N,N,N',N'-tetramethylhexamethylenediamine

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 Jul 1997

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

not specified

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N'-tetramethylhexamethylenediamine

EC Number:

203-842-9

EC Name:

N,N,N',N'-tetramethylhexamethylenediamine

Cas Number:

111-18-2

Molecular formula:

C10H24N2

IUPAC Name:

[6-(dimethylamino)hexyl]dimethylamine

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

Method

Target gene:

Histidine and tryptophan.

Species / strain

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.

Controls

Controls 1

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.

Controls 2

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.

Controls 3

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.

Controls 4

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.

Controls 5

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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	-	-	-	-	-	-	-	-

 

Applicant's summary and conclusion

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.