Reaction products of fatty acids, C18 (unsaturated) alkyl and epoxidized fatty esters, with amines polyethylenepoly-, tetraethylenepentamine

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:

Experimental starting date:15 April 2021 and Experimental completion date: 10 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted June 2012

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:

Higher MW TEPA-fatty acids adducts and TEPA-Epoxidized Fatty esters

IUPAC Name:

Higher MW TEPA-fatty acids adducts and TEPA-Epoxidized Fatty esters

Constituent 2

Reference substance name:

TEPA-fatty acid adducts mw 600 - 1000 Da

IUPAC Name:

TEPA-fatty acid adducts mw 600 - 1000 Da

Constituent 3

Reference substance name:

TEPA-fatty acid adducts MW 350-600

IUPAC Name:

TEPA-fatty acid adducts MW 350-600

Constituent 4

Reference substance name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

EC Number:

292-587-7

EC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Cas Number:

90640-66-7

Molecular formula:

C8H23N5, C10H25N5

IUPAC Name:

Amines, polyethylenepoly-, tetraethylenepentamine fraction

Test material form:

liquid

Method

Target gene:

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-pair substitution to tryptophan independence (Green and Muriel, 1976; and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains

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):

Not applicable

Additional strain / cell type characteristics:

not applicable

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Microsomal Enzyme Fraction
The Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley) used in this study were purchased from Moltox; Lot No.’s 4370 (Experiment 1) and 4359 (Experiment 2) and the protein level was adjusted to 20 mg/mL.

Test concentrations with justification for top dose:

Experiment 1: The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level). Eight concentrations
of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed for each tester strain, using the direct plate incorporation method.

Experiment 2:The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 0.15, 0.50, 1.5, 5, 15, 50, 150 and 500 µg/plate.

Vehicle / solvent:

The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide (DMSO) at the same concentration in solubility checks performed in-house. DMSO was therefore selected as the solvent.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

In the absence of S9-mix: WP2uvrA 3, TA100 and TA1535

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

In the absence of S9-mix: TA1537

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide (4NQO)

Remarks:

In the absence of S9-mix: TA98

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

no

Positive control substance:

other: 2-Aminoanthracene (2AA)

Remarks:

In the presence of S9-mix: TA100, TA1535 TA1537 and WP2uvrA

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

In the presence of S9-mix: TA98

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Experiment 1 – Plate Incorporation Method (triplicate with and triplicate without metabolic activation)
- Experiment 2 – Pre-incubation Method (triplicate with and triplicate without metabolic activation)
- Number of independent experiment : 2 ; experiment 1 and 2


TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable:
- Exposure duration/duration of treatment:
- Harvest time after the end of treatment (sampling/recovery times):


METHODS FOR MEASUREMENTS OF GENOTOXICIY
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). A single manual count was performed due to the light background contamination which prevented an accurate automated count. In this case manual counts are performed which are
considered to be equivalent to the machine counts.

Rationale for test conditions:

The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), Green and Muriel (1976), and Mortelmans and Riccio (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the solvent and untreated controls. Typical published ranges are presented as follows:
Strain TA1535 7 to 40
Strain TA100 60 to 200
Strain TA1537 2 to 30
Strain TA98 8 to 60
Strain WP2uvrA 10 to 60

These values were confirmed against current in-house historical control profiles to further validate acceptability. Although the number of spontaneous revertants can be expected to fall within the ranges, they may occasionally fall outside these.

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the frequency of revertant colonies, both with or without metabolic activation (S9-mix).
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. 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. 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
A test item is considered non-mutagenic (negative) in the test system if the above criteria are
not met.
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:

Statistical significance was not included as part of the result evaluation.
Major Computerized Systems
Perceptive Instruments – Ames Study Manager v1.24 and Ames Sorcerer v3 Veeva QMS – Electronic communication system

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

9-Aminoacridine hydrochloride monohydrate at 80 µg/plate

Test results 2

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 5 µg/plate

Test results 3

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 3 µg/plate and 2-Aminoanthracene (2AA) at 1 µg/plate

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

4-Nitroquinoline-1-oxide (4NQO) at 0.2 µg/plate and Benzo[a]pyrene (BP) at 5 µg/plate

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Remarks:

DMSO

True negative controls validity:

valid

Positive controls validity:

valid

Remarks:

N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG) at 2 µg/plate and 2-Aminoanthracene (2AA) at 10 µg/plate

Additional information on results:

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 were considered to be 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 (dimethyl sulphoxide) control plates were 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 S9-mix.
Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.


Experiment 1 (plate incorporation)
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.
Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains in the
absence and presence of S9-mix ≥150 µg/plate.
No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.


Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the toxic limit.
Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains in the absence of S9-mix ≥ 50 µg/plate and in the presence of S9-mix ≥ 150 µg/plate.
No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix.
There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix.

Conclusion:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and
Escherichia coli (OECD TG 471) the test item 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 was considered to be non-mutagenic.

Remarks on result:

other: All strains used

Any other information on results incl. tables

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

Test Period		From: 22 April 2021					To: 25 April 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	105 101 122	(109) 11.2#	13 17 20	(17) 3.5	30 33 39		(34) 4.6	19 17 17	(18) 1.2	16 11 15	(14) 2.6
	1.5 µg	126 124 116	(122) 5.3	12 13 11	(12) 1.0	15 19 24		(19) 4.5	22 17 17	(19) 2.9	10 17 6	(11) 5.6
	5 µg	131 116 118	(122) 8.1	20 8 13	(14) 6.0	20 22 25		(22) 2.5	19 19 13	(17) 3.5	13 11 12	(12) 1.0
	15 µg	107 110 106	(108) 2.1	13 19 8	(13) 5.5	19 24 24		(22) 2.9	26 16 19	(20) 5.1	4 20 17	(14) 8.5
	50 µg	97 90 96	(94) 3.8	8 13 12	(11) 2.6	25 23 24		(24) 1.0	21 16 17	(18) 2.6	11 16 10	(12) 3.2
	150 µg	66 82 51	(66) 15.5	8 7 9	(8) 1.0	25 20 25		(23) 2.9	17 14 17	(16) 1.7	2 3 3	(3) 0.6
	500 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	14 S 11 S 16 S		(14) 2.5	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
	1500 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V		(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T		(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	566 518 577	(554) 31.4	546 514 596	(552) 41.3	617 546 499		(554) 59.4	147 162 149	(153) 8.1	302 259 212	(258) 45.0

E

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

S              Sparse bacterial background lawn

T              Toxic, no bacterial background lawn

V              Very weak bacterial background lawn

#         Standard deviation

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

Test Period		From: 22 April 2021					To: 25 April 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	126 129 118	(124) 5.7#	12 12 12	(12) 0.0	29 31 29		(30) 1.2	26 19 28	(24) 4.7	16 18 13	(16) 2.5
	1.5 µg	138 125 143	(135) 9.3	10 13 17	(13) 3.5	30 34 20		(28) 7.2	25 15 31	(24) 8.1	21 12 9	(14) 6.2
	5 µg	124 127 130	(127) 3.0	10 20 14	(15) 5.0	24 28 21		(24) 3.5	29 27 26	(27) 1.5	23 14 11	(16) 6.2
	15 µg	103 126 131	(120) 14.9	9 8 5	(7) 2.1	35 30 30		(32) 2.9	34 26 27	(29) 4.4	12 22 14	(16) 5.3
	50 µg	109 110 109	(109) 0.6	6 6 14	(9) 4.6	29 32 25		(29) 3.5	29 15 19	(21) 7.2	16 20 13	(16) 3.5
	150 µg	30 97 79	(69) 34.7	5 7 7	(6) 1.2	25 23 34		(27) 5.9	22 21 23	(22) 1.0	8 9 12	(10) 2.1
	500 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	10 S 13 S 11 S		(11) 1.5	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0
	1500 µg	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0	12 S 8 S 6 S		(9) 3.1	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0
	5000 µg	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T		(0) 0.0	0 T 0 T 0 T	(0) 0.0	0 T 0 T 0 T	(0) 0.0
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	2567 2336 2569	(2491) 133.9	266 278 255	(266) 11.5	229 244 240		(238) 7.8	161 171 177	(170) 8.1	367 310 304	(327) 34.8

BP2AASTV#

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

S           Sparse bacterial background lawn

T           Toxic, no bacterial background lawn

V           Very weak bacterial background lawn

#           Standard deviation

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

Test Period		From: 06 May 2021					To: 09 May 2021
S9-Mix (-)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	129 113 138	(127) 12.7#	8 14 14	(12) 3.5	16 15 22		(18) 3.8	18 18 14	(17) 2.3	8 12 11	(10) 2.1
	0.15 µg	111 139 123	(124) 14.0	17 16 18	(17) 1.0	19 10 19		(16) 5.2	14 12 16	(14) 2.0	9 7 7	(8) 1.2
	0.5 µg	130 131 132	(131) 1.0	8 10 17	(12) 4.7	16 18 15		(16) 1.5	20 21 8	(16) 7.2	10 8 10	(9) 1.2
	1.5 µg	149 131 148	(143) 10.1	10 9 15	(11) 3.2	13 15 20		(16) 3.6	16 34 16	(22) 10.4	9 16 10	(12) 3.8
	5 µg	122 143 144	(136) 12.4	19 8 12	(13) 5.6	24 20 25		(23) 2.6	21 21 18	(20) 1.7	11 7 9	(9) 2.0
	15 µg	110 144 131	(128) 17.2	7 11 11	(10) 2.3	21 8 24		(18) 8.5	14 34 23	(24) 10.0	11 12 7	(10) 2.6
	50 µg	88 S 88 S 116 S	(97) 16.2	13 13 13	(13) 0.0	23 11 18		(17) 6.0	21 S 24 S 24 S	(23) 1.7	9 8 3	(7) 3.2
	150 µg	0 V 0 V 0 V	(0) 0.0	14 S 13 S 12 S	(13) 1.0	18 S 15 S 11 S		(15) 3.5	0 V 0 V 0 V	(0) 0.0	5 S 11 S 4 S	(7) 3.8
	500 µg	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0	14 S 17 S 16 S		(16) 1.5	0 T 0 T 0 T	(0) 0.0	0 V 0 V 0 V	(0) 0.0
Positive controls S9-Mix (-)	Name	ENNG		ENNG		ENNG			4NQO		9AA
	Dose Level	3 µg		5 µg		2 µg			0.2 µg		80 µg
	No. of Revertants	691 458 1130	(760) 341.2	166 171 339	(225) 98.5	764 467 524		(585) 157.6	170 155 171	(165) 9.0	354 359 345	(353) 7.1

ENNG4NQO9AASTV#

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

S              Sparse bacterial background lawn

T              Toxic, no bacterial background lawn

V              Very weak bacterial background lawn

#              Standard deviation

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

Test Period		From: 06 May 2021					To: 09 May 2021
S9-Mix (+)	Dose Level Per Plate	Number of revertants (rounded mean) +/- SD
		Base-pair substitution strains							Frameshift strains
		TA100		TA1535		WP2uvrA			TA98		TA1537
	Solvent Control (DMSO)	154 114 132	(133) 20.0#	11 11 13	(12) 1.2	29 17 13		(20) 8.3	34 29 32	(32) 2.5	9 15 17	(14) 4.2
	0.15 µg	148 133 153	(145) 10.4	12 14 14	(13) 1.2	14 14 19		(16) 2.9	26 24 23	(24) 1.5	7 11 15	(11) 4.0
	0.5 µg	129 135 142	(135) 6.5	9 11 7	(9) 2.0	14 28 32		(25) 9.5	20 22 19	(20) 1.5	8 14 16	(13) 4.2
	1.5 µg	150 154 147	(150) 3.5	10 8 12	(10) 2.0	21 15 25		(20) 5.0	24 32 22	(26) 5.3	4 7 16	(9) 6.2
	5 µg	134 152 131	(139) 11.4	9 13 13	(12) 2.3	34 41 19		(31) 11.2	19 21 27	(22) 4.2	13 11 11	(12) 1.2
	15 µg	142 125 130	(132) 8.7	14 12 8	(11) 3.1	37 36 19		(31) 10.1	25 19 40	(28) 10.8	7 17 15	(13) 5.3
	50 µg	116 143 123	(127) 14.0	12 5 8	(8) 3.5	22 23 30		(25) 4.4	22 28 23	(24) 3.2	9 7 21	(12) 7.6
	150 µg	121 S 95 S 75 S	(97) 23.1	10 S 7 S 7 S	(8) 1.7	23 S 14 S 14 S		(17) 5.2	18 S 20 S 23 S	(20) 2.5	2 S 4 S 5 S	(4) 1.5
	500 µg	0 V 0 V 0 V	(0) 0.0	0 V 0 V 0 V	(0) 0.0	14 S 17 S 14 S		(15) 1.7	0 V 0 V 0 V	(0) 0.0	0 T 0 T 0 T	(0) 0.0
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	1478 1088 1484	(1350) 226.9	198 171 183	(184) 13.5	119 212 214		(182) 54.3	142 143 156	(147) 7.8	358 302 309	(323) 30.5

BP2AASTV#

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

S           Sparse bacterial background lawn

T           Toxic, no bacterial background lawn

V           Very weak bacterial background lawn

#           Standard deviation

Applicant's summary and conclusion

Conclusions:

In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item 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 the test item 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 by the major Japanese Regulatory Authorities including METI, MHLW and MOE, the OECD Guidelines for Testing of Chemicals No. 471 “Bacterial Reverse Mutation Test”, 21 July 1997 as updated in 2020, Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749) and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

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 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 µg/plate. As the overall 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 0.15 to 500 µg/plate. Eight 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, and were selected based on the cytotoxicity noted in Experiment 1, and the potential for a change in the cytotoxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.

Results

The number of revertant counts for the solvent (dimethyl sulphoxide (DMSO)) control plates were 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 metabolic activation (S9- mix) were validated.

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. Toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains in the absence and presence of S9- mix ≥150 µg/plate in the first mutation test (plate incorporation method).

Based on the results of Experiment 1, the toxic limit of the test item was employed as the maximum concentration in the second mutation test (pre-incubation method). Similarly, toxicity, evaluated as a visible reduction in the growth of the bacterial background lawns or a reduction in revertant counts, was observed with test item exposure to all tester strains in the absence of S9-mix ≥ 50 µg/plate and in the presence of S9-mix ≥ 150 µg/plate.

No test item precipitate was observed on the plates at any of the doses tested either in the presence or in the absence of S9-mix in Experiments 1 and 2.

There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 1 (plate incorporation method).

Similarly, no biologically relevant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, 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 Reaction products of fatty acids, C18 (unsaturated) alkyl and epoxidized Fatty esters, with amines polyethylenepoly-, tetraethylenepentamine fraction 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 Reaction products of fatty acids, C18 (unsaturated) alkyl and epoxidized Fatty esters, with amines polyethylenepoly-, tetraethylenepentamine fraction was considered to be non-mutagenic.