Amines, di-C12-18-alkylmethyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 20 July 2022

Experimental completion date 01 August 2022

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 July 1997 as corrected in 2020

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) number 440/2008 of 30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: • The Japanese Ministry of Health, Labour and Welfare (MHLW), Ministry of Economy, Trade and Industry (METI), and Ministry of the Environment (MOE) Guidelines of 31 March 2011

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: • ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Amines, di-C12-18-alkylmethyl
Chemical name N-methyl-N-(C12-18)-alkyl(C12-18)-alkane-1-amine
CAS Number 68439-75-8*
Batch Number 2455431
Expiry Date 01-02-2025
Appearance Liquid
Purity (%) UVCB; stated as 100%
Correction Factor Not Applicable
Storage Conditions: Room temperature in the dark

Target gene:

Histidone operon for Salmonella
Tryptophan operon for Escherichia

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:

The metabolic activation system was lyophilized phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix (MutazymeTM) reconstituted with cold, sterile water to provide a 10% phenobarbital/beta-naphthoflavone induced rat liver S9 and cofactors mix. Lot No. 4509 was used in this study.

Test concentrations with justification for top dose:

Experiment 1:1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
The maximum concentration was 5000 g/plate (the OECD TG 471 maximum recommended dose level).

Experiment 2: 15, 50, 150, 500, 1500 and 5000 µg/ plate
Six test item concentrations were selected in Experiment 2 to ensure the study achieved at least four non toxic dose levels as required by the test guideline. These were selected based on the absence of cytotoxicity noted in Experiment 1 and the potential for a change in the test item cytotoxicity profile following the modification in test methodology from plate incorporation to pre-incubation

Vehicle / solvent:

The solvent control used was as follows:
Identity: THF
Supplier: Sigma-Aldrich
Batch number, (purity), expiry STBK4969, (99.99%), Feb 2023

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rate

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Absence of S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Absence of S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Absence of S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: "-Aminoanthracene (2AA)

Remarks:

Presence of S9 mix

Untreated negative controls:

yes

Remarks:

Spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Presence of S9 mix

Details on test system and experimental conditions:

Test Item Preparation and Analysis
In solubility checks performed in house, the test item was noted as immiscible in sterile distilled water, dimethyl sulphoxide and dimethyl formamide at 50 mg/mL and acetone at 100 mg/mL but was fully miscible in tetrahydrofuran (THF) at 200 mg/mL. THF was therefore selected as the solvent.

The test item was accurately weighed and, on the day of each experiment, approximate
half-log dilutions prepared in high purity THF by vortex mixing. No correction for purity was required. THF is toxic to the bacterial cells at and above 50 µL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 µL (0.025 mL) aliquots. THF is considered an acceptable solvent for use in this test system (Maron et al., 1981). All test item preparation and dosing was performed under yellow safety lighting.

All formulations were used within four hours of preparation and were assumed stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations were not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.025 mL aliquot of the appropriate concentration of test item or solvent or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.

With Metabolic Activation
The procedure was the same as described previously except that untreated controls were not performed and, following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
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 of the background bacterial lawn (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 using the plate incorporation method was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).

Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.025 mL of the appropriate concentration of test item formulation or solvent or 0.1 mL of appropriate positive control were incubated at 37 ± 3°C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Each concentration of the test item, appropriate positive and solvent controls and each bacterial strain, was assayed using triplicate plates. Untreated controls were also performed in triplicate on the same day as the mutation test.

With Metabolic Activation
The procedure was the same as described previously except that untreated controls were not performed and, following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3°C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
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 of the background bacterial lawn (toxicity).


The following sterility controls were performed:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix in triplicate for both experiments;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix in triplicate for both experiments;
The maximum concentration of the test item in the absence of S9-mix only was plated onto a nutrient agar plate (on a single plate prior to Experiment 1).

Evaluation criteria:

3.5 Evaluation Criteria
A test article is considered to have provided a mutagenic response if the assay data are valid, and:
1. Treatments with the test article provide a concentration-related increase in revertant numbers at one or more concentrations in at least one strain with or without metabolic activation system
2. An increase in mean revertant colony numbers per plate is observed which is ≥2-fold (in strains TA98, TA100 and WP2 uvrA pKM101) or ≥3-fold (in strains TA1535 or TA1537) the concurrent vehicle control values
3. Any increase in revertant numbers is reproducible, where applicable.
4. Statistical analysis of data as determined by UKEMS (Mahon et al, 1989) where the values are outside or exceed the in-house historical vehicle\untreated control range.

Results which only partially satisfy the above criteria will be dealt with on a case-by-case basis. Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.
Although most experiments give clear positive or negative results, in some instances the data generated prohibit making a definite judgment about test item activity. Results of this type are reported as equivocal.

Statistics:

The plate scoring system contains built-in statistical analysis using Dunnett’s and statistical significance was included as part of the result evaluation.
At the request of the Sponsor, additional analysis was performed on the positive controls to further support their efficacy and acceptability. Significance was confirmed for the positive control values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent vehicle control.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A test item precipitate (oily in appearance) was noted at and above 500 µg/plate in both the presence and absence of S9-mix. This observation did not prevent the scoring of revertant colonies.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The dose level of the test item was selected as the maximum dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absenceof S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A test item precipitate (oily in appearance) was noted at and above 500 µg/plate in both the presence and absence of S9-mix. This observation did not prevent the scoring of revertant colonies.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The dose level of the test item was selected as the maximum dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absenceof S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A test item precipitate (oily in appearance) was noted at and above 500 µg/plate in both the presence and absence of S9-mix. This observation did not prevent the scoring of revertant colonies.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The dose level of the test item was selected as the maximum dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absenceof S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A test item precipitate (oily in appearance) was noted at and above 500 µg/plate in both the presence and absence of S9-mix. This observation did not prevent the scoring of revertant colonies.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The dose level of the test item was selected as the maximum dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absenceof S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

A test item precipitate (oily in appearance) was noted at and above 500 µg/plate in both the presence and absence of S9-mix. This observation did not prevent the scoring of revertant colonies.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The dose level of the test item was selected as the maximum dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absenceof S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Please refer to "Any other information on results" for the Tables mentioned below

Prior to use, the relevant strains were checked for characteristics (deep rough character, ampicillin resistance, UV light sensitivity and histidine or tryptophan auxotrophy), viability and spontaneous reversion rate (all checks were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments were shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report but are reported in the original data for this study.

Results for the untreated controls (spontaneous mutation rates) and viability are presented in Table 1 and were considered acceptable. These data are for concurrent untreated control plates dosed in the absence of S9 performed on the same day as the Mutation Test.

The number of revertant counts for the solvent (THF) control plates were generally consistent with the laboratory’s historical control ranges. A single count for TA100 (vehicle control dosed in the absence of S9-mix after the second mutation test) was marginally below the minimum level of the in-house historical untreated/solvent control minima for the tester strain. This count was considered acceptable as the other solvent and untreated control counts were within the expected range and the tester strain responded very well to the respective positive controls in both the presence and absence of S9 mix. The positive control chemicals induced increases in mean revertant numbers of >2-fold in strains TA98, TA100, and WP2uvrA pKM101, and >3-fold in strains TA1535 and TA1537 the concurrent vehicle control values, confirming discrimination between different strains and an active S9 preparation.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test item, positive and solvent controls, both with and without S9-mix, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.


Experiment 1 (plate incorporation) – Table 2 and Table 3
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 as recommended for a soluble and non-toxic substance.

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix.

A precipitate of the test item (oily in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix. This precipitate did not prevent the scoring of revertant colonies.
 
There were no meaningful statistically or biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, at any test item dose, either with or without S9-mix in Experiment 1 (plate incorporation method). Three statistically significant values were noted (TA1537 at 5 µg/plate in the absence of S9-mix and at 50 and 150 µg/plate in the presence of S9-mix). However, as the maximum fold increase was only 1.7 times the concurrent vehicle control and the individual colony counts were within the in-house historical vehicle/untreated control range for the relevant strain, the responses were considered of no biological relevance.

Experiment 2 (pre-incubation) – Table 4 and Table 5
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).

There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix.

A test item precipitate (oily in appearance) was noted at and above 500 g/plate in both the presence and absence of S9-mix. This observation did not prevent the scoring of revertant colonies.

There were no statistically or biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, at any test item dose, either with or without S9-mix.

Table 1             Spontaneous Mutation Rates (Concurrent Untreated Controls in the Absence of S9)

Experiment 1

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA pKM101		TA98		TA1537
124		11		111		20		12
152	(138)	16	(12)	95	(101)	30	(22)	6	(8)
138		10		98		17		6
Viability – Bacterial cells 10^9 per mL
2.8		2.6		2.3		1.9		2.3

Experiment 2

Number of revertants (mean number of colonies per plate)
Base-pair substitution type						Frameshift type
TA100		TA1535		WP2uvrA pKM101		TA98		TA1537
103		14		100		20		6
117	(111)	12	(15)	116	(114)	19	(18)	8	(6)
113		18		125		14		4
Viability – Bacterial cells 10^9 per mL
1.5		2.2		4.2		1.7		1.4

 

 

Table 2             Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period		From: 22 July 2022					To: 25 July 2022
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrApKM101			TA98		TA1537
	Solvent Control (THF)	127 123 109	(120) 9.5#	9 10 17	(12) 4.4	107 84 96		(96) 11.5	25 19 25	(23) 3.5	6 7 15	(9) 4.9
	1.5 µg	123 116 125	(121) 4.7	10 9 9	(9) 0.6	106 113 114		(111) 4.4	24 24 25	(24) 0.6	11 10 10	(10) 0.6
	5 µg	116 127 112	(118) 7.8	19 13 9	(14) 5.0	102 145 97		(115) 26.4	23 30 24	(26) 3.8	16 15 15	(15) 0.6 *
	15 µg	142 140 124	(135) 9.9	16 14 13	(14) 1.5	130 81 83		(98) 27.7	35 23 26	(28) 6.2	15 10 10	(12) 2.9
	50 µg	110 122 122	(118) 6.9	15 16 12	(14) 2.1	86 91 172		(116) 48.3	15 15 17	(16) 1.2	11 11 11	(11) 0.0
	150 µg	115 135 113	(121) 12.2	19 11 10	(13) 4.9	107 105 105		(106) 1.2	14 17 20	(17) 3.0	16 16 10	(14) 3.5
	500 µg	132 P 115 P 109 P	(119) 11.9	11 P 10 P 9 P	(10) 1.0	114 P 116 P 114 P		(115) 1.2	27 P 19 P 11 P	(19) 8.0	10 P 14 P 14 P	(13) 2.3
	1500 µg	117 P 120 P 139 P	(125) 11.9	15 P 14 P 15 P	(15) 0.6	140 P 142 P 121 P		(134) 11.6	19 P 21 P 18 P	(19) 1.5	13 P 13 P 14 P	(13) 0.6
	5000 µg	124 P 129 P 131 P	(128) 3.6	14 P 13 P 12 P	(13) 1.0	114 P 145 P 100 P		(120) 23.0	15 P 26 P 21 P	(21) 5.5	11 P 11 P 10 P	(11) 0.6
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		0.5 µg			0.2 µg		80 µg
	No. of Revertants	649 747 739	(712) 54.4 ***	615 1068 1096	(926) 270.0 ***	1159 1169 1181		(1170) 11.0 ***	127 110 125	(121) 9.3 ***	196 125 125	(149) 41.0 ***

 

Table 3             Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period		From: 22 July 2022					To: 25 July 2022
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrApKM101			TA98		TA1537
	Solvent Control (THF)	139 127 133	(133) 6.0#	11 11 14	(12) 1.7	156 152 135		(148) 11.2	24 27 22	(24) 2.5	10 11 10	(10) 0.6
	1.5 µg	134 119 118	(124) 9.0	16 11 6	(11) 5.0	138 143 151		(144) 6.6	34 34 23	(30) 6.4	10 10 10	(10) 0.0
	5 µg	132 139 156	(142) 12.3	15 15 16	(15) 0.6	187 155 150		(164) 20.1	20 37 27	(28) 8.5	16 13 13	(14) 1.7
	15 µg	134 134 153	(140) 11.0	12 10 10	(11) 1.2	143 142 146		(144) 2.1	32 26 31	(30) 3.2	9 9 10	(9) 0.6
	50 µg	130 143 129	(134) 7.8	12 10 9	(10) 1.5	143 139 139		(140) 2.3	23 28 30	(27) 3.6	17 15 17	(16) 1.2 **
	150 µg	143 136 135	(138) 4.4	7 9 7	(8) 1.2	137 139 137		(138) 1.2	23 23 22	(23) 0.6	21 19 14	(18) 3.6 **
	500 µg	141 P 141 P 156 P	(146) 8.7	7 P 13 P 10 P	(10) 3.0	149 P 159 P 149 P		(152) 5.8	32 P 30 P 29 P	(30) 1.5	20 P 11 P 11 P	(14) 5.2
	1500 µg	129 P 135 P 136 P	(133) 3.8	9 P 8 P 15 P	(11) 3.8	157 P 164 P 142 P		(154) 11.2	35 P 38 P 23 P	(32) 7.9	14 P 14 P 14 P	(14) 0.0
	5000 µg	141 P 124 P 122 P	(129) 10.4	7 P 10 P 8 P	(8) 1.5	147 P 148 P 117 P		(137) 17.6	39 P 31 P 22 P	(31) 8.5	8 P 10 P 10 P	(9) 1.2
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	1366 1379 1193	(1313) 103.8 ***	223 200 188	(204) 17.8 ***	731 707 635		(691) 50.0 ***	157 218 179	(185) 30.9 ***	150 170 155	(158) 10.4 ***

 

Table 4             Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period		From: 29 July 2022					To: 01 August 2022
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrApKM101			TA98		TA1537
	Solvent Control (THF)	71 95 89	(85) 12.5#	12 9 20	(14) 5.7	110 107 101		(106) 4.6	19 20 16	(18) 2.1	7 6 8	(7) 1.0
	15 µg	97 87 96	(93) 5.5	11 14 16	(14) 2.5	106 105 107		(106) 1.0	14 20 17	(17) 3.0	5 6 6	(6) 0.6
	50 µg	96 97 96	(96) 0.6	23 25 15	(21) 5.3	110 107 108		(108) 1.5	30 24 22	(25) 4.2	7 8 12	(9) 2.6
	150 µg	104 92 94	(97) 6.4	12 9 16	(12) 3.5	116 118 115		(116) 1.5	17 14 17	(16) 1.7	6 5 6	(6) 0.6
	500 µg	84 P 88 P 103 P	(92) 10.0	11 P 14 P 10 P	(12) 2.1	110 P 107 P 106 P		(108) 2.1	12 P 6 P 15 P	(11) 4.6	13 P 5 P 5 P	(8) 4.6
	1500 µg	95 P 90 P 89 P	(91) 3.2	9 P 9 P 17 P	(12) 4.6	102 P 102 P 132 P		(112) 17.3	14 P 8 P 14 P	(12) 3.5	6 P 11 P 2 P	(6) 4.5
	5000 µg	113 P 95 P 94 P	(101) 10.7	17 P 17 P 18 P	(17) 0.6	91 P 107 P 109 P		(102) 9.9	14 P 18 P 16 P	(16) 2.0	3 P 4 P 9 P	(5) 3.2
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		0.5 µg			0.2 µg		80 µg
	No. of Revertants	1198 1044 1127	(1123) 77.1 ***	4513 4175 4122	(4270) 212.1 ***	899 841 794		(845) 52.6 ***	162 180 189	(177) 13.7 ***	142 100 114	(119) 21.4 ***

 

Table 5             Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period		From: 29 July 2022					To: 01 August 2022
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrApKM101			TA98		TA1537
	Solvent Control (THF)	113 114 144	(124) 17.6#	12 12 13	(12) 0.6	150 138 135		(141) 7.9	26 32 23	(27) 4.6	7 13 6	(9) 3.8
	15 µg	154 141 129	(141) 12.5	11 14 11	(12) 1.7	185 142 141		(156) 25.1	17 28 28	(24) 6.4	9 10 8	(9) 1.0
	50 µg	130 134 129	(131) 2.6	14 12 14	(13) 1.2	163 123 132		(139) 21.0	15 14 23	(17) 4.9	6 13 15	(11) 4.7
	150 µg	117 119 142	(126) 13.9	15 14 17	(15) 1.5	157 138 144		(146) 9.7	21 23 20	(21) 1.5	7 16 9	(11) 4.7
	500 µg	134 P 137 P 140 P	(137) 3.0	10 P 11 P 12 P	(11) 1.0	156 P 182 P 133 P		(157) 24.5	29 P 19 P 23 P	(24) 5.0	9 P 9 P 17 P	(12) 4.6
	1500 µg	121 P 123 P 122 P	(122) 1.0	10 P 9 P 13 P	(11) 2.1	157 P 155 P 155 P		(156) 1.2	25 P 29 P 22 P	(25) 3.5	5 P 16 P 5 P	(9) 6.4
	5000 µg	108 P 117 P 115 P	(113) 4.7	19 P 13 P 13 P	(15) 3.5	158 P 132 P 159 P		(150) 15.3	25 P 32 P 30 P	(29) 3.6	13 P 13 P 10 P	(12) 1.7
Positive controls S9-Mix (+)	Name	2AA		2AA		2AA			BP		2AA
	Dose Level	1 µg		2 µg		10 µg			5 µg		2 µg
	No. of Revertants	899 907 694	(833) 120.7 ***	263 252 226	(247) 19.0 ***	1136 1173 1218		(1176) 41.1 ***	120 110 116	(115) 5.0 ***	110 137 149	(132) 20.0 ***

BP 2AA P***#

 

ENNG          N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO           4-Nitroquinoline-1-oxide

9AA             9-Aminoacridine

P                 Test item precipitate

*                  p ≤ 0.05

***              p ≤ 0.001

#                 Standard deviation

BP            Benzo(a)pyrene

2AA          2-Aminoanthracene

P              Test item precipitate

**             p ≤ 0.01

***                p ≤ 0.001

 

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item, Amines, di-C12-18-alkylmethyl, did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix). Under the conditions of this test, Amines, di-C12-18-alkylmethyl, was considered to be non-mutagenic.

Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA pKM101 were treated with the test item using both the Ames plate incorporation and pre-incubation methods at up to eight 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 the guidance given in OECD TG 471 and was selected as 1.5 to 5000 mg/plate.  As the result of Experiment 1 was Negative, Experiment 2 was performed using the pre-incubation method with fresh cultures of the bacterial strains and fresh test item formulations.  The dose range was modified following the results of Experiment 1 and was 15 to 5000 µg/plate.  Six test item concentrations were selected in Experiment 2 in order to ensure the study achieved at least four non‑toxic dose levels as required by the test guideline.  Test item concentrations for Experiment 2 were selected based on the absence of cytotoxicity noted in Experiment 1 and the potential for a change in test item cytotoxicity profile following the modification in test methodology from plate incorporation to pre-incubation.

Results

The number of revertant counts for the solvent (tetrahydrofuran (THF)) control plates were consistent with the laboratory’s historical control ranges.  The positive control chemicals induced statistically significant increases with mean revertant numbers of >2-fold in strains TA98, TA100, and WP2uvrA pKM101, and >3-fold in strains TA1535 and TA1537 the concurrent vehicle control values, confirming discrimination between different strains and an active S9 preparation.

The dose level of the test item in the first experiment was selected as the OECD TG 471 recommended maximum dose level  of 5000 µg/plate.  There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or in the absence of S9-mix. 

A test item precipitate (oily in appearance) was noted at and above 500 mg/plate in both the presence and absence of S9-mix in Experiments 1 and 2.  The precipitate did not prevent the scoring of revertant colonies.

There were no meaningful statistically or biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, at any test item dose, either with or without S9-mix in Experiment 1 (plate incorporation method).  Three statistically significant values were noted (TA1537 at 5 µg/plate in the absence of S9-mix and at 50 and 150 µg/plate in the presence of S9-mix). However, as the maximum fold increase was only 1.7 times the concurrent vehicle control and the individual colony counts were within the in-house historical vehicle/untreated control range for the relevant strain, the responses were considered of no biological relevance. 

 

Similarly, no statistically or biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any test item dose, either with or without S9-mix in Experiment 2 (pre‑incubation method). 

Conclusion

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item, Amines, di-C12-18-alkylmethyl, did not induce an increase in the frequency of revertant colonies that met the criteria for a positive result, either with or without metabolic activation (S9-mix).  Under the conditions of this test, Amines, di-C12-18-alkylmethyl, was considered to be non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

One study is recorded for this endpoint and was chosen as a key study. The potential of the substance to induce reverse mutation in bacteria was assessed using five strains of Salmonella typhimurium and Escherichia coli Wp2uvrA according to a method comparable to OECD guideline 471. The study was conducted in compliance with the principles of Good Laboratory Practice.

 

The substance was tested in two independent experiments, with and without a metabolic activation system, both performed according to the direct plate incorporation method. Bacteria were exposed to the substance at six dose-levels (three plates/dose-level) selected from a preliminary toxicity test. In both experiments, the dose-levels ranged from 1.5 to 5000 μg/plate for all strains, with and without S9 mix. After 48 to 72 hours of incubation, the revertant colonies were scored.

 

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid.

The substance did not induce any significant increase in the number of revertants, both with and without S9 mix, in any of the five strains of Salmonella typhimurium and Escherichia coli WP2uvrA.

The substance was considered to be non-mutagenic under the conditions of this test.

 

Short description of key information:

The potential of the substance to induce gene mutation in bacteria was investigated using a gene mutation assay on Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and on E. Coli WP2uvrA (method equivalent to OECD guideline 471, GLP).

The test item was found to be not mutagenic in this assay.

 

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance is not mutagenic in the vitro gene mutation test in bacteria. However and according to the criteria laid down in EU regulation (EC) n° 1272/2008 (CLP) and the EU directive 67/548/EEC, the substance cannot be classified for genetic toxicity on a basis of a sole gene mutation test in bacteria.