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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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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 - 02 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 guidelineopen allclose all
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:
30 May 2008
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
not specified
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
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

Constituent 1
Chemical structure
Reference substance name:
Polyethylene polyamine, pentaethylenehexamine fraction
EC Number:
701-266-7
Molecular formula:
Complex UVCB substance
IUPAC Name:
Polyethylene polyamine, pentaethylenehexamine fraction

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 strains TA98 and WP2uvrA (absence of S9 only) and TA100 (absence and presence of S9);
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 and 4217; 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 and at least one toxic dose level 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 employing the plate incorporation method (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 and WP2uvrA (absence of S9 only) and TA100 (absence and presence of S9).

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 resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Experiment 2 & 3: statistically significant increases in the frequency of revertant colonies at 5000 µg/plate -S9 and from 1500 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
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Experiment 2 & 3: 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
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
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
Experiment 2 & 3: 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
Key result
Species / strain:
E. coli WP2 uvr A
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
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
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
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 statistically 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). There was a noticeable increase in TA100 at 5000 µg/plate with metabolic activation but this response did not meet the criteria for a positive response.

Experiment 2 (preincubation): Statistically significant increases in revertant colony frequency were noted initially at 5000 µg/plate for TA100, TA98 and WP2uvrA dosed in the absence of S9 and from 1500 µg/plate for TA100 dosed in the presence of S9. Maximum increases in excess of two-fold when compared to the concurrent vehicle controls of 3.4-fold were noted for WP2uvrA and 2.8-fold for TA98. These responses were also accompanied by individual colony counts in excess of the historical untreated/vehicle control maxima of the lab for both of the strains. The smaller but statistically significant increases noted in TA100 dosed in the absence and presence of S9, whilst these increases did not achieve a 2-fold response over the concurrent vehicle control (1.9-fold in the absence of S9 and 1.7-fold in the presence of S9), the individual revertant colony counts at 5000 µg/plate were in excess of the historical control maxima of the lab. 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 and WP2uvrA revertant colony frequency were again noted at 5000 µg/plate in the absence of S9. The increases at this dose level were in excess of two-fold (4.0x for TA98 and 6.7x for WP2uvrA) when compared to the concurrent vehicle controls. These responses were also accompanied by individual colony counts in excess of the historical untreated/vehicle control maxima of the lab for each strain
with evidence of a weak dose-related response at the lower dose levels. There were also smaller increases noted in TA100 (2.0-fold in the absence of S9 and 1.9-fold in the presence of S9) with individual revertant colony counts above the historical control maxima of the lab at 5000 µg/plate and evidence of a weak dose-related response.

See Tables 1 - 6 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)

107
83
107

(99)
13.9#

19
13
14

(15)
3.2

24
20
22

(22)
2.0

29
22
13

(21)
8.0

14
10
10

(11)
2.3

1.5 µg

92
106
105

(101)
7.8

9
13
11

(11)
2.0

17
28
14

(20)
7.4

25
24
17

(22)
4.4

14
7
4

(8)
5.1

5 µg

102
120
87

(103)
16.5

16
10
8

(11)
2.0

19
15
25

(20)
5.0

24
24
16

(21)
4.6

4
10
4

(6)
3.5

15 µg

101
112
88

(100)
12.0

12
12
15

(13)
1.7

16
22
13

(17)
4.6

12
19
24

(18)
6.0

15
6
9

(10)
4.6

50 µg

87
92
89

(89)
2.5

19
16
10

(15)
4.6

20
13
24

(19)
5.6

18
11
22

(17)
5.6

12
17
5

(11)
6.0

150 µg

90
98
104

(97)
7.0

8
8
11

(9)
1.7

15
23
16

(18)
4.4

18
15
18

(17)
1.7

5
12
12

(10)
4.0

500 µg

103
108
97

(103)
5.5

14
10
13

(12)
2.1

22
17
19

(19)
2.5

19
16
19

(18)
1.7

11
13
14

(13)
1.5

1500 µg

107
99
88

(98)
9.5

8
19
14

(14)
5.5

18
33
31

(27)
8.1

27
17
12

(19)
7.6

5
12
14

(10)
4.7

5000 µg

128
98
87

(104)
21.2
***

11
18
17

(15)
3.8

22
26
26

(25)
2.3

21
23
26

(23)
2.5

11
11
13

(12)
1.2

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

344
547
567

(486)
123.4

180
163
205

(183)
21.1

371
737
689

(599)
198.9

105
100
115

(107)
7.6

281
245
188

(238)
46.9

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)

97
125
109

(110)
14.0#

14
8
7

(10)
3.8

31
39
22

(31)
8.5

20
27
16

(21)
5.6

6
11
12

(10)
3.2

1.5 µg

111
106
113

(110)
3.6

7
12
6

(8)
3.2

37
28
21

(29)
8.0

31
32
185

(27)
7.8

7
11
7

(8)
2.3

5 µg

123
89
126

(113)
20.6

6
7
8

(7)
1.0

29
23
18

(23)
5.5

28
31
16

(25)
7.9

14
14
17

(15)
1.7

15 µg

114
105
111

(110)
4.6

12
10
15

(12)
2.5

28
36
29

(31)
4.4

20
19
17

(19)
1.5

7
18
7

(11)
6.4

50 µg

106
110
104

(107)
3.1

7
8
10

(8)
1.5

27
23
39

(30)
8.3

26
17
29

(24)
6.2

11
11
16

(13)
2.9

150 µg

116
111
97

(108)
9.8

5
6
10

(7)
2.6

20
25
29

(25)
4.5

23
15
26

(21)
5.7

9
8
7

(8)
1.0

500 µg

104
111
109

(108)
3.6

8
6
11

(8)
2.5

23
29
33

(28)
5.0

21
20
19

(20)
1.0

8
7
44

(9)
2.1

1500 µg

101
104
116

(107)
7.9

13
9
10

(11)
2.1

16
16
32

(21)
9.2

22
20
16

(19)
3.1

4
7
13

(8)
4.6

5000 µg

142
138
142

(141)
2.3

19
20
12

(17)
4.4

28
35
31

(31)
3.5
*

16
29
19

(21)
6.8

14
9
16

(13)
3.6

Positive controls +S9

Name

2AA

2AA

2AA

BP

2AA

Dose Level

1 µg

2 µg

10 µg

5 µg

2 µg

No. of Revertants

1294
1393
1645

(1444)
181.0

315
283
240

(279)
37.6

163
162
219

(182)
32.1

102
111
106

(106)
4.5

148
186
279

(238)
47.4

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)

115
129
135

(126)
10.3#

14
14
16

(15)
1.2

18
22
20

(20)
2.0

19
19
24

(21)
2.9

10
8
9

(9)
1.0

15 µg

113
138
111

(121)
15.0

18
18
21

(19)
1.7

21
22
27

(23)
3.2

25
19
27

(24)
4.2

11
8
9

(9)
1.5

50 µg

111
133
145

(130)
17.2

24
11
16

(17)
6.6

24
21
26

(24)
2.5

13
29
18

(20)
8.2

10
10
11

(10)
0.6

150 µg

127
144
137

(136)
8.5

13
16
14

(14)
1.5

28
26
23

(26)
2.5

21
14
16

(17)
3.6

11
8
8

(9)
1.7

500 µg

132
135
140

(136)
4.0

9
13
15

(12)
3.1

22
36
24

(27)
7.6

21
15
18

(18)
3.0

9
7
12

(9)
2.5

1500 µg

144
129
122

(132)
11.2

10
9
15

(11)
3.2

39
25
19

(28)
10.3

8
16
25

(16)
8.5

5
16
14

(12)
5.9

5000 µg

219
268
214

(234)
29.8
***

18
15
20

(18)
2.5

57
59
86

(67)
16.2
***

74
64
39

(59)
18.0
***

8
21
24

(18)
8.5

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

649
764
673

(695)
60.7

406
340
430

(392)
46.6

531
490
573

(531)
41.5

178
235
187

(200)
30.6

191
213
85

(163)
68.4

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)

138
134
120

(131)
9.5#

14
10
9

(11)
2.6

25
28
30

(28)
2.5

27
17
19

(21)
5.3

14
12
11

(12)
1.5

15 µg

130
167
134

(144)
20.3

8
13
7

(9)
3.2

21
36
38

(32)
9.3

15
35
30

(27)
10.4

7
7
8

(7)
0.6

50 µg

119
110
161

(130)
27.2

13
15
7

(12)
4.2

35
33
29

(32)
3.1

28
29
23

(27)
3.2

12
11
18

(14)
3.8

150 µg

138
134
154

(142)
10.6

3
15
6

(8)
6.2

30
16
26

(24)
7.2

18
28
30

(25)
6.4

14
13
9

(12)
2.6

500 µg

133
132
128

(131)
2.6

8
11
8

(9)
1.7

27
19
21

(22)
4.2

19
24
21

(21)
2.5

10
5
11

(9)
3.2

1500 µg

164
180
170

(171)
8.1
*

14
11
10

(12)
2.1

34
26
34

(31)
4.6

23
25
27

(25)
2.0

10
11
9

(10)
1.0

5000 µg

215
240
225

(227)
12.6
***

16
16
8

(13)
4.6

44
37
34

(38)
5.1

24
23
31

(26)
4.4

11
14
19

(15)
4.0

Positive controls +S9

Name

2AA

2AA

2AA

BP

2AA

Dose Level

1 µg

2 µg

10 µg

5 µg

2 µg

No. of Revertants

1751
1488
1768

(1559)
157.0

296
247
255

(266)
26.3

141
105
144

(130)
21.7

99
112
123

(111)
12.0

245
200
243

(229)
25.4

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

* p≤0.05

*** p≤0.001

# Standard deviation

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

Dose Level
Per Plate

Number of revertants (mean) +/- SD

TA100

WP2uvrA

TA98

Solvent Control
(Water)

110
130
119

(120)
10.0#

20
20
24

(21)
2.3

21
24
17

(21)
3.5

500 µg

110
150
132

(131)
20.0

17
16
17

(17)
0.6

19
23
24

(22)
2.6

1500 µg

141
156
157

(151)
9.0

14
25
26

(22)
6.7

25
27
34

(29)
4.7

3000 µg

224
155
135

(171)
46.7

19
28
30

(26)
5.9

23
33
33

(30)
5.8

4000 µg

211
138
188

(179)
37.3

32
44
31

(36)
7.2
*

49
34
37

(40)
7.9
*

5000 µg

288
258
173

(240)
59.7
**

152
139
129

(140)
11.5
***

113
76
64

(84)
25.5
***

Positive controls -S9

Name

ENNG

ENNG

4NQO

Dose Level

3 µg

2 µg

0.2 µg

No. of Revertants

536
563
511

(537)
26.0

626
595
565

(595)
30.5

180
227
223

(210)
26.1

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 6: Results of Experiment 3 (preincubation; confirmatory experiment) - with metabolic activation

Dose Level
Per Plate

Number of revertants (mean) +/- SD

TA100

Solvent Control
(Water)

140
137
116

(131)
13.1#

500 µg

152
141
140

(144)
6.7

1500 µg

148
156
143

(149)
6.6

3000 µg

176
186
158

(173)
14.2

4000 µg

180
208
176

(188)
17.4

5000 µg

385
230
149

(255)
119.9
*

Positive controls +S9

Name

2AA

Dose Level

1 µg

No. of Revertants

1622
1679
1709

(1670)
44.2

2AA: 2 -Aminoanthracene

* p≤0.05

# Standard deviation

Applicant's summary and conclusion

Conclusions:
positive with and without metabolic activation

Polyethylene polyamine, pentaethylenehexamine fraction (PEHA) 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 Polyethylene polyamine, pentaethylenehexamine fraction (PEHA) induced reproducible dose-related and statistically significant increases in the frequency of TA98 and WP2uvrA revertant colonies (absence of S9) and TA100 (presence and absence of S9) employing a 20 minute pre-incubation at 37 °C that met the criteria for a positive result. Under the conditions of this test Polyethylene polyamine, pentaethylenehexamine fraction (PEHA) was considered to be weakly mutagenic.