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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 - 31 August 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:
Amines, polyethylenepoly-, triethylenetetramine fraction
EC Number:
292-588-2
EC Name:
Amines, polyethylenepoly-, triethylenetetramine fraction
Cas Number:
90640-67-8
Molecular formula:
C6H18N4, C8H20N4
IUPAC Name:
(2-aminoethyl)({2-[(2-aminoethyl)amino]ethyl})amine; tris(2-aminoethyl)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:
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)
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 : 1; OECD TG 471 permits non-repetition when a clear, positive response is obtained in the first mutation test (plate Incorporation method); therefore, a second, confirmatory test was not required;

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

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.

TREATMENT AND HARVEST SCHEDULE:
- 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:
maximum increases over the vehicle control of 3.7 fold were noted in the absence of metabolic activation at 1500 µg/plate and 4.4 fold in the presence of metabolic activation at 5000 µg/plate
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 and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
maximum increases over the vehicle control of 18.8 and 13 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
maximum increases over the vehicle control of 3.4 and 1.9 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate
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
Remarks:
minor statistically significant increases were noted in the absence (max = 1.9 fold) and presence (max= 2.5 fold) of metabolic activation; increases did not meet the required criteria (> 3-fold increase), but were considered in the overall assessment;
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
Remarks:
minor statistically significant increases were noted in the absence of metabolic activation (max = 2.5 fold); increase did not meet the required criteria (> 3-fold increase), but were considered in the overall assessment;
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. The revertant colony counts for TA1537 dosed with 9-Aminoacridine in the absence of metabolic activation (Experiment 1) were above the historical values. This is considered not to affect study integrity as the strain responded within acceptable parameters for the vehicle and untreated controls and viability data. 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).
- Results: Dose-related and statistically significant increases in excess of 2-fold were noted in bacterial strains TA100 and WP2uvrA in both the absence and presence of metabolic activation. For TA100, maximum increases over the vehicle control of 3.7 fold were noted in the absence of metabolic activation at 1500 µg/plate and 4.4 fold in the presence of metabolic activation at 5000 µg/plate. For WP2uvrA, maximum increases over the vehicle control of 18.8 and 13 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate. For TA98, maximum increases over the vehicle control of 3.4 and 1.9 fold were noted in the absence and presence of metabolic activation respectively at 5000 µg/plate. All of these increases were also accompanied by individual revertant colony counts above the upper maxima of the historical vehicle and untreated control ranges of the lab. Other, minor statistically significant increases were noted for TA1535 in the absence (max = 1.9 fold) and presence (max= 2.5 fold) of metabolic activation and TA1537 in the absence of metabolic activation (max = 2.5 fold). Whilst these increases did not all meet the required criteria (3-fold for TA1535 and TA1537, and 2-fold for the remaining strains), there were signs of a
mild response, which should also be considered in the overall assessment of the results.
See Tables 1 and 2 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)

113
145
121

(126)
16.7#

20
19
20

(20)
0.6

27
26
28

(27)
1.0

21
27
21

(23)
3.5

10
12
8

(10)
2.0

1.5 µg

126
136
138

(133)
6.4

24
28
15

(22)
6.7

18
21
22

(20)
2.1

32
26
33

(30)
3.8

8
12
15

(12)
3.5

5 µg

143
122
134

(133)
10.5

25
19
20

(21)
3.2

21
25
20

(22)
2.6

22
32
24

(26)
5.3

11
15
8

(11)
3.5

15 µg

146
132
141

(140)
7.1

22
19
20

(20)
1.5

41
31
30

(34)
6.1

30
39
25

(31)
7.1

17
9
11

(12)
4.2

50 µg

207
208
233

(216)
14.7
***

20
26
15

(20)
5.5

116
82
110

(103)
18.1
***

29
40
39

(36)
6.1

9
15
10

(11)
3.2

150 µg

315
285
310

(303)
16.1
***

28
20
34

(27)
7.0

168
159
183

(170)
12.1
***

44
27
42

(38)
9.3

7
7
12

(9)
2.9

500 µg

378
401
416

(398)
19.1
***

41
29
31

(34)
6.4

295
325
307

(309)
15.1
***

39
64
62

(55)
13.9
***

9
17
9

(12)
4.6

1500 µg

465
467
455

(462)
6.4
***

31
32
48

(37)
9.5
*

379
391
364

(378)
13.5
***

46
51
70

(56)
12.7
***

13
10
14

(12)
2.1

5000 µg

468
435
454

(452)
16.6
***

22
40
38

(33)
9.9
*

473
527
523

(508)
30.1
***

63
81
92

(79)
14.6
***

17
24
18

(20)
3.8
*

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
696
624

(656)
36.6

267
269
203

(246)
37.5

152
152
148

(151)
2.3

112
114
137

(121)
13.9

1880
1495
1605

(1660)
198.3

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)

127
131
125

(128)
3.1#

26
16
9

(17)
8.5

17
33
29

(26)
8.3

37
41
37

(38)
2.3

15
9
18

(14)
4.6

1.5 µg

144
127
118

(130)
13.2

11
12
24

(16)
7.2

31
25
22

(26)
4.6

34
43
30

(36)
6.7

7
11
10

(9)
2.1

5 µg

146
142
133

(140)
6.7

15
15
8

(13)
4.0

19
18
27

(21)
4.9

29
25
32

(29)
3.5

19
11
13

(14)
4.2

15 µg

157
176
168

(167)
9.5
**

19
17
26

(21)
4.7

27
25
20

(24)
3.6

46
47
46

(46)
0.6

18
8
16

(14)
5.3

50 µg

234
201
218

(218)
16.5
***

19
19
13

(17)
3.5

67
61
79

(69)
9.2
***

37
31
26

(31)
5.5

15
11
12

(13)
2.1

150 µg

356
384
384

(374)
15.4
***

35
22
17

(25)
9.3

148
124
123

(132)
14.2
***

50
39
36

(42)
7.4

19
11
8

(13)
5.7

500 µg

521
462
471

(485)
31.8
***

21
25
24

(23)
2.1

202
209
214

(208)
6.0
***

44
57
50

(50)
6.5
*

10
13
11

(11)
1.5

1500 µg

503
507
489

(500)
9.5
***

22
25
41

(29)
10.2

269
287
276

(277)
9.1
***

65
68
53

(62)
7.9
***

18
13
12

(14)
3.2

5000 µg

546
539
606

(564)
36.8
***

32
43
30

(35)
7.0
*

357
303
355

(338)
30.6
***

70
73
77

(73)
3.5
***

15
11
15

(14)
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

1531
1677
1733

(1647)
104.3

316
315
268

(300)
27.4

152
174
196

(174)
22.0

116
129
104

(116)
12.5

247
263
269

(260)
11.4

BP: Benzo(a)pyrene

2AA: 2 -Aminoanthracene

* p≤0.05

** p≤0.01

*** p≤0.001

# Standard deviation

Applicant's summary and conclusion

Conclusions:
positive with and without metabolic activation

Amines, polyethylenepoly-, triethylenetetramine fraction (TETA) was positive in the Ames/Salmonella Plate Incorporation 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-, triethylenetetramine fraction (TETA) induced dose-related and statistically significant increases in the frequency of TA100, TA98 and WP2uvrA revertant colonies that met the criteria for a positive result, both with and without metabolic activation (S9-mix). Minor statistically significant increases were noted for TA1535 in the absence and presence of metabolic activation and TA1537 in the absence of metabolic activation. These increases did not meet the required criteria for a positive outcome. They were regarded as signs of a mild response and as such taken into account for the overall assessment. Under the conditions of this test Amines, polyethylenepoly-, triethylenetetramine fraction (TETA) was therefore considered to be mutagenic.