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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 November 2015 - 07 March 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
1-((2-Amino-2-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol
Molecular formula:
C13H22N2O2
IUPAC Name:
1-((2-Amino-2-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol
Constituent 2
Chemical structure
Reference substance name:
1-((2-Amino-1-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol
Molecular formula:
C13H22N2O2
IUPAC Name:
1-((2-Amino-1-methylethyl)amino)-3-(2-methylphenoxy)propan-2-ol
Constituent 3
Chemical structure
Reference substance name:
3,3'-(Propane-1,2-diylbis(azanediyl))bis(1-(2-methylphenoxy)propan-2-ol)
Molecular formula:
C23H34N2O4
IUPAC Name:
3,3'-(Propane-1,2-diylbis(azanediyl))bis(1-(2-methylphenoxy)propan-2-ol)
Constituent 4
Chemical structure
Reference substance name:
1,3-Bis(2-methylphenoxy)propan-2-ol
Molecular formula:
C17H20O3
IUPAC Name:
1,3-Bis(2-methylphenoxy)propan-2-ol
Constituent 5
Reference substance name:
unknown compounds
Molecular formula:
not applicable (unknown compounds)
IUPAC Name:
unknown compounds
Test material form:
liquid

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
Test concentrations with justification for top dose:
Concentrations: 5, 16, 50, 160, 500 and 1000 μg/plate (-S9 mix), 16, 50, 160, 500, 1000 and 1600 μg/plate (+S9 mix)
Justification for top dose: Based on a concentration range finding test. In this test strong cytotoxic effect of the test item (absent background lawn and absent revertant growth) was obtained at the concentrations of 5000 and 1600 μg/plate, without metabolic activation (-S9 Mix) and at 5000 μg/plate, with addition of metabolic activation (+S9 Mix).
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: Based on a solubility test.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene and 4-nitro-1,2-phenylene-diamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes, 37 °C
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 160 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 160 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 160 µg/plate
Vehicle controls validity:
valid
Untreated 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:
cytotoxicity
Remarks:
at ≥ 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at ≥ 500 µg/plate
Vehicle controls validity:
valid
Untreated 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:
cytotoxicity
Remarks:
at ≥ 1600 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed .

RANGE-FINDING/SCREENING STUDIES: The concentration range investigated in the main tests was based on two preliminary range finding tests.

Any other information on results incl. tables

Analytical Measurment:

To verify the test item concentrations samples from the stock solution and vehicle control will be taken and the test item content was determined with a previously validated analytical method. For the analysis of the test item concentrations, representative samples from the stock solutions (with a concentration of 32 mg/mL) and vehicle control were taken and analysed before each main experiment. The two main components of test item were used for the quantitation of the test item. The measured concentrations of components mentioned above varied between 92 % and 102 % in the samples in comparison to the nominal values.

Table 1: Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

19.3

1.07

21.3

0.98

82.0

1.21

91.3

1.02

9.3

0.88

9.0

0.96

4.0

0.67

5.3

0.84

33.0

1.03

42.7

1.24

DMSO Control (100 µL)

17.7

1.00

24.0

1.00

80.7

1.00

8.7

1.00

3.7

1.00

4.7

1.00

33.0

1.00

Ultrapure Water Control

79.7

1.00

9.7

1.00

26.0

1.00

DMSO Control (50 µL)

18.0

1.00

21.7

1.00

68.0

1.00

89.3

1.00

10.7

1.00

9.3

1.00

6.0

1.00

6.3

1.00

32.0

1.00

34.3

1.00

1600

3.7

0.17

2.3

0.03

0.0

0.00

0.0

0.00

16.7

0.49

1000

0.0

0.00

18.3

0.85

0.0

0.00

117.0

1.31

0.0

0.00

15.3

1.64

0.0

0.00

4.3

0.68

13.0

0.41

36.0

1.05

500

6.0

0.33

38.3

1.77

72.7

1.07

138.7

1.55

4.3

0.41

19.7

2.11

0.0

0.00

5.7

0.89

33.3

1.04

40.0

1.17

160

20.0

1.11

34.7

1.60

113.3

1.67

127.3

1.43

11.3

1.06

16.3

1.75

4.3

0.72

8.3

1.32

35.7

1.11

33.0

0.96

50

20.3

1.13

36.3

1.68

89.0

1.31

101.0

1.13

9.0

0.84

13.7

1.46

7.3

1.22

7.0

1.11

35.7

1.11

37.7

1.10

16

14.3

0.80

26.7

1.23

86.7

1.27

101.3

1.13

9.0

0.84

11.0

1.18

5.3

0.89

8.0

1.26

27.0

0.84

39.7

1.16

5

17.3

0.96

79.7

1.17

9.3

0.88

7.3

1.22

34.3

1.07

NPD (4mg)

481.3

27.25

SAZ (2mg)

796.7

10.00

1093.3

113.10

9AA (50mg)

581.3

158.55

MMS (2mL)

562.7

21.64

2AA (2mg)

1290.7

53.78

1629.3

20.20

194.3

22.42

123.0

26.36

2AA (50mg)

215.3

6.53

MR: Mutation Rate; NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: DMSO (50 µL) was applied as vehicle of the test item and DMSO (100 µL) was applied as vehicle of the positive control substances NPD, 9AA and 2AA. The ultrapure water (100 µL) was applied as vehicle of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (50 µL); the mutation rate of the NPD, 9AA and 2AA refers to the DMSO sample (100 µL). The mutation rate of MMS and SAZ refers to ultrapure water (100 µL).

Table 2: Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

21.3

0.93

26.7

1.10

87.3

1.12

100.0

1.12

10.3

0.76

11.7

1.09

7.3

1.05

6.7

1.18

27.3

0.92

32.7

0.96

DMSO Control (100 µL)

16.3

1.00

23.3

1.00

73.0

1.00

9.7

1.00

6.7

1.00

5.3

1.00

33.0

1.00

Ultrapure Water Control

77.3

1.00

9.3

1.00

31.0

1.00

DMSO Control (50 µL)

23.0

1.00

24.3

1.00

78.0

1.00

89.3

1.00

13.7

1.00

10.7

1.00

7.0

1.00

5.7

1.00

29.7

1.00

34.0

1.00

1600

0.0

0.00

0.0

1.00

0.0

0.00

0.0

0.00

10.3

0.30

1000

0.0

0.00

4.0

0.16

0.0

0.00

36.7

0.00

0.0

0.00

8.0

0.75

0.0

0.00

4.0

0.13

26.7

0.78

500

1.7

0.07

31.7

1.30

6.0

0.08

117.3

0.41

2.7

0.20

13.7

1.28

0.0

0.00

6.7

1.18

11.3

0.38

35.7

1.05

300

1.3

0.19

160

11.0

0.48

32.3

1.33

70.7

0.91

112.0

1.25

7.7

0.56

10.3

0.97

2.7

0.38

8.3

1.47

40.3

1.36

38.0

1.12

50

28.7

1.25

31.7

1.30

88.7

1.14

101.7

1.14

11.0

0.80

13.0

1.22

7.0

1.00

7.3

1.29

29.7

1.00

35.3

1.04

16

24.0

1.04

31.7

1.30

95.3

1.22

112.3

1.26

11.7

0.85

14.0

1.31

6.7

0.95

8.0

1.41

32.3

1.09

32.0

0.94

5

27.7

1.20

23.7

0.97

84.0

1.08

130.7

1.46

11.3

0.83

13.3

1.25

8.3

1.19

6.0

1.06

27.0

0.91

40.3

1.19

1.6

23.7

1.03

97.3

1.25

13.3

0.98

7.7

1.10

28.7

0.97

NPD (4mg)

452.0

27.67

SAZ (2mg)

1034.7

13.38

840.0

90.00

9AA (50mg)

445.3

66.80

MMS (2mL)

978.7

31.57

2AA (2mg)

965.3

41.37

1056.0

14.47

117.3

12.14

90.0

16.88

2AA (50mg)

213.3

6.46

MR: Mutation Rate; NPD:4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks: DMSO (50 µL) was applied as vehicle of the test item and DMSO (100 µL) was applied as vehicle of the positive control substances NPD, 9AA and 2AA. The ultrapure water (100 µL) was applied as vehicle of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (50 µL); the mutation rate of the NPD, 9AA and 2AA refers to the DMSO sample (100 µL). The mutation rate of MMS and SAZ refers to ultrapure water (100 µL).

Table 3: Historical Control Values

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

19.7

94.6

10.9

8.9

25.2

SD

1.7

2.4

0.4

1.3

5.2

Minimum

8

67

4

3

11

Maximum

40

133

21

20

52

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

24.5

112.9

11.0

9.2

31.7

SD

1.4

6.1

0.5

1.2

6.4

Minimum

11

74

3

3

13

Maximum

43

159

20

20

60

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

18.1

87.0

10.8

8.5

24.6

SD

1.1

3.6

0.6

1.3

3.3

Minimum

9

58

4

3

10

Maximum

36

131

23

20

54

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

23.0

102.4

11.0

9.2

31.4

SD

0.8

7.7

0.4

1.3

5.8

Minimum

11

69

3

3

12

Maximum

42

148

23

21

59

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

19.5

92.8

11.3

9.1

26.6

SD

1.4

5.0

0.5

1.8

6.6

Minimum

12

62

4

3

10

Maximum

30

139

22

18

52

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

24.4

109.9

11.2

9.5

34.0

SD

1.2

6.7

0.8

1.9

6.1

Minimum

13

83

5

4

16

Maximum

37

149

18

18

63

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

285.9

1214.0

1024.3

594.4

858.4

SD

25.5

80.0

120.3

36.4

164.7

Minimum

152

609

407

136

330

Maximum

598

2272

2597

2048

1760

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1395.7

1727.4

160.7

145.8

204.9

SD

261.4

244.1

30.0

18.9

3.9

Minimum

286

712

91

70

133

Maximum

3211

3435

328

315

367

Abbreviations: TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coliWP2uvrA, SD: Standard deviation

Applicant's summary and conclusion

Conclusions:
The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.
Executive summary:

The test item was examined for its mutagenic activity in two series of in vitro microbial assays employing Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA as indicator organisms. The plate incorporation test with and without addition of liver S9 mix from phenobarbital/p-naphthoflavone-pretreated rats was used. In this study, two experimental series were performed. In the experiments with S9 mix, 10% and 20% S9 in the S9 mix were used. Treatments of all tester strains were performed using test item formulations prepared in DMSO in the absence and in the presence of S9 mix, using final concentrations between 5 and 1000 µg/plate (-S9) and between 16 and 1600 µg/plate (+S9), plus vehicle and positive controls.The concentrations, including the controls, were tested in triplicates. In the performed experiments positive and negative (vehicle) controls were run concurrently. For the analysis of the test item concentrations, representative samples from the stock solutions (with a nominal concentration of 32 mg/mL) and vehicle control were taken and analysed before each main experiment.


In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No biological relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values within the actual historical control data ranges were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. In the performed experiments inhibitory effects of the test item (absent revertants, decreased number of revertant colony numbers and/or affected background lawn development) were observed in all examined strains. The 160 μg/plate was found to be the lowest cytotoxic concentration, observed in confirmatory mutation test in the case of Salmonella typhimurium TA98, TA1535 and TA1537 strains, in the absence of exogenous metabolic activation (-S9 Mix). No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9 Mix) throughout the study.


The reported data of this mutagenicity assay showed, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test item is considered non-mutagenicin this bacterial reverse mutation assay.