N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine

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:

October 2018 - March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

See details below

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

See details below

Principles of method if other than guideline:

A discrepancy has been noted between the General Study Plan and original data regarding the incubation period of the test plates (currently stated in the General Study Plan as ‘approximately 48 hours’). It is acknowledged that this time period is not sufficiently reflected in the original data. In reality, the plates are incubated for significantly longer
(48 to 72 hours) prior to scoring. Initially Bruce Ames, who developed the method in 1975, recommended 48 hour incubation of plates. However, by the 1990’s, with many Ames test being performed, it was considered that the plates could be incubated for 48 to 72 hours without any detriment to the study result. This is confirmed in the current OECD 471 test guideline which states the following “All plates in a given assay should be incubated at
37° C for 48 – 72 hours.” Therefore, the discrepancy detailed above is considered due to an over familiarity to the wording and long term acceptance of the phrase ‘approximately 48 hours’.
This deviation is considered to have no impact on either the result or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazin-1,3,5(2H,4H,6H)-tripropanamine

IUPAC Name:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazin-1,3,5(2H,4H,6H)-tripropanamine

Constituent 2

Reference substance name:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine

EC Number:

240-004-1

EC Name:

N,N,N',N',N'',N''-hexamethyl-1,3,5-triazine-1,3,5(2H,4H,6H)-tripropanamine

Cas Number:

15875-13-5

Molecular formula:

C18H42N6

IUPAC Name:

(3-{3,5-bis[3-(dimethylamino)propyl]-1,3,5-triazinan-1-yl}propyl)dimethylamine

Test material form:

liquid: viscous

Specific details on test material used for the study:

Identification: N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine
Batch Number: 2305905
Purity: 98.62%
Expiry Date: 15 July 2020
Appearance: Clear pale yellow liquid
Storage Conditions: Room temperature, in the dark
No correction for purity was required.

Method

Target gene:

Salmonella typhimurium
Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

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:

S9
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized
in-house procedures (outside the confines of this study). Lot No.’s PB/NF S9 25 May 2018 (Experiments 1 and 2) and 28 October 2018 (Experiment 3 Confirmatory) were used in this study.
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

Test concentrations with justification for top dose:

The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 ug/plate.

Experiment 1 was considered to be weakly positive the plate incorporation method was again employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix) to confirm initial findings. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations with an amended dose range employed of 50, 150, 500, 750 1000, 1500, 2000, 3000 and 5000 µg/plate. Nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both reproducibility and a better dose-related response.

A third (confirmatory) experiment was performed in TA100 and TA98 (with and without metabolic activation) using pre-incubation methodology following the results from Experiments 1 and 2. The dose ranges were 500, 700, 800, 900, 1000, 1250 and 1500 µg/plate for TA100 and 700, 800, 900, 1000, 1250, 1500 and 2000 µg/plate for TA98. As for Experiment 2, intermediate dose levels were selected in the confirmatory experiment in order to confirm and potentially enhance the mutagenic responses observed in both Experiments 1 and 2.

Vehicle / solvent:

Sterile distilled water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in nutrient broth (Oxoid Limited; lot numbers 2104309 04/2022 (Experiments 1 and 2) and 2216012 11/2022 (Experiment 3 only)) and incubated at 37 ± 3 °C for approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity with titres determined by viable count analysis on nutrient agar plates.

All of the plates were incubated at 37 ± 3 C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Rationale for test conditions:

The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These Salmonella typhimurium strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium, revertants may be produced after exposure to a chemical mutagen which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre treated with a mixture known to induce an elevated level of these enzymes.

Evaluation criteria:

Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Experiment 1 at 1500 µg/plate in the presence and absence of metabolic activation only

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Experiment 1 at 500 µg/plate in the presence of metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Experiment 1 @ 1500 µg/plate in the presence of metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Experiment 2

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Experiment 2 1500 µg/plate in the absence of metabolic activation and at 1500 and 2000 µg/plate in the presence of metabolic activation

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 3 @ between 500 and 1000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 3 @ between 900 and 1250 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 8

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

Experiment 3 @ between 500 and 1250 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In Experiment 1 (plate incorporation), the test item induced a greater than two-fold increase in the frequency of TA98 revertant colonies at 1500 µg/plate in the presence of metabolic activation only.
Smaller increases in revertant colony frequency were observed in TA100 at 500 µg/plate in the presence of metabolic activation, TA1535 at 1500 µg/plate in the presence of metabolic activation, TA98 at 1500 µg/plate in the absence of metabolic activation and WP2uvrA at 5000 µg/plate in the absence of metabolic activation and were all considered statistically significant using Dunnets Regression analysis.

In Experiment 2 (plate incorporation), no increase greater than two or three times the concurrent solvent control (depending on bacterial strain type) was observed in any of the strains tested (either in the presence or absence of S9-mix). However, several strains exhibited statistically significant increases in the frequency of revertant colonies in the absence and presence of metabolic activation, particularly TA100 which exhibited increases at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria.

There was also a small dose-response relationship noted between 500 and 1000 µg/plate in both the absence and presence of metabolic activation. The responses were much more clear following the inclusion of intermediate dose levels. Increases in TA98 revertant colony frequency were noted at 1500 µg/plate in the absence of metabolic activation and at 1500 and 2000 µg/plate in the presence of metabolic activation. Smaller responses were also noted for TA1535 (1500 and 2000 µg/plate in the presence of metabolic activation) and TA1537 (1500 µg/plate in the presence of metabolic activation).

In experiment 3, in the absence of metabolic activation, increases greater than two times the concurrent solvent control were observed in TA100 between 500 and 1000 µg/plate and TA98 between 900 and 1250 µg/plate. In the presence of metabolic activation, increases of greater than two times the concurrent solvent control were observed in TA100 only between 500 and 1250 µg/plate. Smaller but statistically significant increases were noted in TA98 at 900 and 1500 µg/plate. The response was much clearer following the inclusion of intermediate dose levels and employing the pre-incubation modification, particularly in TA100 which exhibited increases at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria.

In all three experiments, no test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).

Any other information on results incl. tables

Spontaneous Mutation Rates (Concurrent Negative Controls)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
147		15		27		28		10
150	(143)	22	(16)	27	(29)	24	(29)	17	(14)
133		12		32		34		16

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA		TA98		TA1537
123		22		25		21		15
132	(137)	18	(20)	30	(31)	28	(25)	22	(16)
155		21		37		25		11

 

Experiment 3

Number of revertants (mean number of colonies per plate)
Base-pair substitution type		Frameshift type
TA100		TA98
118		29
97	(107)	24	(24)
107		20

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 04 September 2018					To: 07 September 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	133 138 147	(139) 7.1#	10 19 25	(18) 7.5	26 25 29		(27) 2.1	21 29 25	(25) 4.0	9 11 12	(11) 1.5
	1.5 µg	138 132 124	(131) 7.0	16 16 22	(18) 3.5	23 19 33		(25) 7.2	21 22 25	(23) 2.1	14 4 9	(9) 5.0
	5 µg	131 124 135	(130) 5.6	29 12 23	(21) 8.6	36 28 34		(33) 4.2	22 23 21	(22) 1.0	13 14 14	(14) 0.6
	15 µg	117 138 139	(131) 12.4	11 26 14	(17) 7.9	27 22 25		(25) 2.5	11 21 27	(20) 8.1	20 13 7	(13) 6.5
	50 µg	126 137 117	(127) 10.0	21 15 15	(17) 3.5	36 27 30		(31) 4.6	21 19 26	(22) 3.6	11 14 11	(12) 1.7
	150 µg	118 118 123	(120) 2.9	21 17 14	(17) 3.5	28 21 34		(28) 6.5	32 24 27	(28) 4.0	20 20 5	(15) 8.7
	500 µg	147 150 148	(148) 1.5	19 20 17	(19) 1.5	29 28 19		(25) 5.5	32 34 26	(31) 4.2	9 18 15	(14) 4.6
	1500 µg	109 107 92	(103) 9.3	21 25 28	(25) 3.5	27 29 29		(28) 1.2	40 43 47	*** (43) 3.5	21 S 16 S 13 S	(17) 4.0
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	37 40 43		* (40) 3.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		628 694 757	(693) 64.5	982 1717 1880	(1526) 478.4	1088 1010 969		(1022) 60.5	245 201 194	(213) 27.6	210 611 559	(460) 218.1

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 04 September 2018					To: 07 September 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	133 130 132	(132) 1.5#	18 15 14	(16) 2.1	49 45 42		(45) 3.5	22 21 34	(26) 7.2	14 10 12	(12) 2.0
	1.5 µg	147 137 138	(141) 5.5	26 14 12	(17) 7.6	31 46 46		(41) 8.7	25 39 31	(32) 7.0	8 11 12	(10) 2.1
	5 µg	146 138 112	(132) 17.8	18 11 11	(13) 4.0	25 23 42		(30) 10.4	38 27 24	(30) 7.4	24 15 10	(16) 7.1
	15 µg	129 143 125	(132) 9.5	9 11 14	(11) 2.5	32 25 28		(28) 3.5	26 35 35	(32) 5.2	12 8 15	(12) 3.5
	50 µg	143 144 149	(145) 3.2	14 11 17	(14) 3.0	41 38 47		(42) 4.6	26 27 22	(25) 2.6	14 14 17	(15) 1.7
	150 µg	141 121 154	(139) 16.6	11 15 12	(13) 2.1	48 34 36		(39) 7.6	29 32 35	(32) 3.0	17 20 11	(16) 4.6
	500 µg	183 194 166	*** (181) 14.1	14 12 19	(15) 3.6	44 40 36		(40) 4.0	34 32 28	(31) 3.1	14 9 15	(13) 3.2
	1500 µg	174 S 207 S 169 S	*** (183) 20.6	36 20 37	** (31) 9.5	41 40 41		(41) 0.6	61 65 72	*** (66) 5.6	20 S 14 S 14 S	(16) 3.5
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	42 40 48		(43) 4.2	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1987 1843 1912	(1914) 72.0	396 383 342	(374) 28.2	227 270 304		(267) 38.6	262 241 259	(254) 11.4	337 335 331	(334) 3.1

 

Test Results: Experiment 2 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 11 September 2018					To: 14 September 2018
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	137 131 131	(133) 3.5#	23 29 17	(23) 6.0	40 34 24		(33) 8.1	30 21 23	(25) 4.7	13 17 12	(14) 2.6
	50 µg	144 136 147	(142) 5.7	22 20 17	(20) 2.5	32 37 46		(38) 7.1	30 28 19	(26) 5.9	10 12 16	(13) 3.1
	150 µg	133 150 141	(141) 8.5	31 35 22	(29) 6.7	41 40 33		(38) 4.4	15 39 19	(24) 12.9	16 12 20	(16) 4.0
	500 µg	184 159 151	** (165) 17.2	31 22 36	(30) 7.1	22 44 30		(32) 11.1	27 31 36	(31) 4.5	20 23 24	(22) 2.1
	750 µg	200 200 204	*** (201) 2.3	20 32 35	(29) 7.9	25 30 52		(36) 14.4	36 32 26	(31) 5.0	18 13 15	(15) 2.5
	1000 µg	239 219 242	*** (233) 12.5	29 21 38	(29) 8.5	41 33 30		(35) 5.7	35 33 39	(36) 3.1	19 11 19	(16) 4.6
	1500 µg	163 158 185	** (169) 14.4	20 39 36	(32) 10.2	41 43 43		(42) 1.2	46 38 46	* (43) 4.6	14 12 21	(16) 4.7
	2000 µg	47 S 78 S 54 S	(60) 16.3	25 36 22	(28) 7.4	43 35 40		(39) 4.0	23 43 37	(34) 10.3	4 S 8 S 16 S	(9) 6.1
	3000 µg	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0	25 39 37		(34) 7.6	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	18 43 31		(31) 12.5	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		ENNG		ENNG			4NQO		9AA
		3 µg		5 µg		2 µg			0.2 µg		80 µg
		562 544 616	(574) 37.5	606 623 702	(644) 51.2	424 441 501		(455) 40.5	126 131 125	(127) 3.2	289 319 485	(364) 105.6

 

Test Results: Experiment 2 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 11 September 2018					To: 14 September 2018
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (Water)	122 132 141	(132) 9.5#	10 20 14	(15) 5.0	32 36 32		(33) 2.3	26 39 31	(32) 6.6	13 12 22	(16) 5.5
	50 µg	141 128 120	(130) 10.6	22 15 13	(17) 4.7	35 40 56		(44) 11.0	27 24 30	(27) 3.0	22 24 13	(20) 5.9
	150 µg	141 149 177	(156) 18.9	16 32 17	(22) 9.0	34 50 32		(39) 9.9	32 34 21	(29) 7.0	11 16 13	(13) 2.5
	500 µg	166 183 172	** (174) 8.6	16 20 18	(18) 2.0	43 32 33		(36) 6.1	40 29 35	(35) 5.5	16 17 19	(17) 1.5
	750 µg	226 223 207	*** (219) 10.2	31 26 21	(26) 5.0	34 29 42		(35) 6.6	43 46 43	(44) 1.7	19 18 13	(17) 3.2
	1000 µg	223 280 261	*** (255) 29.0	32 19 21	(24) 7.0	42 40 39		(40) 1.5	37 42 57	(45) 10.4	17 14 19	(17) 2.5
	1500 µg	260 248 255	*** (254) 6.0	36 37 21	** (31) 9.0	35 42 43		(40) 4.4	41 70 47	** (53) 15.3	22 27 21	* (23) 3.2
	2000 µg	71 S 89 S 87 S	(82) 9.9	30 33 25	* (29) 4.0	37 38 48		(41) 6.1	59 51 60	** (57) 4.9	25 S 26 S 20 S	* (24) 3.2
	3000 µg	0 T 0 T 0 T	(0) 0.0	8 S 6 S 2 S	(5) 3.1	45 42 47		(45) 2.5	10 10 8	(9) 1.2	8 S 6 S 9 S	(8) 1.5
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	45 50 40		(45) 5.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		2AA		2AA			BP		2AA
		1 µg		2 µg		10 µg			5 µg		2 µg
		1891 1990 1837	(1906) 77.6	363 362 295	(340) 39.0	238 211 240		(230) 16.2	169 157 168	(165) 6.7	392 370 380	(381) 11.0

Experiment 3 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 29 January 2019		To: 01 February 2019
S9-Mix (-)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strain		Frameshift strain
		TA100		TA98
	Solvent Control (Water)	98 103 77	(93) 13.8#	16 17 14	(16) 1.5
	500 µg	220 219 188	*** (209) 18.2	N/T
	700 µg	211 254 242	*** (236) 22.2	21 29 26	* (25) 4.0
	800 µg	232 201 230	*** (221) 17.3	27 29 34	** (30) 3.6
	900 µg	181 231 210	*** (207) 25.1	33 28 41	*** (34) 6.6
	1000 µg	233 248 250	*** (244) 9.3	27 21 35	** (28) 7.0
	1250 µg	110 S 76 S 76 S	(87) 19.6	39 32 26	*** (32) 6.5
	1500 µg	67 S 71 S 53 S	(64) 9.5	23 S 28 S 28 S	* (26) 2.9
	2000 µg	N/T		0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (-)	Name Dose Level No. of Revertants	ENNG		4NQO
		3 µg		0.2 µg
		589 538 591	(573) 30.0	211 171 211	(198) 23.1

 

Experiment 3 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 29 January 2019		To: 01 February 2019
S9-Mix (+)	Dose Level Per Plate	Number of revertants (mean) +/- SD
		Base-pair substitution strain		Frameshift strain
		TA100		TA98
	Solvent Control (Water)	101 125 93	(106) 16.7#	26 29 13	(23) 8.5
	500 µg	211 222 211	** (215) 6.4	N/T
	700 µg	229 247 244	*** (240) 9.6	32 43 29	(35) 7.4
	800 µg	257 291 254	*** (267) 20.6	36 31 38	(35) 3.6
	900 µg	259 277 276	*** (271) 10.1	54 36 39	* (43) 9.6
	1000 µg	281 255 241	*** (259) 20.3	28 43 49	(40) 10.8
	1250 µg	188 216 236	** (213) 24.1	36 36 38	(37) 1.2
	1500 µg	140 S 108 S 263 S	* (170) 81.8	35 35 56	* (42) 12.1
	2000 µg	N/T		8 S 23 S 6 S	(12) 9.3
Positive controls S9-Mix (+)	Name Dose Level No. of Revertants	2AA		BP
		1 µg		5 µg
		571 601 574	(582) 16.5	89 90 118	(99) 16.5

2AA  2-Aminoanthracene

BP           Benzo(a)pyrene

N/T            Not tested at this dose level

S                Sparse bacterial background lawn

*                p<=0.05

**              p<=0.01

***            p<=0.00

#             Standard deviation

Applicant's summary and conclusion

Conclusions:

N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was considered to be weakly mutagenic under the conditions of this test.

Executive summary:

N,N,N',N',N'',N''-Hexamethyl-1,3,5-Triazine-1,3,5(2H,4H,6H)-Tripropanamine was tested according to OECD Test Guideline 471 "Bacterial Reverse Mutation Test".

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to nine dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).

The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 ug/plate. As Experiment 1 was considered to be weakly positive the plate incorporation method was again employed for Experiment 2 in the presence and absence of metabolic activation (S9-mix) to confirm initial findings.  

The experiment was repeated using fresh cultures of the bacterial strains and fresh test item formulations with an amended dose range employed of 50, 150, 500, 750 1000, 1500, 2000, 3000 and 5000 µg/plate. Nine test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both reproducibility and a better dose-related response.

A third (confirmatory) experiment was performed in TA100 and TA98 (with and without metabolic activation) using pre-incubation methodology following the results from Experiments 1 and 2.  The dose ranges were 500, 700, 800, 900, 1000, 1250 and 1500 µg/plate for TA100 and 700, 800, 900, 1000, 1250, 1500 and 2000 µg/plate for TA98.  As for Experiment 2, intermediate dose levels were selected in the confirmatory experiment in order to confirm and potentially enhance the mutagenic responses observed in both Experiments 1 and 2.

The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with and without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.  In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of metabolic activation from 1500 µg/plate (TA1537) and at 5000 µg/plate (remaining Salmonella strains).

In the presence of metabolic activation, weakened lawns were noted from 1500 µg/plate to TA100 and TA1537 and at 5000 µg/plate to TA98 and TA1535.  

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (plate incorporation method).  The test item induced a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in both the presence and absence of metabolic activation, initially from 2000 µg/plate (TA100 and TA1537).  Toxicity was also observed in the confirmatory experiment from 1250 µg/plate in the absence of metabolic activation and 1500 µg/plate in the presence of metabolic activation.  

No weakened bacterial background lawns were observed in the E.coli strain, WP2uvrA in any of the experiments performed.

No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in any of the experiments performed.

In Experiment 1 (plate incorporation method), the test item induced a greater than two-fold increase in the frequency of TA98 revertant colonies at 1500 µg/plate in the presence of metabolic activation only.  Smaller, statistically significant increases in revertant colony frequency were observed in TA100 at 500 µg/plate in the presence of metabolic activation, TA1535 (1500 µg/plate in the presence of metabolic activation), TA98 (1500 µg/plate in the absence of metabolic activation) and WP2uvrA (5000 µg/plate in the absence of metabolic activation).

In Experiment 2, no increase greater than two or three times the concurrent solvent control (depending on bacterial strain type) was observed in any of the strains tested (either in the presence or absence of S9-mix).  However, several strains exhibited statistically significant increases in the frequency of revertant colonies in the absence and presence of metabolic activation, particularly TA100 which exhibited increases in colony frequency at sub-toxic dose concentrations in excess of the maximum value stated in the acceptability criteria outlined in Section 3.4.  There was also a small dose-response relationship noted between 500 and 1000 µg/plate in both the absence and presence of metabolic activation.

In the Confirmatory Experiment, in the absence of metabolic activation, increases greater than two times the concurrent solvent control were observed in TA100 between 500 and 1000 µg/plate and TA98 between 900 and 1250 µg/plate.  In the presence of metabolic activation, increases of greater than two times the concurrent solvent control were observed in TA100 only between 500 and 1250 µg/plate.  Smaller but statistically significant increases were noted in TA98 at 900 and 1500 µg/plate.  

The observed increases in revertant colony frequency which were two times greater than the concurrent solvent control were inconsistent between Experiment 1 and 2.  In Experiment 1, only TA98 at 1500 µg/plate in the presence of S9-mix achieved a twofold increase and no strain achieved  twofold increases over the solvent control in Experiment 2.  However, both experiments exhibited statistically significant increases that approached two times the concurrent solvent control, particularly in TA98 and TA100, both in the presence and absence of metabolic activation. The number of revertant colonies observed in these strains also approached or exceeded the upper limit of the expected range for these strains.

 

A third, confirmatory experiment using the pre-incubation method was performed in TA100 and TA98, both in the presence and absence of metabolic activation and a narrowed dose range.  The results of this experiment showed that the test item exhibited increases in revertant colony frequency of greater than two times the concurrent solvent control over all of the sub-toxic dose levels in TA100 both with and without metabolic activation.  In TA98, increases in revertant colony frequency of greater than two times the concurrent solvent control were observed at 900 and 1250 µg/plate only and none in the presence of S9 mix (although fold increases were noted from 1.6 to 1.9 times the concurrent vehicle control).

 

As it is, there is no concordance between the experiments other than there are small but reproducible increases in revertant colony frequency in TA100 and TA98 in particular.  However, these statistically significant increases were observed consistently over all three experiments and, where the change in methodology to the pre-incubation method was implemented, the increased sensitivity of the assay exhibited a much clearer response, particularly in the frequency of TA100 revertant colonies. Consequently, the overall conclusion of the study must be that the test substance should be considered weakly mutagenic.

The weakly mutagenic results of the Ames test were evaluated against historical control data for the period 2017 -2018 (attached ). It was observed that for TA98 only one value was out of the historical control range (Experiment 1 – with metabolic activation, 1500 µg). The mean values of the negative control data from the Ames test were within the range of 2017, but near to the maximum values and some single values are also out of the range. For TA 100, with and without metabolization and increase of 32% in the number of mutations within the negative control data from 2017 to 2018, which was not observed for the other strains. Historical data for 2019 were not yet available, so a trend could not yet be confirmed.