1,3-Propanediaminium, N1-[3-[[2-[dimethyl[3-[(2-methyl-1-oxo-2-propen-1-yl)amino]propyl]ammonio]acetyl]amino]propyl]-2-hydroxy-N1,N1,N3,N3,N3-pentamethyl-, chloride (1:3)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 April to 08 May, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP, guidline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2R)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium [3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2S)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium hexachloride

EC Number:

700-055-7

Cas Number:

630113-05-2

Molecular formula:

C22 H48 N5 O3.3Cl

IUPAC Name:

[3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2R)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium [3-(2-{dimethyl[3-(2-methylprop-2-enamido)propyl]azaniumyl}acetamido)propyl][(2S)-2-hydroxy-3-(trimethylazaniumyl)propyl]dimethylazanium hexachloride

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Napthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Doses adjusted for content of the main constituent
Experiment 1: 3; 10; 33; 100; 333; 1000; 2500; and 5000; µg/plate
Experiment 2: 33; 100; 333; 1000; 2500; and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: acceptable solubility of test substance

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535; TA 100 without metabolic activation

Controls 2

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

TA 1537; TA 98 without metabolic activation

Controls 3

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

WP2 uvrA without metabolic activation

Controls 4

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA 1535; TA 1537; TA 98, TA100, Wp2 uvrA with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: experiment 1: plate incorporation; experiment 2: preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

There were no toxic effects. Plates incubated with the test item showed normal background growth up to 5000µg/plate with and without S9 mix in both experiments.

No substantial increase in revertant colony numbers of any of the 5 tester strains was observed following treatment with TRIQUAT MONOMER at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and these showed a distinct increase of induced revertant colonies.

Results of Gene Mutation Assay: Pre-experiment and Experiment 1:

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			14 ± 2	15 ± 4	37 ± 3	150 ± 12	49 ± 10
	Untreated			14 ± 4	16 ± 2	39 ± 5	129 ± 20	50 ± 1
	Triquat	3 µg		14 ± 3	18 ± 3	33 ± 5	153 ± 18	35 ± 4
	Monomer	10 µg		17 ± 2	17 ± 2	35 ± 2	157 ± 8	48 ± 4
		33 µg		15 ± 1	15 ± 3	34 ± 1	143 ± 9	46 ± 3
		100 µg		11 ± 2	15 ± 2	36 ± 1	150 ± 8	43 ± 6
		333 µg		13 ± 1	18 ± 4	28 ± 2	148 ± 10	37 ± 6
		1000 µg		14 ± 5	15 ± 5	29 ± 3	145 ± 6	44 ± 8
		2500 µg		14 ± 3	18 ± 3	33 ± 7	167 ± 11	50 ± 2
		5000 µg		19 ± 4	19 ± 4	30 ± 3	187 ± 10	54 ± 2
	NaN3	10 µg		2121 ± 70			2568 ± 110
	4-NOPD	10 µg				543 ± 23
	4-NOPD	50 µg			106 ± 13
	MMS	3.0 µL						1283 ± 21
With Activation	Deionised water			17 ± 2	24 ± 5	45 ± 5	171 ± 5	70 ± 7
	Untreated			17 ± 3	21 ± 8	46 ± 4	160 ± 7	66 ± 3
	Triquat	3 µg		17 ± 8	24 ± 4	43 ± 8	142 ± 11	56 ± 7
	Monomer	10 µg		18 ± 1	23 ± 3	39 ± 5	156 ± 16	67 ± 5
		33 µg		21 ± 4	20 ± 1	34 ± 7	177 ± 9	66 ± 9
		100 µg		17 ± 5	21 ± 1	41 ± 2	159 ± 19	49 ± 13
		333 µg		17 ± 3	21 ± 1	36 ± 1	157 ± 7	59 ± 12
		1000 µg		14 ± 2	25 ± 2	46 ± 5	152 ± 6	65 ± 2
		2500 µg		14 ± 1	26 ± 3	44 ± 8	147 ± 8	65 ± 5
		5000 µg		24 ± 3	22 ± 4	39 ± 5	138 ± 14	60 ± 8
	2-AA	2.5 µg		403 ± 10	297 ± 13	2145 ± 42	2447 ± 104
	2-AA	10.0 µg						297 ± 5

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Results of Gene Mutation Assay: Experiment 2:

Metabolic Activation	Test Group	Dose Level (µg/plate)		Revertant Colony Counts (Mean ±SD)
				TA 1535	TA 1537	TA 98	TA 100	WP2 uvrA
Without Activation	Deionised water			16 ± 4	12 ± 4	32 ± 9	137 ± 12	58 ± 5
	Untreated			17 ± 0	17 ± 7	27 ± 4	134 ± 6	55 ± 7
	Triquat	33 µg		16 ± 6	9 ± 1	30 ± 7	135 ± 12	49 ± 2
	Monomer	100 µg		17 ± 3	12 ± 5	30 ± 9	136 ± 11	53 ± 3
		333 µg		17 ± 3	13 ± 3	30 ± 10	128 ± 11	58 ± 10
		1000 µg		13 ± 4	10 ± 3	29 ± 7	134 ± 7	55 ± 6
		2500 µg		17 ± 2	11 ± 2	34 ± 3	132 ± 12	60 ± 3
		5000 µg		16 ± 8	15 ± 1	31 ± 4	131 ± 8	60 ± 9
	NaN3	10 µg		2040 ± 40			2152 ± 55
	4-NOPD	10 µg				491 ± 55
	4-NOPD	50 µg			124 ± 5
	MMS	3.0 µL						635 ± 85
With Activation	Deionised water			21 ± 6	16 ± 4	39 ± 3	172 ± 12	59 ± 8
	Untreated			16 ± 3	14 ± 1	46 ± 11	161 ± 25	58 ± 9
	Triquat	33 µg		22 ± 2	16 ± 4	35 ± 4	176 ± 13	57 ± 8
	Monomer	100 µg		23 ± 6	16 ± 3	39 ± 8	174 ± 12	61 ± 9
		333 µg		22 ± 4	18 ± 3	43 ± 11	176 ± 12	66 ± 5
		1000 µg		18 ± 5	19 ± 0	38 ± 5	159 ± 12	64 ± 6
		2500 µg		24 ± 4	15 ± 7	35 ± 10	159 ± 14	61 ± 4
		5000 µg		23 ± 1	15 ± 6	36 ± 12	163 ± 11	63 ± 10
	2-AA	2.5 µg		245 ± 7	125 ± 10	1041 ± 58	977 ± 93
	2-AA	10.0 µg						227 ± 25

Key to Positive Controls
NaN3 2-AA 4-NOPD MMS	sodium azide 2-aminoanthracene 4-nitro-o-phenylene-diamine methyl methane sulfonate

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Executive summary:

This study was performed to investigate the potential of TRIQUAT MONOMER to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, and TA 100, and Escherichia coli strain WP2 uvrA.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations of the active ingredient (main constituent):

Pre-Experiment/Experiment I:   3, 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate

Experiment II:  33; 100; 333; 1000; 2500; and 5000 µg/plate

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without metabolic activation in both independent experiments.

No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in the test groups with or without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with TRIQUAT MONOMER at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, in the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, TRIQUAT MONOMER is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.