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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 October 2009 to 13 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Method

Target gene:
Not applicable.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: These mutant strains of Salmonella are incapable of synthesising histidine
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Escherichia coli (WP2uvrA-) requires tryptophan and which can be reverse mutated by base substitution to tryptophan independence. This strain also has a deletion in an excision repair gene.
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/ ß­naphthoflavone induced rate liver S9
Test concentrations with justification for top dose:
Mutation Test - Experiment 1
Five concentrations of the test material (50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 ml) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 ml of molten, trace histidine or tryptophan supplemented, top agar, 0.1 ml of the test material formulation, vehicle or positive control and either 0.5 ml of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37°C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.
Mutation Test - Experiment 2
The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was expanded to 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate.
Additional dose levels (1.5, 5 and 15 µg/plate) and an expanded dose range were selected for Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test material.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Other positive controls were: 9-Aminoacridine (9AA), 4-Nitroquinoline-1-oxide (4NQO), 2-Aminoanthracene (2AA) and Benzo(a)pyrene
Details on test system and experimental conditions:
See below
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
Acceptance Criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls.
The appropriate characteristics for each tester strain have been confirmed, eg rfa cell-wall mutation and pKM101 plasmid R-factor etc.
All tester strain cultures should be in the approximate range of 1 to 9.9 x 10^9 bacteria per ml.
Each mean positive control value should be at least twice the respective vehicle control value for each strain, thus demonstrating both the intrinsic sensitivity of the tester strains to mutagenic exposure and the integrity of the S9-mix.
There should be a minimum of four non-toxic test material dose levels.
There should be no evidence of excessive contamination.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Mutation Test:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9 mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
In Experiment 1, the test material did not cause a visible reduction in the growth of the bacterial background lawn at any dose level. However, in the second (pre-incubation) experiment, the test material caused a visible reduction in the growth of the bacterial background lawn in all tester strains, both with and without metabolic activation, initially at 500 µg/plate. The test material was, therefore, either tested up to the maximum recommended dose level of 5000 µg/plate or the toxic limit, depending on the Experiment number. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.
No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.
All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 17 October 2009

To: 20 October 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

117

128

125

(123)

5.7#

30

28

30

(29)

1.2

19

27

37

(28)

9.0

22

25

16

(21)

4.6

10

10

20

(13)

5.8

-

50

117

120

132

(123)

7.9

35

27

31

(31)

4.0

21

24

24

(23)

1.7

25

21

24

(23)

2.1

10

16

15

(14)

3.2

-

150

106

113

124

(114)

9.1

31

25

29

(28)

3.1

32

29

30

(30)

1.5

25

25

12

(21)

7.5

14

12

21

(16)

4.7

-

500

134

108

117

(120)

13.2

24

20

24

(23)

2.3

23

22

18

(21)

2.6

23

29

16

(23)

6.5

10

15

10

(12)

2.9

-

1500

102

106

142

(117)

22.0

20

33

28

(27)

6.6

20

21

21

(21)

0.6

19

19

20

(19)

0.6

13

8

8

(10)

2.9

-

5000

126

124

117

(122)

4.7

26

28

29

(28)

1.5

15

13

16

(15)

1.5

20

19

21

(20)

1.0

10

9

11

(10)

1.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

675

662

705

(681)

22.1

574

526

471

(524)

51.5

867

883

907

(886)

20.1

164

168

150

(161)

9.5

866

398

617

(627)

234.2

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

#        Standard deviation

Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 17 October 2009

To: 20 October 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

121

139

123

(128)

9.9#

16

22

15

(18)

3.8

35

24

27

(29)

5.7

27

29

32

(29)

2.5

12

18

16

(15)

3.1

+

50

118

121

102

(114)

10.2

21

16

13

(17)

4.0

25

31

21

(26)

5.0

26

23

23

(24)

1.7

15

18

16

(16)

1.5

+

150

92

122

114

(109)

15.5

22

18

13

(18)

4.5

27

29

34

(30)

3.6

26

26

23

(25)

1.7

19

15

13

(16)

3.1

+

500

101

98

107

(102)

4.6

18

24

21

(21)

3.0

29

26

21

(25)

4.0

26

26

23

(25)

1.7

9

12

10

(10)

1.5

+

1500

87

86

106

(93)

11.3

20

15

15

(17)

2.9

16

24

29

(23)

6.6

25

20

27

(24)

3.6

13

15

11

(13)

2.0

+

5000

97

107

87

(97)

10.0

8

15

14

(12)

3.8

15

23

16

(18)

4.4

20

18

20

(19)

1.2

15

10

7

(11)

4.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(µg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1344

2044

1499

(1629)

367.7

139

198

197

(178)

33.8

265

282

249

(265)

16.5

192

197

199

(196)

3.6

301

407

448

(385)

75.9

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

#        Standard deviation

Experiment 2 – Without Metabolic Activation

Test Period

From: 10 November 2009

To: 13 November 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

-

0

106

121

119

(115)

8.1#

20

23

27

(23)

3.5

23

26

23

(24)

1.7

22

19

19

(20)

1.7

14

13

12

(13)

1.0

-

1.5

101

121

95

(106)

13.6

21

21

22

(21)

0.6

24

23

26

(24)

1.5

20

20

20

(20)

0.0

15

16

12

(14)

2.1

-

5

119

98

136

(118)

19.0

22

27

23

(24)

2.6

25

24

27

(25)

1.5

24

19

20

(21)

2.6

11

13

14

(13)

1.5

-

15

98

102

90

(97)

6.1

24

22

24

(23)

1.2

26

22

26

(25)

2.3

19

24

15

(19)

4.5

11

15

11

(12)

2.3

-

50

99

120

110

(110)

10.5

26

22

18

(22)

4.0

22

20

18

(20)

2.0

23

20

20

(21)

1.7

14

15

13

(14)

1.0

-

150

107

102

106

(105)

2.6

20

26

24

(23)

3.1

27

22

22

(24)

2.9

18

23

20

(20)

2.5

13

15

11

(13)

2.0

-

500

89 S

71 S

85 S

(82)

9.5

20 S

19 S

15 S

(18)

2.6

0 V

0 V

0 V

(0)

0.0

11 S

13 S

3 S

(9)

5.3

10 S

2 S

3 S

(5)

4.4

-

1500

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(µg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

771

739

551

(687)

118.9

267

278

332

(292)

34.8

1027

967

975

(990)

32.6

122

95

122

(113)

15.6

690

338

370

(466)

194.6

 

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 10 November 2009

To: 13 November 2009

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

+

0

88

102

91

(94)

7.4#

15

21

18

(18)

3.0

31

36

32

(33)

2.6

24

22

22

(23)

1.2

14

13

15

(14)

1.0

+

1.5

84

82

102

(89)

11.0

15

22

18

(18)

3.5

30

22

30

(27)

4.6

21

23

19

(21)

2.0

15

11

11

(12)

2.3

+

5

101

90

89

(93)

6.7

12

15

12

(13)

1.7

27

34

36

(32)

4.7

20

14

19

(18)

3.2

15

15

10

(13)

2.9

+

15

89

103

101

(98)

7.6

20

14

18

(17)

3.1

33

30

29

(31)

2.1

19

19

21

(20)

1.2

14

13

15

(14)

1.0

+

50

95

87

85

(89)

5.3

14

13

15

(14)

1.0

35

27

30

(31)

4.0

18

19

18

(18)

0.6

16

15

13

(15)

1.5

+

150

99

113

97

(103)

8.7

14

21

19

(18)

3.6

34

25

23

(27)

5.9

21

18

19

(19)

1.5

13

13

16

(14)

1.7

+

500

98

82

90

(90)

8.0

15 S

15 S

14 S

(15)

0.6

21

29

24

(25)

4.0

19

24

22

(22)

2.5

11

15

11

(12)

2.3

+

1500

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

0 T

0 T

0 T

(0)

0.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(µg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1393

1269

1372

(1345)

66.4

200

205

172

(192)

17.8

503

486

514

(501)

14.1

173

139

123

(145)

25.5

388

374

383

(382)

7.1

 

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

S        Sparse bacterial background lawn

T        Toxic, no bacterial background lawn

V        Very weak bacterial background lawn

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results :
negative

The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The test material was considered to be non-mutagenic under the conditions of this test conducted according to EU Method B13/14.