Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation test (plate incorporation test) according to OECD TG 471 the test item was not mutagenic in the absence and presence of a rat liver metabolizing system (S9 mix) (reference 7.6.1-1).

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 17,2019 - August 19, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
21st July 1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Due to migration, the value was transferred to one of the current document's attachments
Test concentrations with justification for top dose:
5, 15.8, 50, 158, 500, 1580, 5000 µg/plate (with and without S9 mix)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium (DMSO)

- Justification for choice of solvent/vehicle: The selection of the solvent for this assay was based on the available information from a preliminary solubility test. DMSO showed best performance and was thus used for this experiment at a maximum concentration of 100 µL/plate.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
H2O
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2 uvrA, without S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene
Remarks:
TA98, TA100, TA 1535, TA 1537, WP2uvrA, with S9 mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-o-phenylenediamine
Remarks:
TA98, TA1537, without S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
- Number of independent experiments : 2

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

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- background growth inhibition

Evaluation criteria:
A test material was to be defined as positive or mutagenic in this assay if
- the assay is considered valid and
- a biologically relevant increase in the mean number of revertants above a threshold of 2-fold (TA 98, TA 100, WP2 uvrA) or 3-fold (TA 1535, TA 1537) as compared to the concurrent negative controls is observed
- an increase exceeding the threshold at only one concentration is considered as biologically meaningful if reproduced in a second independent experiment
- a concentration-dependent increase is considered biologically meaningful if the threshold is exceeded at more than one concentration
A test material is defined as negative or non-mutagenic in this assay if
- the assay is considered valid and none of the above-mentioned criteria are met
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation, at the top concentration of 5000 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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation, at the top concentration of 5000 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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation, at the top concentration of 5000 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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
without metabolic activation, at the top concentration of 5000 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 and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate.

Toxicity was observed in TA98, TA100 and TA1535 in the 1st series, and in TA98 and TA 1537 in the 2nd series, without metabolic activation, at the top concentration of 5000 µg/plate. The individual and summarized data are presented in the tables section.

Ames test:
- Individual plate counts : see table 1

HISTORICAL CONTROL DATA: see table 2

Table 1: Summary 1st Series

Metabolic

Activation

Test

Material

Concentr.

[µg/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

Without activation

DMSO

 

37 ± 9

107 ± 11

34 ± 6

12 ± 5

24 ± 4

Art. 851009

5.00

41 ± 7

104 ± 1

32 ± 4

11 ± 2

22 ± 1

15.8

38 ± 6

104 ± 7

38 ± 11

10 ± 4

22 ± 6

50.0

37 ± 10

110 ± 19

36 ± 4

11 ± 4

28 ± 5

158

36 ± 4

107 ± 3

39 ± 7

10 ± 2

25 ± 4

500

41 ± 7

103 ± 17

28 ± 9

10 ± 4

27 ± 7

1580

46 ± 6

105 ± 4

24 ± 5

12 ± 3

25 ± 7

5000

11 ± 3 E T

84 ± 6 T E

19 ± 6 E T

10 ± 3 E

26 ± 4 E

NaN3

2.00

 

1675 ± 293

1027 ± 29

 

 

NQO

2.00

 

 

 

 

1030 ± 96

4-NOPD

60.0

 

 

 

102 ± 7

 

4-NOPD

20.00

685 ± 59

 

 

 

 

 

 

 

 

 

 

 

 

With activation

DMSO

 

48 ± 1

133 ± 14

36 ± 3

11 ± 4

31 ± 5

Art. 851009

5.00

44 ± 8

128 ± 7

30 ± 4

14 ± 7

34 ± 8

15.8

40 ± 7

122 ± 23

28 ± 6

7 ± 1

30 ± 2

50.0

44 ± 7

120 ± 22

34 ± 6

13 ± 5

36 ± 9

158

41 ± 2

142 ± 14

30 ± 4

7 ± 2

32 ± 5

500

52 ± 9

130 ± 11

29 ± 4

13 ± 5

37 ± 2

1580

56 ± 7

140 ± 18

37 ± 7

12 ± 4

34 ± 6

5000

72 ± 6 E

111 ± 4 E

28 ± 7 E

10 ± 4 E

37 ± 4 E

2-AA

2.00

909 ± 165

1947 ± 81

 

 

 

2-AA

10.0

 

 

 

 

312 ± 13

2-AA

5.00

 

 

188 ± 33

650 ± 89

 

Key to Positive controls
NQO              4-Nitroquinoline-N-oxide

2-AA              2-Aminoanthracene

4-NOPD         4-Nitro-o-phenylendiamin

NaN3              Sodium azide

 

Key to Plate Postfix Codes

E                   Precipitation until end of experiment

T                    Toxicity = red. bact. background lawn

Table 2: Summary 2nd Series  

Metabolic

Activation

Test

Material

Concentr.

[ug/plate]

Revertants per plate (Mean ± SD)

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

Without activation

DMSO

 

29 ± 6

111 ± 18

25 ± 4

11 ± 4

31 ± 5

Art. 851009

50.0

35 ± 6

101 ± 12

24 ± 5

8 ± 4

19 ± 6

158

30 ± 4

106 ± 7

26 ± 3

7 ± 1

28 ± 2

500

28 ± 13

98 ± 12

26 ± 4

11 ± 1

29 ± 11

1580

43 ± 11

107 ± 8

32 ± 6

9 ± 2

25 ± 2

5000

12 ± 4 E T

82 ± 11 E

17 ± 4 E

5 ± 3 E

29 ± 9 E

NaN3

2.00

 

1821 ± 111

949 ± 20

 

 

NQO

2.00

 

 

 

 

1878 ± 94

4-NOPD

20.0

573 ± 66

 

 

 

 

4-NOPD

60.0

 

 

 

89 ± 12

 

 

 

 

 

 

 

 

 

With activation

DMSO

 

36 ± 4

124 ± 14

23 ± 4

13 ± 2

33 ± 4

 

50.0

39 ± 4

124 ± 5

22 ± 4

10 ± 2

33 ± 8

158

42 ± 9

119 ± 20

22 ± 3

8 ± 3

38 ± 3

500

33 ± 7

125 ± 11

21 ± 7

11 ± 3

35 ± 11

1580

43 ± 10

111 ± 6

20 ± 1

17 ± 4

40 ± 9

5000

43 ± 3 E

136 ± 11 E

18 ± 6 E

11 ± 3 E

34 ± 3 E

2-AA

5.00

 

 

161 ± 29

176 ± 52

 

2-AA

2.00

339 ± 8

706 ± 90

 

 

 

2-AA

10.00

 

 

 

 

152 ± 38

 Key to Positive controls
NQO              4-Nitroquinoline-N-oxide

2-AA              2-Aminoanthracene

4-NOPD         4-Nitro-o-phenylendiamin

NaN3              Sodium azide

 

Key to Plate Postfix Codes

E                   Precipitation until end of experiment

T                    Toxicity = red. bact. background lawn

Table 3:Individual Values 1st Series

Without metabolic activation

Strain

Test Material

Concentr.

[µg/plate]

Mean revertants per plate

Standard Deviation

Ratio treated / solvent

Individual revertants per plate

TA 98

Art 851009

5.00

40.7

6.5

1.1

34

47

41

 

 

 

15.8

37.7

6.4

1.0

45

34

34

 

 

 

50.0

36.7

10.4

1.0

40

45

25

 

 

 

158

36.3

3.5

1.0

36

40

33

 

 

 

500

41.0

6.6

1-1

47

42

34

 

 

 

1580

46.0

6.1

1.3

42

43

53

 

 

 

5000

11.0

2.6

0.3

12 E T

8 E T

13 E T

 

 

 

DMSO

 

36.5

9.4

 

28

45

43

29

47

27

 

 

 

 

 

 

 

 

 

 

 

 

TA 100

Art 851009

5.00

103.7

0.6

1.0

104

104

103

 

 

 

15.8

104.0

6.6

1.0

103

98

111

 

 

 

50.0

109.7

19.4

1.0

132

97

100

 

 

 

158

106.7

3.2

1.0

109

103

108

 

 

 

500

103.3

16.6

1.0

119

86

105

 

 

 

1580

105.3

3.8

1.0

101

107

108

 

 

 

5000

84.0

6.1

0.8

81 T E

91 T E

80 T E

 

 

 

DMSO

 

106.5

11.0

 

122

90

111

103

112

101

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

Art 851009

5.00

31.7

4.2

0.9

33

35

27

 

 

 

15.8

38.3

11.2

1.1

48

26

41

 

 

 

50.0

35.7

4.0

1.1

35

32

40

 

 

 

158

38.7

6.7

1.2

37

46

33

 

 

 

500

27.7

8.7

0.8

35

18

30

 

 

 

1580

24.3

4.9

0.7

21

22

30

 

 

 

5000

19.3

5.5

0.6

13 E T

23 E T

22 E T

 

 

 

DMSO

 

33.5

6.3

 

35

34

23

30

40

39

 

 

 

 

 

 

 

 

 

 

 

 

TA 1537

Art 851009

5.00

11.0

1.7

0.9

9

12

12

 

 

 

15.8

10.3

4.0

0.9

6

14

11

 

 

 

50.0

10.7

3.8

0.9

8

9

15

 

 

 

158

9.7

2.3

0.8

7

11

11

 

 

 

500

10.3

4.0

0.9

14

6

11

 

 

 

1580

11.7

2.5

1.0

12

14

9

 

 

 

5000

10.0

2.6

0.8

11 E

12 E

7 E

 

 

 

DMSO

 

11.8

4.8

 

14

8

8

20

13

8

 

 

 

 

 

 

 

 

 

 

 

 

WP2 uvrA

Art 851009

5.00

22.3

0.6

0.9

22

23

22

 

 

 

15.8

21.7

6.1

0.9

15

27

235

 

 

 

50.0

27.7

5.1

1.2

32

29

22

 

 

 

158

25.0

4.0

1.0

21

25

29

 

 

 

500

27.0

6.6

1.1

21

34

26

 

 

 

1580

25.3

6.5

1.1

25

19

32

 

 

 

5000

26.0

3.6

1.1

25 E

30 E

23 E

 

 

 

DMSO

 

23.8

4.3

 

21

30

18

26

26

22

 

 

 

 

 

 

 

 

 

 

 

 

TA 98

4-NOPD

20.0

685.3

59.0

18.8

737

621

698

 

 

 

TA 100

NaN3

2.00

1675.0

292.9

15.7

1868

1819

1338

 

 

 

TA 1535

NaN3

2.00

1026.7

28.6

30.6

994

1039

1047

 

 

 

TA 1537

4-NOPD

60.0

102.3

7.4

8.6

105

94

108

 

 

 

WP2 uvrA

NQO

2.00

1029.7

96.0

43.2

1000

952

1137

 

 

 

 Key to Positive controls

4-NOPD         4-Nitro-o-phenylendiamin

NaN3              Sodium azide

NQO              4-Nitroquinoline-N-oxide

 

Key to Plate Postfix Codes

E                   Precipitation until end of experiment

T                    Toxicity = red. bact. background lawn


With metabolic activation

Strain

Test Material

Concentr.

[ug/plate]

Mean revertants per plate

Standard Deviation

Ratio treated / solvent

Individual revertants per plate

TA 98

Art 851009

5.00

43.7

8.1

0.9

39

39

53

 

 

 

 

15.8

39.7

7.0

0.8

47

33

39

 

 

 

 

50.0

43.7

7.1

0.9

36

50

45

 

 

 

 

158

41.3

1.5

0.9

41

40

43

 

 

 

 

500

52.0

8.9

1.1

59

42

55

 

 

 

 

1580

56.0

6.6

1.2

49

57

62

 

 

 

 

5000

72.0

5.6

1.5

71 E

67 E

78 E

 

 

 

 

DMSO

 

47.5

1.0

 

48

49

46

47

47

48

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 100

Art 851009

5.00

128.0

7.0

1.0

136

123

125

 

 

 

 

15.8

122.3

23.5

0.9

148

102

117

 

 

 

 

50.0

120.3

22.1

0.9

141

97

123

 

 

 

 

158

142.3

13.6

1.1

155

144

128

 

 

 

 

500

130.0

10.6

1.0

138

134

118

 

 

 

 

1580

140.3

18.1

1.1

121

157

143

 

 

 

 

5000

111.3

3.5

0.8

115 E

111 E

108 E

 

 

 

 

DMSO

 

132.7

14.0

 

128

119

159

124

131

135

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

Art 851009

5.00

30.0

3.6

0.8

34

27

29

 

 

 

 

15.8

28.3

5.7

0.8

22

30

33

 

 

 

 

50.0

34.3

5.5

1.0

29

40

34

 

 

 

 

158

29.7

3.8

0.8

27

28

34

 

 

 

 

500

29.0

3.6

0.8

32

25

30

 

 

 

 

1580

37.0

7.0

1.0

42

29

40

 

 

 

 

5000

28.3

6.8

0.8

26 e

23 E

36 E

 

 

 

 

DMSO

 

35.7

2.8

 

35

41

33

35

36

34

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1537

Art 851009

5.00

13.7

7.4

1.2

8

22

11

 

 

 

 

15.8

6.7

0.6

0.6

6

7

7

 

 

 

 

50.0

12.7

5.0

1.2

8

18

12

 

 

 

 

158

6.7

1.5

0.6

7

8

5

 

 

 

 

500

13.0

5.3

1.2

11

9

19

 

 

 

 

1580

12.0

3.6

1.1

16

11

9

 

 

 

 

5000

10.0

3.6

0.9

13 E

11 E

6 E

 

 

 

 

DMSO

 

11.0

4.1

 

6

16

12

6

12

14

 

 

 

 

 

 

 

 

 

 

 

 

 

 

WP2 uvrA

Art 851009

5.00

34.3

8.0

1.1

26

35

42

 

 

 

 

15.8

30.3

1.5

1.0

30

32

29

 

 

 

 

50.0

35.7

9.1

1.2

32

29

46

 

 

 

 

158

32.0

4.6

1.0

33

27

36

 

 

 

 

500

37.0

1.7

1.2

36

39

36

 

 

 

 

1580

33.7

6.0

1.1

40

28

33

 

 

 

 

5000

37.0

4.0

1.2

33 E

37 E

41 E

 

 

 

 

DMSO

 

30.7

4.7

 

34

25

37

27

28

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 98

2-AA

2.00

908.7

165.1

19.1

732

1059

935

 

 

 

 

TA 100

2-AA

2.00

1946.7

80.5

14.7

1916

1886

2038

 

 

 

 

TA1535

2-AA

5.00

188.0

32.7

5.3

225

163

176

 

 

 

 

TA 1537

2-AA

5.00

649.7

88.8

59.1

629

747

573

 

 

 

 

WP2 uvrA

2-AA

10.00

312.3

13.2

10.2

298

324

315

 

 

 

 

 Key to Positive controls

2-AA              2-Aminoanthracene

 

Key to Plate Postfix Codes

E                   Precipitation until end of experiment

Table 4:Individual Values 2nd Series

Without metabolic activation

Strain

Test Material

Concentr.

[µg/plate]

Mean revertants per plate

Standard Deviation

Ratio treated / solvent

Individual revertants per plate

TA 98

Art 851009

50.0

34.7

5.5

1.2

29

40

35

 

 

 

158

30

4.4

1.0

25

33

32

 

 

 

500

27.7

12.7

1.0

36

34

13

 

 

 

1580

42.7

11.0

1.5

42

32

54

 

 

 

5000

11.7

3.5

0.4

12 E T

15 E T

8 E T

 

 

 

DMSO

 

29.0

6.0

 

32

37

32

25

28

20

 

 

 

 

 

 

 

 

 

 

 

 

TA 100

Art 851009

50.0

101.3

11.6

0.9

107

109

88

 

 

 

158

106.3

6.8

1.0

104

101

114

 

 

 

500

98.0

11.8

0.9

111

88

95

 

 

 

1580

107.0

7.9

1.0

116

101

104

 

 

 

5000

81.7

10.7

0.7

76 E

75 E

94 E

 

 

 

DMSO

 

110.8

18.3

 

102

139

90

116

96

122

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

Art 851009

50.0

23.7

4.7

0.9

22

20

29

 

 

 

158

26.0

2.6

1.0

23

27

28

 

 

 

500

26.0

3.6

1.0

25

30

23

 

 

 

1580

31.7

5.8

1.3

25

35

35

 

 

 

5000

16.7

5.1

0.7

11 E

21 E

18 E

 

 

 

DMSO

 

25.2

4.0

 

29

29

26

22

26

19

 

 

 

 

 

 

 

 

 

 

 

 

TA 1537

Art 851009

50.0

8.0

3.6

0.7

11

4

9

 

 

 

158

7.3

1.2

0.6

8

6

8

 

 

 

500

11.3

0.6

1.0

11

11

12

 

 

 

1580

9.3

2.3

0.8

8

12

8

 

 

 

5000

5.0

3.0

0.4

2 E

5 E

8 E

 

 

 

DMSO

 

11.3

4.0

 

9

12

8

19

11

9

 

 

 

 

 

 

 

 

 

 

 

 

WP2 uvrA

Art 851009

50.0

19.3

6.0

0.6

25

20

13

 

 

 

158

28.0

1.7

0.9

26

29

29

 

 

 

500

29.0

10.6

0.9

25

41

21

 

 

 

1580

25.3

2.1

0.8

23

26

27

 

 

 

5000

29.3

9.3

0.9

19 E

32 E

37 E

 

 

 

DMSO

 

31.3

5.5

 

29

26

41

32

33

27

 

 

 

 

 

 

 

 

 

 

 

 

TA 98

4-NOPD

20.0

573.0

66.3

19.8

647

553

519

 

 

 

TA 100

NaN3

2.00

1821.0

110.8

16.4

1898

1871

1694

 

 

 

TA 1535

NaN3

2.00

949.0

20.2

37.7

957

964

926

 

 

 

TA 1537

4-NOPD

60.0

88.7

12.0

7.8

88

101

77

 

 

 

WP2 uvrA

NQO

2.00

1878.3

93.5

59.9

1791

1977

1867

 

 

 

 

Key to Positive controls

4-NOPD         4-Nitro-o-phenylendiamin

NaN3              Sodium azide

NQO              4-Nitroquinoline-N-oxide

 

Key to Plate Postfix Codes

E                   Precipitation until end of experiment

T                    Toxicity = red. bact. background lawn

With metabolic activation

Strain

Test Material

Concentr.

[µg/plate]

Mean revertants per plate

Standard Deviation

Ratio treated / solvent

Individual revertants per plate

TA 98

Art 851009

50.0

38.7

3.5

1.1

35

39

42

 

 

 

 

158

42.3

8.5

1.2

36

52

39

 

 

 

 

500

32.7

6.5

0.9

26

39

33

 

 

 

 

1580

43.3

9.7

1.2

35

41

54

 

 

 

 

5000

43.3

2.5

1.2

46 E

43 E

41 E

 

 

 

 

DMSO

 

35.5

3.6

 

36

41

34

35

37

30

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 100

Art 851009

50.0

123.7

5.1

1.0

125

128

118

 

 

 

 

158

118.7

19.6

1.0

98

137

121

 

 

 

 

500

125.3

10.5

1.0

115

136

125

 

 

 

 

1580

110.7

5.5

0.9

105

111

116

 

 

 

 

5000

135.7

11.0

1.1

132 E

127 E

148 E

 

 

 

 

DMSO

 

124.0

14.3

 

151

124

119

121

121

108

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1535

Art 851009

50.0

22.3

4.0

1.0

20

27

20

 

 

 

 

158

22.0

3.0

0.9

25

19

22

 

 

 

 

500

20.7

7.0

0.9

28

20

14

 

 

 

 

1580

20.0

1.0

0.9

20

19

21

 

 

 

 

5000

18.3

5.9

0.8

25 E

16 E

14 E

 

 

 

 

DMSO

 

23.2

4.3

 

23

30

21

21

18

26

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 1537

Art 851009

50.0

10.0

1.7

0.8

11

8

11

 

 

 

 

158

8.0

3.0

0.6

8

5

11

 

 

 

 

500

11.0

3.5

0.8

9

15

9

 

 

 

 

1580

16.7

3.8

1.3

15

21

14

 

 

 

 

5000

11.0

3.0

0.8

11 E

8 E

14 E

 

 

 

 

DMSO

 

13.0

1.7

 

15

11

12

13

12

15

 

 

 

 

 

 

 

 

 

 

 

 

 

 

WP2 uvrA

Art 851009

50.0

33.3

7.6

1.0

30

42

28

 

 

 

 

158

38.0

2.6

1.1

41

37

36

 

 

 

 

500

34.7

10.8

1.0

47

27

30

 

 

 

 

1580

39.7

9.0

1.2

34

50

35

 

 

 

 

5000

33.7

2.9

1.0

32 E

37 E

32 E

 

 

 

 

DMSO

 

33.2

4.0

 

37

33

33

37

33

26

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TA 98

2-AA

2.00

339.0

7.5

9.5

331

340

346

 

 

 

 

TA 100

2-AA

2.00

706.3

89.8

5.7

698

800

621

 

 

 

 

TA1535

2-AA

5.00

160.7

28.9

6.9

194

143

145

 

 

 

 

TA 1537

2-AA

5.00

175.7

52.0

13.5

216

117

194

 

 

 

 

WP2 uvrA

2-AA

10.00

152.0

38.1

4.6

108

175

173

 

 

 

 

Key to Positive controls

2-AA              2-Aminoanthracene

 

Key to Plate Postfix Codes

E                   Precipitation until end of experiment


Table 5: Historical Data

The historical data have been obtained in experiments between 01/2018 and 12/2018.

 

Negative Controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Solvent

Total Plates

618

608

624

612

461

456

480

473

576

575

Number of Values

121

119

121

121

82

82

86

85

111

111

Minimum

9

11

85

96

11

7

5

5

19

19

Maximum

46

51

144

158

37

44

18

16

51

58

Mean

28

33

112

124

20

18

9

10

33

38

Standard Deviation

6.5

7.8

11.5

12.9

5.7

6.2

2.4

2.5

5.8

7.6

 

 

Postive controls

Strain

TA 98

TA 100

TA 1535

TA 1537

WP2 uvrA

S9 Mix

Without

With

Without

With

Without

With

Without

With

Without

With

Compound

DAUN

2-AA

NaN3

2-AA

NaN3

2-AA

9-AA

2-AA

NQO

2-AA

Total Plates

311

304

312

306

231

228

240

238

288

288

Number of Values

121

119

121

121

82

82

86

85

111

111

Minimum

67

107

363

447

162

74

85

72

294

124

Maximum

1243

1862

2910

5235

2637

298

4040

660

3170

1494

Mean

365

616

1603

1801

853

166

1403

282

1881

364

Standard Deviation

232.7

380.7

369.1

1045.8

259.6

53.8

838.8

137.6

386.3

157.3

Conclusions:
Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.
Executive summary:

The test item was examined according to OECD TG 471 for its mutagenic activity in an in vitro bacterial reverse mutation test employing Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/(beta-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 5000 µg/plate, vehicle and positive controls.

The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.

The strain-specific positive controls, namely sodium azide, 4-nitroquinolin-N-oxide and 4-Nitro-o-phenylenediamine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the study is considered valid.

Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate. Toxicity was observed in TA98, TA100 and TA1535 in the 1st series, and in TA98 and TA 1537 in the 2nd series, without metabolic activation, at the top concentration of 5000 µg/plate.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.

Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames Test

The test item was examined according to OECD TG 471 for its mutagenic activity in an in vitro bacterial reverse mutation test employing Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA as indicator organisms.

The plate incorporation test with and without addition of liver S9 mix from Phenobarbital/(beta-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 5000 µg/plate, vehicle and positive controls.

The mean numbers of revertant colonies of the current negative controls were within the range of historical negative control values.

The strain-specific positive controls, namely sodium azide, 4-nitroquinolin-N-oxide and 4-Nitro-o-phenylenediamine in the absence of S9 mix, yielded the expected mutant frequencies that were greatly exceeding the negative controls. The genotype of the tester strains used was thus confirmed. 2-Aminoanthracene which requires metabolic activation was strongly mutagenic. This indicates that the exogenous metabolizing system used in the present investigation (S9 mix) was active. Thus, the study is considered valid.

Following treatment with the test item, precipitation of the test material on the agar plates occurred at concentrations > 5000 µg/plate. Toxicity was observed in TA98, TA100 and TA1535 in the 1st series, and in TA98 and TA 1537 in the 2nd series, without metabolic activation, at the top concentration of 5000 µg/plate.

Under the conditions described, there were no relevant increases in revertant numbers observed after exposure to the test item in the absence and presence of S9 mix.

Under the experimental conditions reported here, the test item did not induce gene mutations by base-pair or frameshift changes in the genome of the strains used. Therefore, it was concluded that with and without addition of S9 mix as the exogenous metabolizing system, the test item was not mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation test.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.