Amines, polyethylenepoly-, tetraethylenepentamine fraction

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:

12 August 2020 - 07 September 2020

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 as corrected in 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

August 1998

Deviations:

not specified

Test guideline 4

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

Version / remarks:

31 March 2011

Deviations:

not specified

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH S2(R1) Federal Register

Version / remarks:

June 2012

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

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:

(2-aminoethyl)[2-({2-[(2-aminoethyl)amino]ethyl}amino)ethyl]amine; bis(2-aminoethyl)({2-[(2-aminoethyl)amino]ethyl})amine

Method

Target gene:

his / trp operon

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:

other:

Remarks:

TA1537: his C 3076; rfa-; uvrB; TA98: his D 3052; rfa-; uvrB-; R-factor; TA1535: his G 46; rfa-; uvrB-; TA100: his G 46; rfa-; uvrB-; R-factor; WP2uvrA trp-; uvrA-;

Cytokinesis block (if used):

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Phenobarbitone / β-Naphthoflavone induced S9 Microsomal fractions (Sprague-Dawley);
- source of S9: purchased from Moltox; Lot No. 4222; the protein level was adjusted to 20 mg/mL;
- method of preparation of S9 mix: The S9-mix was prepared before using sterilized co-factors and maintained on ice for the duration of the test (S9: 5.0 mL, 1.65 M KCl/0.4 M MgCl2: 1.0 mL, 0.1 M glucose-6-phosphate: 2.5 mL, 0.1 M NADP: 2.0 mL, 0.2 M sodium phosphate buffer (pH 7.4): 25.0 mL, sterile distilled water 14.5 mL);
- concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL S9 mix (i.e. 0.05 mL S9)
- quality controls of S9: A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of the experiment.

Test concentrations with justification for top dose:

- Experiment 1 (plate incorporation): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate (5000 µg/plate is the maximum recommended dose level according to OECD TG 471);
- Experiment 2 (preincubation): 15, 50, 150, 500, 1500, 3000 and 5000 µg/plate
Dose range used for Experiment 2 was determined by the results of Experiment 1. Seven 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 lack of 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. An intermediate dose level (3000 µg/plate) was also included following the observation of small but statistically significant increases in revertant colony frequency noted in the first mutation test.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water

- Justification for choice of solvent/vehicle:
The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks.

The test item was accurately weighed and, on the day of the experiment, approximate half-log dilutions prepared in sterile distilled water by mixing on a vortex mixer. Formulated concentrations were adjusted to allow for test item purity with a correction factor of 1.02 employed. All test item preparation and dosing was performed under yellow safety lighting. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations was not determined.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 9-Aminoacridine hydrochloride monohydrate: -S9; in DMSO; TA1537: 80 µg/plate; 2-Aminoanthracene: +S9; in DMSO; TA100: 1 µg/plate; TA1537 / TA1535: 2 µg/plate; WP2uvrA: 10 µg/plate;

Remarks:

In addition, sterility controls (top agar and histidine / biotin or tryptophan -S9, top agar and histidine / biotin or tryptophan +S9, maximum dosing solution of the test item -S9) were performed in singular prior to mutation test.

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate;
- Number of independent experiments: 2;

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation; Experiment 1); preincubation (Experiment 2);

Experiment 1:
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle 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 or S9-mix and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test.

Experiment 2:
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer or S9-mix and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method (see above, Experiment 1).

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period (Experiment 2): 20 minutes;
- Exposure duration/duration of treatment: between 48 and 72 hours;

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition (the plates were viewed microscopically for evidence of thinning of the background bacterial lawn);


METHODS FOR MEASUREMENTS OF GENOTOXICIY: Plates were scored for the presence of revertant colonies using an automated colony counting system after incubation at 37 +/- 3 °C.

Rationale for test conditions:

according to OECD TG 471

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.
2. A reproducible increase at one or more concentrations.
3. Biological relevance against historical control ranges of the lab.
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).
5. Statistical analysis of data as determined by UKEMS.

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 confirmed by using Dunnett’s 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:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Experiment 1: Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate; Experiment 2: statistically significant, dose-related increases from 500 µg/plate -S9 and from 3000 µg/plate +S9;

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:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate (Experiment 1) and at / above 1500 µg/plate (Experiment 2) +/-S9; counts at the upper limit or in excess of the historical control maxima;

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:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate (Experiment 1) +S9 and at / above 3000 µg/plate (Experiment 2) +/-S9; counts at the upper limit or in excess of the historical control maxima;

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:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Small but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate (Experiment 1) +S9 and at / above 3000 µg/plate (Experiment 2) +/-S9; counts at the upper limit or in excess of the historical control maxima;

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:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Remarks:

Experiment 2: Small but statistically significant increases in the frequency of revertant colonies at / above 3000 µg/plate -S9; individual revertant colony counts at the upper limit or in excess of the historical control maxima;

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:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was fully miscible in sterile distilled water at 50 mg/mL in solubility checks.
- Precipitation and time of the determination: 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 Experiment 1 and 2.
- Other confounding effects:
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 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.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Results for the negative controls (spontaneous mutation rates) and viability were considered to be acceptable. The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the historical control range of the lab in both the absence and presence of S9. 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. All of the acceptability criteria were considered to be met. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Ames test:
- Signs of toxicity: 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 Experiment 1 and 2.
- Results:
Experiment 1 (plate incorporation): Small but statistically significant increases in the frequency of revertant colonies were recorded with doses of the test item at 5000 µg/plate for bacterial strains TA100 and WP2uvrA (absence and presence of S9) and TA1535 and TA98 in the presence of S9. Whilst a two or three-fold increase was not achieved for any of the bacterial strains, individual revertant colony counts were at the upper limit or in excess of the historical untreated/vehicle control maxima of the lab.

Experiment 2 (preincubation): Statistically significant and dose-related increases in WP2uvrA revertant colony frequency were noted from 500 µg/plate in the absence of S9 and 3000 µg/plate in the presence of S9. Maximum increases in excess of two-fold (of 6.3-fold and 3.6-fold) when compared to the concurrent vehicle controls were noted in the absence and presence of S9 respectively. These responses were also accompanied by individual colony counts in excess of the historical untreated/vehicle control maxima of the lab for the strain with clear evidence of dose-related increase. There were also smaller statistically significant increases noted in TA100, TA1535, TA98 (absence and presence of S9) and TA1537 in the absence of S9 at and/or above 1500 µg/plate. Whilst these increases did not achieve a 2 or 3-fold response (depending on tester strain type) over the concurrent vehicle control, the individual revertant colony counts, particularly in the case of TA100 dosed in the presence of S9, were in excess of the in-house
historical control maxima.

See Tables 1 - 4 under Any other information on results incl. tables for individual plate counts & mean number of revertant colonies per plate and standard deviation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and the number of data)
- Positive historical control data (2018):
TA100, -S9: 220 - 1525, mean 606, SD 213.6, n = 306
TA100, +S9: 422 - 3928, mean 1726, SD 528.7, n = 300
TA1535, -S9: 74 - 2601, mean 653, SD 484.4, n = 272
TA1535, +S9: 113 - 481, mean 301, SD 57.2, n = 271
WP2uvrA, -S9: 111 - 1420, mean 706, SD 235.8, n = 254
WP2uvrA, +S9: 105 - 697, mean 230, SD 74.8, n = 251
TA98, -S9: 97 - 461, mean 212, SD 77.1, n = 292
TA98, +S9: 79 - 342, mean 158, SD 49.3, n = 292
TA1537, -S9: 86 - 833, mean 274, SD 150.4, n = 276
TA1535, +S9: 116 - 541, mean 294, SD 86.8, n = 272
- Positive historical control data (2019):
TA100, -S9: 205 - 2322, mean 622, SD 294.0, n = 239
TA100, +S9: 318 - 2561, mean 1381, SD 442.8, n = 234
TA1535, -S9: 69 - 4595, mean 790, SD 825.3, n = 230
TA1535, +S9: 112 - 1976, mean 268, SD 124.0, n = 230
WP2uvrA, -S9: 117 - 1391, mean 629, SD 245.6, n = 204
WP2uvrA, +S9: 99 - 790, mean 175, SD 70.8, n = 202
TA98, -S9: 92 - 477, mean 186, SD 71.4, n = 253
TA98, +S9: 88 - 719, mean 165, SD 75.5, n = 246
TA1537, -S9: 76 - 830, mean 266, SD 142.4, n = 230
TA1535, +S9: 109 - 1964, mean 232, SD 127.9, n = 224
- Negative (combined vehicle / untreated) historical control data (2018):
TA100, -S9: 67 - 170, mean 122, SD 18.8, n = 301
TA100, +S9: 64 - 187, mean 125, SD 21.5, n = 297
TA1535, -S9: 7 - 33, mean 17, SD 4.2, n = 542
TA1535, +S9: 9 - 28, mean 14, SD 3.1, n = 279
WP2uvrA, -S9: 11 - 44, mean 27, SD 5.3, n = 511
WP2uvrA, +S9: 20 - 53, mean 36, SD 6.2, n = 253
TA98, -S9: 11 - 41, mean 22, SD 4.5, n = 583
TA98, +S9: 15 - 50, mean 27, SD 5.1, n = 300
TA1537, -S9: 5 - 25, mean 12, SD 3.3, n = 550
TA1535, +S9: 3 - 22, mean 13, SD 3.2, n = 280
- Negative (combined vehicle / untreated) historical control data (2019):
TA100, -S9: 75 - 168, mean 116, SD 18.1, n = 240
TA100, +S9: 81 - 181, mean 122, SD 18.8, n = 234
TA1535, -S9: 9 - 38, mean 17, SD 4.8, n = 462
TA1535, +S9: 7 - 31, mean 14, SD 3.6, n = 237
WP2uvrA, -S9: 12 - 47, mean 25, SD 5.9, n = 410
WP2uvrA, +S9: 16 - 55, mean 32, SD 6.7, n = 205
TA98, -S9: 12 - 39, mean 23, SD 5.4, n = 508
TA98, +S9: 13 - 46, mean 28, SD 5.9, n = 249
TA1537, -S9: 4 - 25, mean 12, SD 3.4, n = 462
TA1535, +S9: 6 - 25, mean 13, SD 3.3, n = 227

Any other information on results incl. tables

Table 1: Results of Experiment 1 (plate incorporation) - without metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	128 132 115	(125) 8.9#	37 22 21	(27) 9.0	26 26 27	(26) 0.6	25 21 27	(24) 3.1	13 9 14	(12) 2.6
1.5 µg	142 115 139	(132) 14.8	27 24 18	(23) 4.6	16 13 32	(20) 10.2	24 25 27	(25) 1.5	13 17 12	(14) 2.6
5 µg	140 110 133	(128) 15.7	28 18 24	(23) 5.0	20 19 35	(25) 9.0	28 16 27	(24) 6.7	10 13 13	(12) 1.7
15 µg	127 140 123	(130) 8.9	34 39 19	(31) 10.4	21 29 23	(24) 4.2	23 28 22	(24) 3.2	17 11 12	(13) 3.2
50 µg	134 133 110	(126) 13.6	32 34 25	(30) 4.7	22 19 23	(21) 2.1	33 28 28	(30) 2.9	12 11 12	(12) 0.6
150 µg	159 114 130	(134) 22.8	23 38 18	(26) 10.4	28 25 22	(25) 3.0	29 17 37	(28) 10.1	10 12 14	(12) 2.0
500 µg	125 126 122	(124) 2.1	18 15 28	(20) 6.8	29 30 16	(25) 7.8	25 13 24	(21) 6.7	9 23 13	(15) 7.2
1500 µg	126 131 137	(131) 5.5	29 35 29	(31) 3.5	35 41 21	(32) 10.3	31 15 27	(24) 8.3	8 18 8	(11) 5.8
5000 µg	175 179 168	(174) 5.6 ***	36 35 38	(36) 1.5	46 46 43	(45) 1.7 *	30 36 41	(36) 5.5	16 16 11	(14) 2.9
Positive controls -S9
Name	ENNG		ENNG		ENNG		4NQO		9AA
Dose Level	3 µg		5 µg		2 µg		0.2 µg		80 µg
No. of Revertants	651 691 703	(682) 27.2	405 514 456	(458) 54.5	745 732 669	(715) 40.6	115 107 101	(108) 7.0	263 452 232	(316) 119.1

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

* p≤0.05

*** p≤0.001

# Standard deviation

Table 2: Results of Experiment 1 (plate incorporation) - with metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	126 138 139	(134) 7.2#	15 23 13	(17) 5.3	29 30 22	(27) 4.4	30 30 25	(28) 2.9	11 10 13	(11) 1.5
1.5 µg	128 127 137	(131) 5.5	18 11 12	(14) 3.8	24 29 22	(25) 3.6	32 35 35	(34) 1.7	9 12 6	(9) 3.0
5 µg	132 132 138	(134) 3.5	16 13 19	(16) 3.0	21 23 31	(25) 5.3	27 30 33	(30) 3.0	9 9 10	(9) 0.6
15 µg	105 121 148	(125) 21.7	19 26 17	(21) 4.7	20 27 16	(21) 5.6	40 34 29	(34) 5.5	10 9 9	(9) 0.6
50 µg	105 115 96	(105) 9.5	17 17 7	(14) 5.8	23 23 33	(26) 1.7	31 31 39	(34) 4.6	8 12 6	(9) 3.1
150 µg	131 111 137	(126) 13.6	19 13 25	(19) 6.0	28 25 25	(26) 1.7	31 31 39	(34) 4.6	8 14 7	(10) 3.8
500 µg	140 120 161	(140) 20.5	12 19 29	(20) 8.5	23 20 38	(27) 9.6	26 35 34	(32) 4.9	8 12 6	(9) 3.1
1500 µg	142 149 131	(141) 9.1	30 20 21	(24) 5.5	34 29 25	(29) 4.5	29 32 32	(31) 1.7	13 5 12	(10) 4.4
5000 µg	229 208 218	(218) 10.5 ***	21 35 41	(32) 10.3 *	43 48 38	(43) 5.0 *	49 45 46	(47) 2.1 ***	18 14 14	(15) 2.3
Positive controls +S9
Name	2AA		2AA		2AA		BP		2AA
Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
No. of Revertants	1528 1582 1782	(1631) 133.8	278 267 288	(278) 10.5	154 178 181	(171) 14.8	228 221 226	(225) 3.6	303 256 247	(269) 30.1

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

* p≤0.05

*** p≤0.001

# Standard deviation

Table 3: Results of Experiment 2 (preincubation) - without metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	144 145 148	(146) 2.1#	18 15 21	(18) 3.0	15 20 18	(18) 2.5	18 20 18	(19) 1.2	7 14 10	(10) 3.5
15 µg	140 142 138	(140) 2.0	21 16 15	(17) 3.2	13 22 17	(17) 4.5	19 19 23	(20) 2.3	10 14 10	(11) 2.3
50 µg	106 111 135	(117) 15.5	15 16 16	(16) 0.6	15 22 18	(18) 3.5	20 17 24	(20) 3.5	11 14 12	(12) 1.5
150 µg	122 132 129	(128) 5.1	12 12 12	(12) 0.0	19 15 20	(18) 2.6	23 21 23	(22) 1.2	8 9 9	(9) 0.6
500 µg	143 163 145	(150) 11.0	16 19 16	(17) 1.7	36 27 31	(31) 4.5 *	16 15 14	(15) 1.0	8 6 7	(7) 1.0
1500 µg	148 143 140	(144) 4.0	24 16 12	(17) 6.1	36 42 57	(45) 10.8 ***	17 26 18	(20) 4.9	7 8 9	(8) 1.0
3000 µg	143 173 139	(152) 18.6	22 25 32	(26) 5.1 *	61 43 68	(57) 12.9 ***	42 24 40	(35) 9.9 ***	25 14 19	(19) 5.5 **
5000 µg	188 179 178	(182) 5.5 **	28 22 30	(27) 4.2 *	123 107 110	(113) 8.5 ***	31 35 38	(35) 3.5 ***	21 26 24	(24) 2.5 ***
Positive controls -S9
Name	ENNG		ENNG		ENNG		4NQO		9AA
Dose Level	3 µg		5 µg		2 µg		0.2 µg		80 µg
No. of Revertants	879 931 585	(798) 186.6	519 475 665	(553) 99.5	843 844 796	(828) 27.4	238 216 219	(224) 11.9	342 317 346	(335) 15.7

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

* p≤0.05

** p≤0.01

*** p≤0.001

# Standard deviation

Table 4: Results of Experiment 2 (preincubation) - with metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA100		TA1535		WP2uvrA		TA98		TA1537
Solvent Control (Water)	147 144 132	(141) 7.9#	15 19 19	(18) 2.3	33 17 27	(26) 8.1	25 26 36	(29) 6.1	13 5 16	(11) 5.7
15 µg	141 145 144	(143) 2.1	13 8 17	(13) 4.5	29 20 34	(28) 7.1	34 20 28	(27) 7.0	12 8 12	(11) 2.3
50 µg	134 138 126	(133) 6.1	26 11 17	(18) 7.5	23 26 20	(23) 3.0	32 22 25	(26) 5.1	8 15 11	(11) 3.5
150 µg	157 141 132	(143) 12.7	15 8 9	(11) 3.8	25 29 21	(25) 4.0	16 27 31	(25) 7.8	10 11 13	(11) 1.5
500 µg	165 142 149	(152) 11.8	14 7 13	(11) 3.8	30 28 29	(29) 1.0	30 38 32	(33) 4.2	10 18 6	(11) 6.1
1500 µg	196 178 192	(189) 9.5 ***	15 9 18	(14) 4.6	42 46 28	(39) 9.5	37 22 35	(31) 8.1	10 7 15	(11) 4.0
3000 µg	248 246 218	(237) 16.8 ***	26 29 31	(29) 2.5	44 49 53	(49) 4.5 **	41 31 38	(37) 5.1	12 13 12	(12) 0.6
5000 µg	250 257 267	(258) 8.5	32 35 46	(38) 7.4 **	88 109 83	(93) 13.8 ***	59 57 54	(57) 2.5 ***	38 9 23	(23) 14.5
Positive controls +S9
Name	2AA		2AA		2AA		BP		2AA
Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
No. of Revertants	1269 1194 1146	(1203) 62.0	223 235 233	(230) 6.4	134 144 163	(147) 14.7	130 126 533	(263) 233.8	238 249 220	(236) 14.6

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

** p≤0.01

*** p≤0.001

# Standard deviation

Applicant's summary and conclusion

Conclusions:

positive with and without metabolic activation

Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) was positive in the Ames assay under the conditions and according to the criteria of the test protocol.

Executive summary:

In the reverse mutation assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli according to OECD TG 471 the test item, Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) met the criteria for a positive result, both with and without metabolic activation (S9-mix). Under the conditions of this test Amines, polyethylenepoly-, tetraethylenepentamine fraction (TEPA) was considered to be mutagenic