N,N,N',N'-tetramethylhexamethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Studies of bacterial reverse mutation (Ames test), mammalian cell mutation and mammalian cell cytogenicity in vitro are available for the substance and reveal no evidence for genetic toxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8th September, 2011 to 17th November 2011.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

30 May 2008

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

Aug 1998

Deviations:

not specified

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

- Name of test material (as cited in study report): N,N,N´,N´-Tetramethyl-1,6-hexanediamine
- Physical state: Clear, colourless liquid.
- Analytical purity: 98.9 +/- 0.3 g/100 g
- Lot/batch No.: 000STD77L0
- Storage condition of test material: At room temperature.
- Other: The homogeneity of the test substance was ensured by mixing before preparation of the test substance solutions.
The stability of the test substance under storage conditions throughout the study period was guaranteed until 05 Aug 2013

Target gene:

Histidine and tryptophan.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Deep-frozen bacterial cultures of Salmonella typhimurium and Escherichia coli were thawed at room temperature and 0.1 mL of this bacterial suspension was inoculated in nutrient broth solution (8 g/L Difco nutrient broth + 5 g/L NaCl) and incubated in a shaking water bath at 37°C for 12 - 16 hours. The cultures grown overnight were kept in iced water from the beginning of the experiment until the end to prevent further growth.

The Salmonella strains were checked at regular intervals for deep rough character (rfa), UV sensitivity (∆uvrB) and ampicillin resistance (R factor plasmid).
The E. coli WP2 uvrA was checked for UV sensitivity.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and ß-naphthoflavone induced rat liver S9 mix.

Test concentrations with justification for top dose:

Experiment 1: 0; 33; 100; 333; 1000; 2500 and 5 000 μg/plate
Experiment 2: 0; 33; 100; 333; 1000; 2500 and 5 000 μg/plate
Experiement 3: 0; 1; 3.3; 10; 33; 100 and 333 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Test material has good solubility in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Remarks:

2.5 μg/plate for TA1535, TA100, TA1537and TA98 and 60 μg/plate for Escherichia coli WP2 uvrA. Dissolved in DMSO.

Positive control substance:

other: 2-aminoanthracene

Remarks:

In the presence of S9 mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 μg/plate for TA1535 and TA100. Dissolved in DMSO.

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9 mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Remarks:

10 μg/plate for TA98. Dissolved in DMSO.

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

In the absence of S9 mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Remarks:

100 μg/plate for TA1537. Dissolved in DMSO.

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9 mix.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Remarks:

5 μg/plate for E. coli WP2 uvrA. Dissolved in DMSO.

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

In the absence of S9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and plate incubation.

For the plate incorporation test, For Salmonella typhimurium, test tubes containing 2-mL portions of soft agar (overlay agar), consisting of 100 mL agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within 30 seconds and incubated at 37 °C for 48 – 72 hours in the dark, after which the number of bacterial colonies were counted.

For Escherichia coli test tubes containing 2-mL portions of soft agar (overlay agar), consisting of 100 mL agar (0.8 % [w/v] agar + 0.6 % [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) are kept in a water bath at about 42 - 45 °C, and the remaining components are added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation) or 0.5 mL phosphate buffer (without metabolic activation)

After mixing, the samples are poured onto Vogel-Bonner agar plates (minimal glucose agar plates) within 30 seconds and incubated at 37 °C for 48 – 72 hours in the dark, after which the number of bacterial colonies were counted.

For the pre-incubation test, 0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) are incubated at 37 °C for the duration of about 20 minutes using a shaker. 2 mL of soft agar is then added and, after mixing, the samples are poured onto the agar plates within 30 seconds.

After incubation at 37 °C for 48 - 72 hours in the dark, the bacterial colonies are counted.

NUMBER OF REPLICATIONS: 3 test plates were used per dose and control.

Evaluation criteria:

Toxicity was detected by a decrease in the number of revertants, clearing or diminution of the background lawn (= reduced his- or trp- background growth) or a reduction in the titer.

The test substance is considered positive in this assay if the following criteria are met:

A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester straineither without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:

The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Statistics:

No information available.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Following experimentation by both the plate incorporation test and the pre-incubation method, there was no relevant in the number of histidine or tryptophan revertants, with and without metabolic activation.

In the plate incorporation test, a weak bacteriotoxic effect in the form of a slight decrease in the number of histidine revertants and a reduction in the
titer, was occasionally observed depending on the strain and test conditions at 2500 μg/plate.

In the pre-incubation assay, bacteriotoxicity in the form of reduced histidine or tryptophan background growth, a decrease in the number of his+ or trp+ revertants and a reduction in the titer was observed, depending on the strain and test conditions, from 333 μg/plate onward.

No test substance precipitation was found with and without S9 mix.

Standard Plate test

Dose/plate	TA 1535		TA 100		TA 1537		TA 98		E. coli WP2 uvrA
	Revertants per plate		Revertants per plate		Revertants per plate		Revertants per plate		Revertants per plate
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Water	14 12 16	15 11 14	81 71 70	111 94 104	7 6 5	8 5 6	14 19 20	25 17 24	45 31 44	45 50 40
33 ug	17 12 14	11 16 15	74 64 85	87 99 86	4 7 8	9 7 10	14 19 15	27 19 17	43 52 63	36 41 46
100 ug	16 11 11	13 12 16	93 88 87	106 108 99	5 4 7	9 3 7	14 24 15	33 25 32	32 55 49	44 37 47
333 ug	17 16 13	15 18 15	71 91 86	96 112 114	5 11 6	8 12 11	12 18 15	26 32 22	47 44 40	45 40 45
1000 ug	12 11 12	12 19 11	69 74 85	102 121 111	8 7 6	7 9 11	16 16 18	29 22 25	44 42 40	44 41 44
2500 ug	12 11 12	11 14 10	85 74 91	116 110 158	4 6 8	5 10 4	15 17 11	21 33 32	56 35 45	45 49 43
5000 ug	11 9 13	12 8 10	60 50 52	104 167 154	5 2 2	8 5 7	12 17 7	22 25 13	56 53 37	43 48 44
MNNG	775 659 731	-	778 621 731	-	335 421 387	-	-	-	-	-
2-AA	-	135 184 152	-	887 965 821	-	114 133 128	-	621 587 663	-	224 232 208
NOPD	-	-	-	-	-	-	558 532 528	-	-	-
4-NQO	-	-	-	-	-	-	-	-	721 732 680	-

 

  Pre-Incubation test:

Dose/plate	TA 1535		TA 100		TA 1537		TA 98		E. coli WP2 uvrA
	Revertants per plate		Revertants per plate		Revertants per plate		Revertants per plate		Revertants per plate
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Water	12 12 10	17 15 19	102 75 89	85 100 95	5 7 5	8 5 7	21 15 17	27 24 29	20 34 28	30 27 26
33 ug	10 11 9	21 19 12	73 89 104	80 91 78	3 5 9	8 5 5	16 19 16	31 24 23	20 23 38	25 31 37
100 ug	12 10 13	17 23 18	96 78 101	85 92 108	7 7 4	6 9 6	14 20 23	24 32 21	28 22 28	21 27 35
333 ug	14 8 14	10 9 12	100 36 85	109 100 97	8 2 2	9 11 4	0B 0B 0B	32 23 36	13 27 19	20 37 31
1000 ug	8B 7B 9B	9B 15B 10B	55B 42B 39B	85B 55B 60B	0B 0B 0B	7B 5B 7B	0B 0B 0B	21B 10B 11B	17B 12B 10B	34B 21B 24B
2500 ug	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	18B 12B 14B	21B 25B 19B
5000 ug	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B	0B 0B 0B
MNNG	775 559 698	-	778 736 658	-		-	-	-	-	-
AAC	-	-	-	-	335 387 364	-	-
2-AA	-	155 127 119	-	1181 1225 1134	-	134 173 152	-	674 775 716	-	214 226 254
NOPD	-	-	-	-	-	-	447 532 576	-	-	-
4-NQO	-	-	-	-	-	-	-	-	674 599 521	-

 

Pre-Incubation test:

Dose/plate	TA 1535		TA 100		TA 1537		TA 98
	Revertants per plate		Revertants per plate		Revertants per plate		Revertants per plate
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
Water	13 11 14	16 11 13	78 78 87	95 81 79	4 8 6	8 8 7	15 21 18	29 20 20
1 ug	11 14 10	16 15 11	70 85 84	83 82 101	6 4 7	7 5 7	20 17 19	26 27 22
3.3 ug	11 9 15	12 13 12	77 82 105	86 97 101	4 6 8	6 8 7	21 19 16	20 21 19
10 ug	10 14 13	13 10 12	80 84 91	91 95 107	5 5 7	9 7 5	21 19 14	24 20 23
33 ug	13 11 12	10 14 13	87 84 82	94 88 103	6 7 4	7 6 5	17 17 17	28 17 24
100 ug	7 12 13	15 11 12	84 75 76	95 89 90	6 4 5	4 8 7	18 18 11	22 14 25
333 ug	8 10 6	9 8 11	64 51 48	92 79 78	3 4 4	8 5 4	2B 1B 4B	17 21 19
MNNG	721 608 682	-	1055 982 1038	-	-	-	-	-
AAC	-	-	-	-	315 351 328	-	-
2-AA	-	144 130 158	-	996 1057 933	-	118 142 109	-	774 831 761
NOPD	-	-	-	-	-	-	447 465 508	-
4-NQO	-	-	-	-	-	-	-	-

 

Conclusions:

Under the conditions of this study, N,N,N´,N´-Tetramethyl-1,6-hexanediamine does not induce mutagenic activity when tested on strains of Salmonella typhimurium and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation.

Executive summary:

In a study conducted in accordance with GLP and OECD 471, the mutagenic potential of N,N,N´,N´-Tetramethyl-1,6-hexanediamine was determined, based on its ability to induce point mutations in selected loci of several bacterial strains in a reverse mutation assay. The study was conducted in the presence and absence of S9 mix and was conducted by plate incorporation and pre-incubation methods. The bacterial strains tested were TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA. In Experiment 1, the doses ranged from 33 μg - 5 000 μg/plate, in Experiment 2, the doses were 1 μg - 5 000 μg/plate and in Experiment 3, the doses were 33 μg - 5 000 μg/plate. Under the conditions of this study, N,N,N´,N´-Tetramethyl-1,6-hexanediamine does not induce mutagenic activity when tested on strains of Salmonella typhimurium and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

22 Jul 2010

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 640/2012; B.49

Version / remarks:

06 July 2012

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- 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
- Storage condition of test material: Stored at room temperature.
- 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.

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:

phenobarbital and ß-naphthoflavone induced rat liver 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)

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.

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.

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 [x10^5/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 [x10^5/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 [x10^5/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 [x10^5/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

Conclusions:

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.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3rd February 2012 to 27th September 2012.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

21 Jul 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

Aug 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- 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: 98.9 g/100 g
- Lot/batch No.: 000STD77L0
- Storage condition of test material: At room temperature.
- 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 was guaranteed until 05 Aug 2013.

Target gene:

HGPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line is a permanent cell line derived from the Chinese hamster and has a
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90%)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO 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 mutagenicity testing was checked for mycoplasma contamination.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital / beta-naphthoflavone induced male Wistar rat liver S9 fraction

Test concentrations with justification for top dose:

1st Experiment without S9 mix: 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0μg/mL
1st Experiment with S9 mix: 26.6, 53.1, 106.3, 212.5, 425.0, 850.0 and 1700μg/mL

2nd Experiment without S9 mix: 106.3, 212.5, 425.0, 850.0 and 1700.0μg/mL

3rd Experiment without S9 mix: 53.1, 106.3, 212.5, 425.0, 850.0 and 1700.0μg/mL
3rd Experiment with S9 mix: 250.0, 500.0, 1000.0 and 1700.0μg/mL

4th Experiment without S9 mix: 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1000.0μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium (Ham's F12)
- Justification for choice of solvent/vehicle: Culture medium was used as the vehicle due to the good solubility of the test substance in water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Culture medium (Ham's F12)

True negative controls:

no

Positive controls:

yes

Remarks:

300 μg/mL

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Culture medium (Ham's F12)

True negative controls:

no

Positive controls:

yes

Remarks:

20 μg/mL

Positive control substance:

other: Methylcholanthrene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
Preparation of test cultures:
Cell stocks (1.0-mL portions) stored in liquid nitrogen were thawed at 37°C in a water bath. 0.5 mL of stock cultures were pipetted into 25 cm2 plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). After 24 hours the medium was replaced to remove any dead cells. At least 2 passages were performed before cells were taken for the experiment. A further passage was also necessary in order to prepare test cultures.

Pretreatment of cells with "HAT" medium:
During the week prior to treatment, any spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5x10E5 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. A subsequent passage in Ham's F12 medium incl. 10% (v/v) FCS was incubated for a further 3 - 4 days.

Attachment period:
For each test group, about 1x10E6 logarithmically growing cells per flask (175 cm²) were seeded into about 20 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours. Two flasks were used for each test group.

Exposure period:
After the attachment period, the medium was removed from the flasks 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.

Expression period:
The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).

Selection period:
For selection of the mutants, six 75 cm2 flasks with 3x10E5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF REPLICATIONS: Duplicate

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency (survival and viability), Mutant frequency
Cloning Efficiency 1 (CE1 survival): For the determination of the influence of the test substance directly after the exposure period, about 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test substance or positive control for 4 hours or 24 hours. After exposure, the cells were rinsed several times with HBSS. Then, the cells were cultured in 5 mL Ham's F12 medium incl. 10% (v/v) FCS.

Cloning Efficiency 2(CE2 viability): For the determination of the mutation rate after the expression period, two aliquots of about 200 cells each were reserved from the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm2) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
In all cases, after seeding the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.
The absolute and relative cloning efficiencies (%) were calculated for each test group.

Mutant Frequency:
The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized per every 10E6 cells seeded.

OTHER EXAMINATIONS:
Changes in pH, osmolarity, solubility and cell morphology were also recorded.

Evaluation criteria:

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Mutant Frequency:
No relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In all four experiments, after 4 and 24 hours treatment with the test substance, the values for the corrected mutation frequencies (MFcorr.: 0.00 – 5.85 per 10E6 cells) were close to the respective vehicle control values (MFcorr.: 1.18 – 2.53 per 10E6 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 16.43 per 10E6 cells; with S9 mix: MFcorr.: 0.00 – 15.83 per 10E6 cells).

The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced a clear increase in mutation frequencies, as expected. The values of the corrected mutant frequencies (without S9 mix: MFcorr.: 124.66 – 819.10 per 10E6 cells; with S9 mix: MFcorr.: 97.92 – 133.59 per 10E6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 47.35 – 1 338.10 per 10E6 cells; with S9 mix: MFcorr.: 26.29 – 413.54 per 10E6 cells).

Cytotoxicity:
Cytotoxic effects, as indicated by clearly reduced cloning efficiencies of 20% below controls were only observed in the 3rd and 4th Experiment in the absence of S9 mix after an exposure period of 24 hours, at least at the highest applied concentrations.
After an exposure period of 24 hours without S9 mix, there was a decrease in the number of colonies from 425.0 μg/mL (CE1 relative: 8.4%) onward in the 3rd Experiment and from 500.0 μg/mL (CE1 relative: 5.9%) onward in the 4th Experiment. The cell densities were distinctly reduced.
In all other experimental parts of this study, in the absence and presence of S9 mix, no decrease in the number of colonies was observed up to the highest applied concentration. The cell densities were not distinctly reduced.

Cell Morphology:
There were no adverse observations on cell morphology (cell attachment) in the 1st Experiment in the absence and presence of S9 mix after 4 hours treatment and in the 3rd Experiment in the presence of S9 mix after 4 hours treatment.
In the absence of S9 mix, after 4 hours treatment in the 2nd Experiment and after 24 hours treatment in the 3rd and 4th Experiment the morphology and attachment of the cells was adversely influenced at least at the highest applied concentration.

Summary of results - experimental parts with S9 mix

Exposure	Exposure period (h)	Test Groups (μg/mL)	S9 mix	Prec.*	Genotoxicity**	Cytotoxicity***
						CE1 (%)	CE2 (%)
1	4	Negative control 26.6 53.1 106.3 212.5 425.0 850.0 1700.0 Positive control	+ + + + + + + + +  +	- - - - - - - - - -	2.24 n.c.^1 n.c.^1 n.c.^1 1.68 0.97 0.66 1.54 97.92	100.0 127.5 115.0 107.5 111.6 114.2 111.8 117.4 106.0	100.0 n.c.^1 n.c.^1 n.c.^1 94.2 94.7 90.2 85.7 97.3
3	4	Negative control 250.0 500.0 1000.0 1700.0 Positive control	+ + + + + +  +	- - - - - - -	2.53 0.61 2.20 0.82 3.24 133.59	100.0 103.4 101.2 101.6 98.7 105.8	100.0 92.4 102.8 106.6 96.1 100.0

 

n.c.¹Culture was not continued since a minimum of only four analysable concentrations are required

n.c.²Culture was not continued due to strong cytotoxicity

n.c.³Culture was not continued due to missing the OECD recommendations

*     Precipitation in culture medium at the end of exposure period

**   Mutant frequency MFcorr.: mutant colonies per 10⁶cells corrected with the CE₂value

*** Cloning efficiency related to the respective vehicle control

 

Summary of results - experimental parts without S9 mix

Exposure	Exposure period (h)	Test Groups (μg/mL)	S9 mix	Prec.*	Genotoxicity**	Cytotoxicity***
						CE1 (%)	CE2 (%)
1	4	Negative control 15.6 31.3 62.5 125.0 250.0 500.0 1000.0 Positive control	- - - - - - - - - -	- - - - - - - - - -	n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3	100.0 125.4 104.7 101.4 117.5 114.6 103.9 89.9 104.1	n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3 n.c.^3
2	4	Negative control 106.3 212.5 425.0 850.0 1700.0 Positive control	- - - - - - - -	- - - - - - - -	1.25 n.c.^1 1.19 0.00 1.68 2.92 124.66	100.0 102.0 95.5 93.5 85.5 72.2 93.5	100.0 n.c.^1 98.8 106.8 101.5 99.3 88.2
3	24	Negative control 53.1 106.3 212.5 425.0 850.0 1700.0 Positive control	- - - - - - - -	- - - - - - - -	n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2	100.0 98.1 72.7 58.1 8.4 2.9 0.0 55.2	n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2 n.c.^2
4	24	Negative control 15.6 31.3 62.5 125.0 250.0 500.0 1000.0 Positive control	- - - - - - - - - -	- - - - - - - - - -	1.18 3.04 5.85 1.15 3.62 0.00 n.c.^2 n.c.^2 819.10	100.0 101.3 97.7 105.3 64.1 50.7 5.9 2.6 83.2	100.0 108.1 95.1 97.0 92.9 99.5 n.c.^2 n.c.^2 51.2

 

n.c.¹Culture was not continued since a minimum of only four analysable concentrations are required

n.c.²Culture was not continued due to strong cytotoxicity

n.c.³Culture was not continued due to missing the OECD recommendations

*     Precipitation in culture medium at the end of exposure period

**   Mutant frequency MFcorr.: mutant colonies per 10⁶cells corrected with the CE₂value

*** Cloning efficiency related to the respective vehicle control

Conclusions:

Under the conditions employed in this study, it can be concluded that N,N,N´,N´-Tetramethyl-1,6-hexanediamine is not a mutagenic substance in the HPRT locus assay using CHO cells.

Executive summary:

To determine the mutagenic potential of N,N,N´,N´-Tetramethyl-1,6-hexanediamine to induce gene mutations at the HPRT locus, a study was conducted on Chinese Hamster Ovary (CHO) cells in accordance with OECD Guideline 476, EU Method B.17 and OPPTS 870.5300. Four independent experiments were conducted in duplicate, in the presence and absence of S9 mix. Appropriate vehicle and positive and negative controls were also used.

The first experiment was conducted with a four hour exposure period, in the presence and absence of S9 mix at the following concentrations:
without S9 mix: 0, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1 000.0 μg/mL

with S9 mix: 0, 26.6, 53.1, 106.3, 212.5, 425.0, 850.0 and 1 700.0 μg/mL

Due to the unexpected weak cytotoxicity observed in the first experiment in the absence of S9 mix, a second experiment was conducted in the absence of S9 mix, again for a four hour exposure period at concentrations of 0, 106.3, 212.5, 425.0, 850.0 and 1700.0 μg/mL.

The third experiment was conducted with a four hour exposure period in the presence of S9 mix and a 24 hour exposure period in the absence of S9 mix at the following concentrations:
without S9 mix: 0, 53.1, 106.3, 212.5, 425.0, 850.0 and 1 700.0 μg/mL

with S9 mix: 0, 250.0, 500.0, 1 000 and 1 700.0 μg/mL

Due to the invalid results in the third experiment in the absence of S9 mix due to unexpectedly strong cytotoxicity results observed, a fourth experiment was conducted in the absence of S9 with an exposure period of 24 hours at concentrations of 0, 15.6, 31.3, 62.5, 125.0, 250.0, 500.0 and 1 000.0 μg/mL.

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

In the 1st Experiment in the absence and presence of metabolic activation, in the 2nd Experiment in the absence of metabolic activation and in the 3rd Experiment in the presence of S9 mix no cytotoxicity was observed up to the highest required concentration evaluated for gene mutations. Due to lacking cytotoxicity in the 1st Experiment in the absence of metabolic activation, this experimental part was discontinued. Besides, after 24 hours treatment in the 3rd Experiment, in the absence of metabolic activation, unexpected strong cytotoxicity occurred. Therefore this experiment was also discontinued. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in four experiments performed independently of each other.

Under the conditions in this study, it can be concluded that N,N,N´,N´-Tetramethyl-1,6-hexanediamine is not a mutagenic substance in the HPRT locus assay using CHO cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No evidence for the induction of reverse mutation was observed in an Ames test (BASF SE, 2011) performed in S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and in E. coli strain WP2 uvrA. No evidence for the induction of reverse mutation was observed in a second Ames test (Anonymous) performed inS. typhimuriumstrains TA 98, TA 100, TA 1535 and TA 1537. In both studies, the substance was tested up to the limit concentration in the absence and presence of metabolic activation.

No evidence for the induction of forward mutation was seen in a study (BASF SE, 2012) performed in cultured CHO cells at concentrations up to 1700 µg/mL and in the absence and presence of metabolic activation.

No evidence for the induction of micronuclei formation was observed in a study performed in V79 cells at concentrations up to 1700 µg/mL and in the absence and presence of metabolic activation.

Justification for classification or non-classification

There is no evidence for genetic toxicity; no classification for genetic toxicity is therefore proposed according to the CLP Regulation (Regulation (EC) No. 1272/2008) or the Dangerous Substances Directive (Directive 67/548/EEC).