S9 mix and at 1 300 μg/mL (28.4% of control) in the presence of S9 mix In the 2nd experiment after 4 hours treatment at 18 hours preparation interval.
Strong growth inhibition was observed in the 3rd experiment at 650 μg/mL (14.4% of control) and above after 18 hours continuous exposure in the absence of S9 mix. In addition, after 18 hours exposure at 28 hours sampling time in the absence of S9 mix and after 4 hours exposure at 28 hours sampling time in the presence of metabolic activation, cytotoxicity was observed at 1300 μg/mL (52.5% or 12.7% of control, respectively). However, in the 3rd experiment concentrations showing clear cytotoxicity were not scorable for cytogenetic damage.
Distinct changes in cell attachment and/or cell morphology were observed in the 2nd experiment in the absence of S9 mix at 1 300 μg/mL only. Changes in cell attachment and/or cell morphology were found at 325 μg/mL and above at 18 and 28 hours preparation interval.
In the 3rd experiment in the absence of S9 mix. In the presence of S9 mix at 28 hours preparation interval no changes in cell attachment and/or cell morphology were found up to the highest applied concentration.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of the results without S9 mix

 

Exp.

Schedule

Test groups

P

Genotoxicity [%]

Cytotoxicity

Exposure/ preparation period

Aberrant cells

Polyploid cells

Cell number [%]

Mitotic index [%]

incl. gaps

excl. gaps

with exchanges

 

 

 

2

 

 

 

4/18 h

Medium

-

6.5

3.5

0.5

0.0

100.0

100.0

40.6 µg/mL

-

n.d.

n.d.

n.d.

n.d.

104.3

n.d.

81.3 µg/mL

-

n.d.

n.d.

n.d.

n.d.

98.0

n.d.

162.5 µg/mL

-

6.0

1.5

1.0

0.0

111.8

122.1

325.0 µg/mL

-

5.5

2.0

0.5

0.0

89.1

106.3

650.0 µg/mL

+

4.5

3.5

1.0

0.0

37.4

85.8

1300.0 µg/mL

+

n.s.

n.s.

n.s.

n.s.

11.2

n.s.

Positive control

-

22.0

18.0

9.0

0.0

n.t.

93.2

 

 

 

 

 

3

 

 

 

 

18/18 h

Medium

-

6.0

4.0

0.5

0.5

100.0

100.0

40.6 µg/mL

-

4.5

2.0

0.5

0.0

106.4

139.6

81.3 µg/mL

-

4.0

0.5

0.5

0.0

99.7

111.1

162.5 µg/mL

-

4.0

3.0

0.5

0.0

100.2

84.5

325.0 µg/mL

-

n.s.

n.s

n.s

n.s

72.6

n.s

650.0 µg/mL

-

n.d.

n.s.

n.s.

n.s.

14.4

n.s.

1300.0 µg/mL

+

n.d.

n.s.

n.s.

n.s.

1.3

n.s.

Positive control

-

31.0

28.0

18.0

0.0

n.t.

78.7

 

 

 

 

 

3

 

 

 

 

18/28 h

Medium

-

5.5

3.0

0.0

0.0

100.0

100.0

162.5 µg/mL

-

n.d.

n.d.

n.d.

n.d.

89.9

n.d.

325.0 µg/mL

-

3.5

1.5

0.5

1.0

68.2

109.7

650.0 µg/mL

-

n.s.

n.s.

n.s.

n.s.

67.7

n.s.

1300.0 µg/mL

+

n.s.

n.s.

n.s.

n.s.

52.5

n.s.

Positive control

-

34.0

33.0

26.0

0.0

n.t.

99.0

P: precipitation determined at the end of exposure period (macroscopic); cytotoxicity expressed as relative values compared with the respective vehicle control; aberrant cells excluding/including gaps and including cells carrying exchanges; n.d.: not determined; n.s.: not scorable due to strong cytotoxicity and/or poor metaphase quality; n.t.: not tested; only sample of 100 metaphases evaluated in positive control due to strong clastogenicity

 

Table 2: Summary of the results with S9 mix

Exp.

Schedule

Test groups

P

Genotoxicity [%]

Cytotoxicity

Exposure/ preparation period

Aberrant cells

Polyploid cells

Cell number [%]

Mitotic index [%]

incl. gaps

excl. gaps

with exchanges

 

 

 

2

 

 

 

4/18 h

Medium

-

5.5

2.0

0.5

0.0

100.0

100.0

81.3 µg/mL

-

n.d.

n.d.

n.d.

n.d.

113.2

n.d.

162.5 µg/mL

-

n.d.

n.d.

n.d.

n.d.

108.8

n.d.

325.0 µg/mL

-

6.0

2.0

0.5

0.0

112.9

112.2

650.0 µg/mL

-

7.5

3.5

1.0

0.0

73.6

109.9

1300.0 µg/mL

-

6.0

3.0

1.0

0.0

28.4

57.3

Positive control

-

28.0

23.0

18.0

0.0

n.t.

72.3

 

 

 

 

 

3

 

 

 

18/18 h

Medium

-

4.0

2.5

1.5

0.0

100.0

100.0

81.3 µg/mL

-

n.d.

n.d.

n.d.

n.d.

91.6

n.d.

162.5 µg/mL

-

2.5

0.5

1.5

0.0

118.0

112.2

325.0 µg/mL

-

4.0

2.0

0.5

0.5

114.3

93.3

650.0 µg/mL

-

4.0

2.5

0.5

0.5

60.3

107.1

1300.0 µg/mL

-

n.s.

n.s.

n.s.

n.s.

12.7

n.s.

Positive control

-

33.0

32.0

19.0

0.0

n.t.

101.0

P: precipitation determined at the end of exposure period (macroscopic); cytotoxicity expressed as relative values compared with the respective vehicle control; aberrant cells excluding/including gaps and including cells carrying exchanges; n.d.: not determined; n.s.: not scorable due to strong cytotoxicity and/or poor metaphase quality; n.t.: not tested; only sample of 100 metaphases evaluated in positive control due to strong clastogenicity

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative