1,3,5-Triazine-2,4,6-triamine, N2,N2'-1,6-hexanediylbis[N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethyl-4-piperidinyl)-

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:

04 Nov 2014 - 13 Nov 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N2-[6-({4,6-bis[butyl(2,2,6,6-tetramethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}(2,2,6,6-tetramethylpiperidin-4-yl)amino)hexyl]-N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

EC Number:

629-754-4

Cas Number:

210988-99-1

Molecular formula:

C82 H156 N18

IUPAC Name:

N2-[6-({4,6-bis[butyl(2,2,6,6-tetramethylpiperidin-4-yl)amino]-1,3,5-triazin-2-yl}(2,2,6,6-tetramethylpiperidin-4-yl)amino)hexyl]-N4,N6-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine

Details on test material:

- Physical state: Solid / white to beige
- Storage condition of test material: Room temperature

Method

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from phenobarbital and β-naphthoflavone induced rats

Test concentrations with justification for top dose:

Standard plate test: 0; 0.1; 0.33; 1.0; 3.3; 10 and 33 μg/plate (without S9 mix); 0; 0.33; 1.0; 3.3; 10; 33 and 100 μg/plate (with S9 mix)
Preincubation test with and without S9 mix: 0; 0.1; 0.33; 1.0; 3.3; 10 and 33 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Controls

Untreated negative controls:

yes

Remarks:

Sterility control

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

see below for details

Positive control substance:

other: see below

Remarks:

with and without S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: plate incorporation; preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: at 37°C for 48 – 72 hours in the dark

NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn

POSITIVE CONTROLS
The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) (Sigma-Aldrich; 96%)
- 2.5 μg/plate, dissolved in DMSO, with strains: TA 1535, TA 100, TA 1537, TA 98
- 60 μg/plate, dissolved in DMSO, with strain: Escherichia coli WP2 uvrA
Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) (Fluka; 97%)
- 5 μg/plate, dissolved in DMSO, with strains: TA 1535, TA 100
• 4-nitro-o-phenylenediamine (NOPD) (Sigma-Aldrich; 98%)
- 10 μg/plate, dissolved in DMSO, with strain: TA 98
• 9-aminoacridine (AAC) (Sigma-Aldrich; 98%)
- 100 μg/plate, dissolved in DMSO, with strain: TA 1537
• 4-nitroquinoline-N-oxide (4-NQO) (Sigma-Aldrich; 98%)
- 5 μg/plate, dissolved in DMSO, with strain: E. coli WP2 uvrA

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance was generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control data range under all experimental conditions in at least two experiments carried out independently of each other.

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:

cytotoxicity

Remarks:

strong bacteriotoxicity was observed depending on the strain and test conditions from about 1.0 μg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test substance precipitation was found at 5000 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
In this study with and without S9 mix, the number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain. In addition, the positive control substances both with and without S9 mix induced a significant increase in the number of revertant colonies within the range of the historical positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A strong bacteriotoxic effect (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed in the standard plate test depending on the strain and test conditions from about 33 μg/plate onward.
In the preincubation assay strong bacteriotoxicity (reduced his- or trp- background growth, decrease in the number of his+ or trp+ revertants) was observed depending on the strain and test conditions from about 1.0 μg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Experimental Result

Experiment I (Standard Plate Test):

	Mean revertants per plate		Mean revertants per plate		Mean revertants per plate		Mean revertants per plate		Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	19.3	27.0	29.0	45.0	10.0	26.3	8.0	8.0	52.3	64.0
33	15.7	23.3	11.0	11.0	8.3	18.7	5.0	7.7	45.7	57.7
100	0.0	0.0	0.0	0.0	0.0	12.0	0.0	0.0	0.0	40.3
333	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
1000	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
2500	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
5000	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0	0.0
positive control	369.3	1898.7	3138.3	457.0	4254.0	200.3	1323.3	189.7	986.3	269.3

Experiment II (Standard Plate Test):

	Mean revertants per plate		Mean revertants per plate		Mean revertants per plate		Mean revertants per plate		Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	23.3		31.7		11.7		10.0		58.7
0.1	25.3	25.3	31.0	34.3	10.7	12.3	9.3	11.0	60.7	67.0
0.33	23.0	24.7	32.7	38.3	10.3	10.0	11.0	11.7	57.7	70.3
1.0	30.3	25.7	43.7	33.3	9.0	9.0	13.3	12.3	56.0	62.3
3.3	26.3	21.3	35.0	30.3	10.7	8.3	9.0	9.7	62.3	65.7
10	24.0	23.3	26.0	23.0	8.0	12.0	10.0	10.7	51.7	64.7
33	10.7	17.7	7.0	16.7	6.0	8.0	1.3	6.0	42.3	59.3
100		0.0		0.0		2.7		0.0		13.0
positive control	389.7	1986.0	4390.3	594.3	4387.3	214.7	881.0	149.0	1034.0	240.7

Experiment III (Preincubation test):

	Mean revertants per plate		Mean revertants per plate		Mean revertants per plate		Mean revertants per plate		Mean revertants per plate
	TA 98		TA 100		TA 1535		TA 1537		WP2uvrA
Dose (µg/plate)	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	19.0	20.3	32.3	30.7	11.3	10.0	11.0	12.0	45.7	49.0
0.1	21.7	23.7	31.7	35.3	10.0	10.7	9.3	10.3	43.7	50.7
0.33	16.3	27.0	30.7	29.7	10.0	8.0	8.7	9.7	41.3	52.3
1.0	11.0	22.7	32.0	31.7	10.7	10.3	6.0	10.0	26.0	48.7
3.3	10.7	20.0	24.0	37.7	10.3	10.0	5.7	9.0	20.3	49.3
10	11.7	23.7	12.7	26.0	4.3	9.0	6.7	11.7	26.0	51.7
33	0.0	16.3	0.0	3.0	0.0	4.3	0.0	3.3	17.3	45.0
positive control	343.0	1337.7	2146.3	691.7	2203.0	216.7	678.3	126.3	456.7	206.0

Applicant's summary and conclusion

Conclusions:

Under the experimental conditions chosen here, it is concluded that the test article is not a mutagenic test substance in the bacterial reverse mutation test in the absence and the presence of metabolic activation.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of bacterial strains Salmonella typhimurium TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA in a reverse mutation assay. Dose range was 0.1 μg - 5000 μg/plate in the standard plate test and 0.1 μg - 33 μg/plate in the preincubation test. Both tests were performed with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found at 5000 μg/plate with and without S9 mix. A strong bacteriotoxic effect was observed depending on the strain and test conditions from about 1.0 μg/plate onward. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.