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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22nd August 2012 - 20th March 2013.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline and GLP compliant
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: OECD Guideline 487
Deviations:
not specified
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 640/2012; B.49
Deviations:
not specified
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Clear colourless liquid.
Details on test material:
- Name of test material (as cited in study report): N,N,N´,N´-Tetramethyl-1,6-hexanediamine
- Molecular weight (if other than submission substance): 172.31 g/mol.
- Physical state: Clear colourless liquid.
- Analytical purity: 98.9g/100g
- Lot/batch No.: 000STD77L0
- Stability under test conditions:- Storage condition of test material: Stored at room temperature.
- Other:
Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance preparations.
Storage stability: The stability of the test substance under storage conditions throughout the study period was guaranteed until 05 Aug 2013.

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Stocks of the V79 cell line (1-mL portions) were maintained at -196°C in liquid nitrogen using 7% (v/v) DMSO in culture medium as a cryoprotectant. Each batch used for the cytogenetic experiments was checked for
− mycoplasma contamination,
− karyotype stability,
− plating efficiency (=colony forming ability) incl. vital staining.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix.
Test concentrations with justification for top dose:
Experiment 1: 4 hour exposure without S9 mix: 27.3, 54.7, 109.4, 218.8, 437.5 and 875.0μg/mL
Experiment 1: 4 hour exposure with S9 mix: 27.3, 54.7, 109.4, 218.8, 437.5, 875.0 and 1750.0μg/mL
Experiment 2: 24 hour exposure without S9 mix: 13.7, 27.3, 54.7, 109.4, 218.8 and 437.5μg/mL
Experiment 2: 4 hour exposure with S9 mix: 437.5, 875.0, 1312.5 and 1750.0μg/mL
Vehicle / solvent:
Culture medium (Minimum Essential Medium)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
500 and 600μg/mL
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Remarks:
2.5μg/mL
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
A single cell suspension was prepared, and about 5 mL MEM supplemented with 10% (v/v) FCS containing 3 – 8E04 cells were seeded on sterile glass slides in each chamber of the Quadriperm dishes using a dispenser. Two chambers of a Quadriperm dish were used for one test culture.
The Quadriperm dishes were incubated at 37°C, 5% (v/v) CO2 and ≥ 90% humidity. The cells were visually checked for attachment before treatment.

After the attachment period, about 6 hours after seeding, the medium was removed from the slides and the treatment medium was added. The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% humidity

At the end of the exposure period, the medium was removed and the cultures were rinsed twice with 5 mL HBSS (Hanks Balanced Salt Solution). Subsequently, 5 mL MEM (incl. 10% [v/v] FCS) was added and incubated at 37°C, 5% (v/v) CO2 and ≥ 90% humidity for the respective recovery time. In the case of continuous treatment, the cell preparation was started directly at the end of exposure.

At the harvest time, 24 hours after start of exposure, the medium was completely removed. For hypotonic treatment, 5 mL of prewarmed 1.5% (w/v) Sodium citrate solution (37°C) was added for about 5 minutes. Then the hypotonic solution was removed and 5 mL cold fixative (ethanol:glacial acetic acid, ratio 3:1; +4°C) was added. After 5 minutes the fixative was removed and 5 mL of fresh cold fixative was added. Then the dishes were kept at room temperature for at least another 5 minutes for complete fixation.
The slides were taken out of the Quadriperm chambers, briefly allowed to drip off and then rapidly passed through a Bunsen burner flame.

After drying, the slides were stained in Wrights solution (modified May-Grünwald solution) for about 3 minutes. After being rinsed once in Titrisol solution pH 7.2, the slides were counterstained with 2.6% (v/v) Giemsa/Titrisol solution pH 7.2 for about 20 minutes. After being rinsed twice in Titrisol solution pH 7.2 and clarified in xylene, the slides were mounted using Corbit-Balsam.

For additional information about the cytotoxic potential of the test substance cells were seeded in flasks (2.5x105 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 recovery period single cell suspensions were prepared from each test group (all test groups, except the positive controls) and the cells were counted using a cell counter.

NUMBER OF CELLS EVALUATED: At least 1 000 cells per culture, means at least 2 000 cells per dose group, were evaluated and the number of micronucleus-containing cells was recorded.

DETERMINATION OF CYTOTOXICITY
- Method: other: cell count.

OTHER EXAMINATIONS:
- Other:
Cell morphology: The test cultures of all test groups were checked microscopically for cell morphology and attachment to the slides as further parameters of toxicity.
pH value: Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). T he pH was measured, at least for the two top doses and for the negative/ control with and without S9 mix.
Osmolarity: Osmolarity was measured, at least for the top dose and for the negative control with and without S9 mix.
Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).
Proliferation Index: For the assessment of test substance induced cytotoxicity the proliferation index as measure of the proliferative activity of the c ells was determined. The proliferation index was determined in at least 1 000 cells per culture (corresponding to at least 2 000 cells per dose group) in all test groups. The number of clones (packs) containing 1, 2, 3 - 4 or 5 - 8 cells was recorded.
Evaluation criteria:
A test substance is considered "positive" if the following criteria are met:
• A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
• The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.

A test substance generally is considered "negative" if the following criteria are met:
• The number of micronucleated cells in the dose groups is not significant increased above the concurrent negative control value and is within the range of the historical negative control data.
Statistics:
The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one-sided.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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 values were not influenced by test substance treatment.
- Precipitation: No precipitation of the test substance in culture medium was observed.

In this study, no biologically relevant increase in the number of micronucleated cells was observed either without S9 mix or after the addition of a metabolizing system.
In both experiments in the absence and presence of metabolic activation after 4 and 24 hours treatment with the test substance the values (0.6 – 1.5% micronucleated cells) were close to the concurrent negative control values (0.5 – 1.5% micronucleated cells) and clearly within the historical negative control data range (0.1 - 1.8% micronucleated cells).

In the 1st Experiment in the presence of S9 mix statistically significant values compared to the respective vehicle control values were obtained at 875 and 1 750 μg/mL (1.3 and 1.5% micronucleated cells, respectively). However, these observations occurred due to the low rate of micronucleated cells in the concurrent vehicle control group (0.5% micronucleated cells). In addition, in both experiments in the presence of metabolic activation a dose-related increase in the micronucleus frequencies was observed. But, all values were were within the range of the concurrent negative control values of this study and clearly within the historical negative control data range and, therefore, these findings have to be regarded as biologically irrelevant.
The positive control substances EMS (without S9 mix; 500 μg/mL) and CPP (with S9 mix; 2.5 μg/mL) induced statistically significant increased micronucleus frequencies in both independently performed experiments. In this study, in the absence and presence of metabolic activation the frequency of micronucleated cells (2.5 – 14.5% micronucleated cells) was clearly above the range of our historical negative control data range (0.1 - 1.8% micronucleated
cells) and within our historical positive control data range (2.3 – 26.6% micronucleated cells)

In this study, in the absence and the presence of S9 mix no cytotoxicity indicated by clearly reduced PI values was observed at the test groups scored for cytogenetic damage.

In the 1st Experiment without metabolic activation clear cytotoxicity indicated by reduced RICC of below 40 – 50% was observed at 875 μg/mL (13.3%). In the
2nd Experiment after 24 hours exposure in the absence of S9 mix the cell counts were reduced to 50.0% RICC at 218.8 μg/mL and above.
In the presence of S9 mix after 4 hours exposure a RICC of 58.0% was obtained in the 1st Experiment at the highest required concentration (1 750 μg/mL). In the 2nd Experiment with metabolic activation the RICC was strongly reduced at 1 312.5 μg/mL (30.7%) and above.

In the 1st Experiment cell attachment was clearly influenced (≥ grade 3) from 437.5 μg/mL onward in the absence of S9 mix and at 1 750 μg/mL in the presence of S9 mix. In the 2nd Experiment in the absence of S9 mix the attachment was strongly reduced (≥ grade 3) from 54.7 μg/mL. In addition, in this experimental part slides were not scorable for cytogenetic damage due to quality reasons from 109.4 μg/mL onward.

COMPARISON WITH HISTORICAL CONTROL DATA: All recorded results were within the historical control ranges.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Experiment 1: 4 hour exposure without S9 mix:

Dose

Cells

Cells including miconuclei

PI

ug/ml

n

n

%

 

Negative control

2000

12

0.6

2.39

218.8 ug/ml

2000

25

1.3

2.26

437.5 ug/ml

2000

12

0.6

2.14

875 ug/ml

2000

25

1.3

2.01

EMS 500 ug/ml

2000

49

2.5**

2.36

 

 

 

 

 

**: p<=0.01

Experiment 1: 4 hour exposure with S9 mix:

Dose

Cells

Cells including miconuclei

PI

ug/ml

n

n

%

 

Negative control

2000

9

0.5

2.35

437.5 ug/ml

2000

11

0.6

2.31

875 ug/ml

2000

26

1.3**

2.09

1750 ug/ml

2000

30

1.5**

2.09

CPP 2.5 ug/ml

2000

262

13.1**

1.91

 

 

 

 

 

**: p<=0.01

Experiment 2: 24 hours exposure without S9 mix:

Dose

Cells

Cells including miconuclei

PI

ug/ml

n

n

%

 

Negative control

2000

29

1.5

2.55

13.7 ug/ml

2000

27

1.4

2.57

27.3 ug/ml

2000

24

1.2

2.58

54.7 ug/ml

2000

27

1.4

2.33

EMS 500 ug/ml

2000

157

7.9**

2.01

 

 

 

 

 

**: p<=0.01

Experiment 2: 4 hours exposure with S9 mix:

Dose

Cells

Cells including miconuclei

PI

ug/ml

n

n

%

 

Negative control

2000

14

0.7

2.47

437.5 ug/ml

2000

19

1.0

2.33

875 ug/ml

2000

24

1.2

2.42

1750 ug/ml

2000

27

1.4

2.29

CPP 2.5 ug/ml

2000

290

14.5**

2.01

 

 

 

 

 

**:p<=0.01

Relative increase in cell count - 1st Experiment; 4 hours exposure, 24 hours harvest time, without S9 mix:

Test Groups

Cell Count

Relative Increase in cell count (RICC) [%]

 

Absolute value [x105/ml]

Relative value [%]

 

Negative control

27.3 ug/ml

54.7 ug/ml

109.4 ug/ml

218.8 ug/ml

437.5 ug/ml

875.0 ug/ml

5.68

4.39

4.57

4.73

3.76

3.52

1.19

100.0

77.3

80.5

83.3

66.2

62.0

21.0

100.0

75.1

78.6

81.7

62.9

58.3

13.3

Relative increase in cell count - 1st Experiment; 4 hours exposure, 24 hours harvest time, with S9 mix:

Test Groups

Cell Count

Relative Increase in cell count (RICC) [%]

 

Absolute value [x105/ml]

Relative value [%]

 

Negative control

27.3 ug/ml

54.7 ug/ml

109.4 ug/ml

218.8 ug/ml

437.5 ug/ml

875.0 ug/ml

1750.0 ug/ml

5.60

5.81

5.54

5.63

5.78

6.02

5.31

3.46

100.0

103.8

98.9

100.5

103.2

107.5

94.8

61.8

100.0

104.1

98.8

100.6

103.5

108.2

94.3

58.0

Relative increase in cell count – 2nd Experiment; 24 hours exposure, 24 hours harvest time, without S9 mix:

Test Groups

Cell Count

Relative Increase in cell count (RICC) [%]

 

Absolute value [x105/ml]

Relative value [%]

 

Negative control

13.7 ug/ml

27.3 ug/ml

54.7 ug/ml

109.4 ug/ml

218.8 ug/ml

437.5 ug/ml

4.52

4.99

4.33

3.45

2.76

2.51

1.18

100.0

110.4

95.8

76.3

61.1

55.5

26.1

100.0

111.7

95.3

73.4

56.2

50.0

16.9

Relative increase in cell count – 2nd Experiment; 4 hours exposure, 24 hours harvest time, with S9 mix:

Test Groups

Cell Count

Relative Increase in cell count (RICC) [%]

 

Absolute value [x105/ml]

Relative value [%]

 

Negative control

437.5 ug/ml

875.0 ug/ml

1312.5 ug/ml

1750.0 ug/ml

4.08

3.28

3.26

1.60

0.60

100.0

80.4

79.9

39.2

14.7

100.0

77.7

77.1

30.7

2.8

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions of this study, the test material does not have the potential to induce miconuclei (clastogenic and/or aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation and does not require classification according to CLP Regulation.
Executive summary:

A study was conducted to determine the potential of N,N,N´,N´-Tetramethyl-1,6-hexanediamine to induce micronuclei in V79 cells in vitro. Two independent experiments were carried out, both with and without meabolic activation in the form of S9 mix.

In the 1st Experiment without S9 mix (4 hours exposure; 24 hours harvest time), the following doses were used:

0; 27.3; 54.7; 109.4; 218.8; 437.5; 875 μg/mL

In the 1st Experiment with S9 mix (4 hours exposure, 24 hour harvest time), the following doses were used:

0; 27.3; 54.7; 109.4; 218.8; 437.5; 875; 1 750 μg/mL

In the 2nd Experiment without S9 mix (24 hours exposure, 24 hours harvest time), the following doses were used:

0; 13.7; 27.3; 54.7; 109.4; 218.8; 437.5 μg/mL

In the 2nd Experiment with S9 mix (4 hours exposure, 24 hours harvest time), the following doses were used:

0; 437.5; 875; 1 312.5; 1 750 μg/mL

A sample of 1000 cells for each culture were analyzed for micronuclei, (2000 cells for each test group). Positive control substances EMS and cyclophosphamide were also used, with and without metabolic activation.

All micronucleus rates obtained after test substance treatment were either close to the concurrent vehicle control data range or within the range of historical negative control data, and therefore, the statistical significances and the dose-dependencies observed in the experimental parts in the presence of metabolic activation have to be considered as biologically irrelevant.

Under the conditions of this study and based on the results obtained, N,N,N´,N´-Tetramethyl-1,6-hexanediamine is considered to have the potential to induce chromosomal-damaging effects. It also does not have the ability to induce numerical chromosomal aberrations under in vitro conditions in V79 cells in the absence and presence of metabolic activation.