Amines, polyethylenepoly-

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 - 01 October 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-

EC Number:

268-626-9

EC Name:

Amines, polyethylenepoly-

Cas Number:

68131-73-7

Molecular formula:

Not applicable (i.e., complex UVCB substance)

IUPAC Name:

Amines, polyethylenepoly-

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

Remarks:

Experiment 1 (plate incorporation) & Experiment 2 (preincubation): all strains were tested, Experiment 3 (preincubation; confirmatory experinment): only with the strain TA98 (absence of S9 only);

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, and 5000 µg/plate
Dose range used for Experiment 2 was determined by the results of Experiment 1. 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, 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.
- Experiment 3 (preincubation; confirmatory experiment): 500, 1500, 3000, 4000 and 5000 µg/plate

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. No correction for purity was required. 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: 3;

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation; Experiment 1); preincubation (Experiment 2 and 3);

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 and 3:
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 (see above, Experiment 1) .

As the results between Experiments 1 (negative) and 2 (positive) were considered to be contradictory, a third, confirmatory experiment was performed using the pre-incubation method and bacterial strains TA98 (absence of S9 only).

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:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

Experiment 2 & 3: small, but statistically significant increases in the frequency of revertant colonies at 5000 µg/plate -S9, meeting the criteria for a positive result;

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 98

Metabolic activation:

with

Genotoxicity:

negative

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:

negative

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 1537

Metabolic activation:

with and without

Genotoxicity:

negative

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 100

Metabolic activation:

with and without

Genotoxicity:

negative

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 6

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

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).
- 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): There were no significant 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 metabolic activation (S9-mix).

Experiment 2 (preincubation): Small but statistically significant increases in TA98 revertant colony frequency were noted at 5000 µg/plate in the absence of S9. The increase at this dose level was in excess of two-fold (2.9x) when compared to the concurrent vehicle control. This response was also accompanied by individual colony counts marginally in excess of the historical untreated/vehicle control maxima of the lab for the strain. There were also smaller but statistically significant increases noted in TA1535 at 1500 and 5000 µg/plate dosed in the presence of S9. However, these increases did not achieve a 3-fold response over the concurrent vehicle control (2.2x) and the individual revertant colony counts were within the in-house historical control range for the tester strain. No significant increases in the frequency of revertant colonies were recorded for any of the remaining bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 3 (preincubation; confirmatory experiment): Small but statistically significant increases in TA98 revertant colony frequency were again noted at 5000 µg/plate in the absence of S9. The increase at this dose level was in excess of two-fold (3.4x) when compared to the concurrent vehicle control. This response was also accompanied by individual colony counts marginally in excess of the in-house historical untreated/vehicle control maxima for the strain. There were also smaller (not statistically significant) increases noted at 3000 and 4000 µg/plate.

See Tables 1 - 5 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)	115 127 116	(119) 6.7#	24 16 16	(19) 4.6	29 25 15	(23) 7.2	27 21 15	(21) 6.0	13 13 10	(12) 1.7
1.5 µg	135 151 142	(143) 8.0	16 17 27	(20) 6.1	21 20 31	(24) 6.1	13 16 26	(18) 6.8	8 14 20	(14) 6.0
5 µg	97 148 126	(124) 25.6	9 25 19	(18) 8.1	29 20 20	(23) 5.2	23 20 19	(21) 2.1	12 12 8	(11) 2.3
15 µg	116 154 102	(124) 26.9	17 25 17	(20) 4.6	19 17 18	(18) 1.0	32 32 20	(28) 6.9	10 16 13	(13) 3.0
50 µg	115 107 115	(112) 4.6	14 17 19	(17) 2.5	24 17 23	(21) 3.8	23 31 26	(27) 4.0	17 15 8	(13) 3.0
150 µg	129 111 127	(122) 9.9	25 14 13	(17) 6.7	21 13 25	(20) 6.1	28 30 29	(29) 1.0	14 11 10	(12) 2.1
500 µg	135 111 135	(127) 13.9	25 13 19	(19) 6.0	25 20 23	(23) 2.5	15 28 20	(21) 6.6	13 14 8	(12) 3.2
1500 µg	114 113 135	(121) 12.4	20 7 14	(14) 6.5	25 20 22	(22) 2.5	25 21 18	(21) 3.5	12 9 8	(10) 2.1
5000 µg	136 133 131	(133) 2.5	13 20 17	(17) 3.5	33 14 23	(23) 9.5	18 26 16	(20) 5.3	8 17 14	(13) 4.6
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	492 464 511	(489) 23.6	194 245 285	(241) 45.6	555 513 471	(513) 42.0	154 102 105	(120) 29.2	147 101 108	(119) 24.8

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

# 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)	104 140 125	(123) 18.1#	16 11 12	(13) 2.6	30 29 23	(27) 3.8	31 23 28	(27) 4.0	10 11 14	(12) 2.1
1.5 µg	121 121 117	(120) 2.3	14 8 16	(13) 4.2	20 15 29	(21) 7.1	30 23 30	(28) 4.0	13 13 11	(12) 1.2
5 µg	138 129 122	(130) 8.0	6 12 10	(9) 3.1	32 24 19	(25) 6.6	24 23 17	(21) 3.8	12 12 12	(12) 0.0
15 µg	139 137 131	(136) 4.2	8 12 11	(10) 2.1	16 36 12	(21) 12.9	23 27 12	(21) 7.8	7 12 7	(9) 2.9
50 µg	127 135 135	(132) 4.6	13 11 8	(11) 2.5	27 28 24	(26) 2.1	22 15 21	(19) 3.8	6 7 11	(8) 2.6
150 µg	113 135 129	(126) 11.4	24 12 11	(16) 7.2	29 18 23	(23) 5.5	22 22 15	(20) 4.0	8 16 14	(13) 4.2
500 µg	121 123 118	(121) 2.5	19 14 13	(15) 3.2	28 25 25	(26) 1.7	13 15 17	(15) 2.0	16 13 11	(13) 2.5
1500 µg	140 121 134	(132) 9.7	17 16 18	(17) 1.0	27 22 26	(25) 2.6	17 22 23	(21) 3.2	17 12 11	(13) 3.2
5000 µg	159 176 138	(158) 19.0	19 21 14	(18) 3.6	30 26 32	(29) 3.1 *	18 22 24	(21) 3.1	14 14 14	(14) 0.0
Positive controls +S9
Name	2AA		2AA		2AA		BP		2AA
Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
No. of Revertants	1919 1763 1846	(1843) 78.1	321 311 299	(310) 11.0	200 206 208	(205) 4.2	152 104 137	(131) 24.6	135 183 195	(171) 31.7

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

# 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)	114 130 138	(127) 12.2#	14 23 20	(19) 4.6	21 31 35	(29) 7.2	19 19 23	(20) 2.3	12 15 12	(13) 1.7
15 µg	151 153 129	(144) 13.3	15 10 21	(15) 5.5	22 22 21	(22) 0.6	25 13 19	(19) 6.0	15 10 15	(13) 2.9
50 µg	125 115 128	(123) 6.8	11 27 10	(16) 9.5	27 31 26	(28) 2.6	19 16 27	(21) 5.7	11 7 13	(10) 3.1
150 µg	116 152 109	(126) 23.1	8 16 18	(14) 5.3	16 17 11	(15) 3.2	24 16 19	(20) 4.0	21 14 14	(16) 4.0
500 µg	140 102 129	(124) 19.6	9 15 13	(12) 3.1	35 30 37	(34) 3.6	10 14 19	(14) 4.5	3 9 7	(6) 3.1
1500 µg	137 122 116	(125) 10.8	18 11 10	(13) 4.4	27 31 39	(32) 6.1	14 20 20	(18) 3.5	9 12 7	(9) 2.5
5000 µg	174 118 161	(151) 29.3	24 26 9	(20) 9.3	28 46 37	(37) 9.0	52 44 75	(57) 16.1 ***	11 9 6	(9) 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	540 540 592	(557) 30.0	234 229 325	(263) 54.0	424 353 369	(382) 37.2	189 144 189	(174) 26.0	302 428 479	(403) 91.1

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

*** 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)	114 120 134	(123) 10.3#	11 10 9	(10) 1.0	41 36 36	(38) 2.9	21 26 24	(24) 2.5	9 13 9	(10) 2.3
15 µg	140 111 125	(125) 14.5	10 8 9	(9) 1.0	26 36 26	(29) 5.8	37 29 27	(31) 5.3	14 10 10	(11) 2.3
50 µg	114 129 127	(123) 8.1	4 13 9	(9) 4.5	30 31 31	(31) 0.6	31 17 20	(23) 7.4	9 16 9	(11) 4.0
150 µg	100 121 129	(117) 15.0	14 8 8	(10) 3.5	30 30 35	(32) 2.9	24 27 19	(23) 4.0	13 10 7	(10) 3.0
500 µg	121 124 126	(124) 2.5	15 8 10	(11) 3.6	47 45 39	(44) 4.2	26 22 35	(28) 6.7	14 13 13	(13) 0.6
1500 µg	121 144 120	(128) 13.6	21 18 20	(20) 1.5 *	41 33 40	(38) 4.4	21 26 25	(24) 2.6	7 8 6	(7) 1.0
5000 µg	151 109 97	(119) 28.4	23 27 17	(22) 5.0 **	37 46 44	(42) 4.7	28 34 30	(31) 3.1	6 10 17	(11) 5.6
Positive controls +S9
Name	2AA		2AA		2AA		BP		2AA
Dose Level	1 µg		2 µg		10 µg		5 µg		2 µg
No. of Revertants	1427 1401 1356	(1395) 35.9	232 251 239	(241) 9.6	140 159 173	(157) 16.6	136 188 136	(153) 30.0	227 200 193	(207) 18.0

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

* p≤0.05

** p≤0.01

# Standard deviation

Table 5: Results of Experiment 3 (preincubation; confirmatory experiment) - without metabolic activation

Dose Level Per Plate	Number of revertants (mean) +/- SD
	TA98
Solvent Control (Water)	20 22 26	(23) 3.1#
500 µg	16 20 17	(18) 2.1
1500 µg	19 20 17	(19) 1.5
3000 µg	35 38 29	(34) 4.6
4000 µg	34 36 41	(37) 3.6
5000 µg	59 67 109	(78) 26.9 ***
Positive controls -S9
Name	4NQO
Dose Level	0.2 µg
No. of Revertants	267 224 183	(225) 42.0

4NQO: 4-Nitroquinoline-1-oxide

*** p≤0.001

# Standard deviation

Applicant's summary and conclusion

Conclusions:

positive with metabolic activation

Amines, polyethylenepoly (PEPA) was considered to be weakly mutagenic 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 (PEPA) induced a
small but reproducible dose-related and statistically significant increase in the frequency of TA98 revertant colonies that met the criteria for a positive result, without metabolic activation (S9-mix) employing a 20 minute pre-incubation at 37 °C. Under the conditions of this test Amines, polyethylenepoly (PEPA) was considered to be weakly mutagenic.