Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: June 2015; signature: September 2015
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: Approximately 4 °C, in the dark
- Other: clear colourless

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Up to eight test item dose levels were selected in Experiment 2 in order to achieve both a minimum of four non-toxic doses and the toxic/guideline limit of the test item following the change in test methodology. The dose levels were selected based on the results of Experiment 1.

Salmonella strain TA100 and E.coli strain WP2uvrA (presence and absence of S9 mix) and Salmonella strain TA98 (presence of S9-mix only):
0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strains TA1535 and TA1537 (presence and absence of S9-mix) and Salmonella strain TA98 (absence of S9-mix only):
0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (DMSO)
- Justification for choice of solvent/vehicle: The test item 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. Dimethyl sulphoxide was selected as the vehicle.
- Other: Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test substance.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment 1. in medium; in agar (plate incorporation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1. All of the plates were incubated at 37 ± 3 ºC for approximately 48 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). A number of manual counts were performed, predominantly due to colonies spreading, thus distorting the actual plate count.

Experiment 2. 0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer OR S9-mix (as appropriate) and 0.1 mL of the test item formulation, vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 ºC for 20 minutes (with shaking) prior to addition of 2 mL of molten amino-acid supplemented media Subsequently, the procedure for incubation and duration was the same as in Experiment 1.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
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 (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item is considered non-mutagenic (negative) in the test system if the above criteria are not met.
In instances of data prohibiting definitive judgement about test item activity are reported as equivocal.
Statistics:
Statistical methods (Mahon, et al.); as recommended by the UKEMS Subcommittee on Guidelines for Mutagenicity Testing, Report - Part III (1989).

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
See table 1 and 2
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 : Test Results: Experiment 1 with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

108

108

85

(100)

13.3#

23

23

25

(24)

1.2

34

25

26

(28)

4.9

22

21

29

(24)

4.4

8

12

21

(14)

6.7

1.5 µg

92

97

119

(103)

14.4

14

23

23

(20)

5.2

33

22

31

(29)

5.9

22

30

26

(26)

4.0

10

14

8

(11)

3.1

5 µg

97

113

102

(104)

8.2

11

27

15

(18)

8.3

20

33

31

(28)

7.0

22

12

18

(17)

5.0

7

13

7

(9)

3.5

15 µg

111

85

98

(98)

13.0

23

18

17

(19)

3.2

18

18

18

(18)

0.0

18

25

30

(24)

6.0

16

9

21

(15)

6.0

50 µg

92

128

119

(113)

18.7

13

21

19

(18)

4.2

27

31

22

(27)

4.5

22

21

16

(20)

3.2

12

16

16

(15)

2.3

150 µg

80

93

111

(95)

15.6

17

18

20

(18)

1.5

27

22

16

(22)

5.5

27

31

30

(29)

2.1

10

10

21

(14)

6.4

500 µg

61

100

78

(80)

19.6

0 V

0 V

0 V

(0)

0.0

18

20

16

(18)

2.0

16 S

17 S

17 S

(17)

0.6

0 V

0 V

0 V

(0)

0.0

1500 µg

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

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

5000 µg

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 T

0 T

0 T

(0)

0.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

448

341

393

(394)

53.5

293

289

314

(299)

13.4

720

815

740

(758)

50.1

134

171

194

(166)

30.3

354

349

344

(349)

5.0

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

120

167

95

(127)

36.6#

22

9

21

(17)

7.2

21

33

38

(31)

8.7

23

21

17

(20)

3.1

8

8

17

(11)

5.2

1.5 µg

114

92

88

(98)

14.0

12

23

17

(17)

5.5

31

33

21

(28)

6.4

26

25

20

(24)

3.2

8

10

9

(9)

1.0

5 µg

84

80

87

(84)

3.5

21

12

13

(15)

4.9

38

25

22

(28)

8.5

19

21

19

(20)

1.2

10

14

7

(10)

3.5

15 µg

84

69

75

(76)

7.5

18

17

16

(17)

1.0

30

27

36

(31)

4.6

21

16

24

(20)

4.0

20

8

5

(11)

7.9

50 µg

87

81

97

(88)

8.1

22

14

13

(16)

4.9

26

29

20

(25)

4.6

22

16

19

(19)

3.0

9

9

17

(12)

4.6

150 µg

99

91

75

(88)

12.2

15

9

9

(11)

3.5

29

22

17

(23)

6.0

17

14

21

(17)

3.5

12

14

8

(11)

3.1

500 µg

87

76

78

(80)

5.9

9 S

9 S

14 S

(11)

2.9

27

18

13

(19)

7.1

21

14

17

(17)

3.5

7 S

4 S

10 S

(7)

3.0

1500 µg

68 S

67 S

25 S

(53)

24.5

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

5000 µg

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 T

0 T

0 T

(0)

0.0

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

432

363

445

(413)

44.1

296

280

332

(303)

26.6

228

406

357

(330)

91.9

159

189

220

(189)

30.5

434

367

393

(398)

33.8

 

Table 2 : Test Results: Experiment 2 with and without metabolic activation and results of concurrent positive controls

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

78

90

68

(79)

11.0#

22

13

21

(19)

4.9

21

15

24

(20)

4.6

18

24

20

(21)

3.1

12

6

12

(10)

3.5

0.15 µg

N/T

 

21

13

16

(17)

4.0

N/T

 

18

18

31

(22)

7.5

9

4

10

(8)

3.2

0.5 µg

68

77

62

(69)

7.5

22

20

8

(17)

7.6

17

21

16

(18)

2.6

21

24

19

(21)

2.5

3

4

4

(4)

0.6

1.5 µg

73

78

71

(74)

3.6

8

11

11

(10)

1.7

16

24

16

(19)

4.6

31

15

16

(21)

9.0

7

7

7

(7)

0.0

5 µg

83

75

75

(78)

4.6

9

20

22

(17)

7.0

14

15

27

(19)

7.2

12

30

19

(20)

9.1

16

20

5

(14)

7.8

15 µg

65

71

89

(75)

12.5

24

8

11

(14)

8.5

24

19

25

(23)

3.2

26

11

13

(17)

8.1

9

12

4

(8)

4.0

50 µg

72

85

67

(75)

9.3

12

13

13

(13)

0.6

13

14

14

(14)

0.6

18

23

28

(23)

5.0

11

8

21

(13)

6.8

150 µg

71

72

80

(74)

4.9

12 S

8 S

6 S

(9)

3.1

19

9

9

(12)

5.8

23

23

27

(24)

2.3

8 S

4 S

4 S

(5)

2.3

500 µg

34 S

50 S

28 S

(37)

11.4

7 S

8 S

4 S

(6)

2.1

12 S

13 S

13 S

(13)

0.6

13 S

7 S

7 S

(9)

3.5

0 V

0 V

0 V

(0)

0.0

1500 µg

0 T

0 T

0 T

(0)

0.0

N/T

 

0 T

0 T

0 T

(0)

0.0

N/T

 

N/T

 

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

229

277

427

(311)

103.3

99

94

132

(108)

20.6

351

402

345

(366)

31.3

149

127

187

(154)

30.4

122

240

197

(186)

59.7

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

74

92

117

(94)

21.6#

11

15

11

(12)

2.3

24

21

25

(23)

2.1

21

21

22

(21)

0.6

13

7

12

(11)

3.2

0.15 µg

N/T

 

11

11

11

(11)

0.0

N/T

 

N/T

 

12

11

7

(10)

2.6

0.5 µg

109

82

86

(92)

14.6

24

11

11

(15)

7.5

15

24

14

(18)

5.5

17

22

18

(19)

2.6

16

15

10

(14)

3.2

1.5 µg

76

100

108

(95)

16.7

16

22

18

(19)

3.1

18

23

24

(22)

3.2

17

29

22

(23)

6.0

6

12

10

(9)

3.1

5 µg

86

96

101

(94)

7.6

10

9

13

(11)

2.1

17

18

15

(17)

1.5

16

26

20

(21)

5.0

22

8

19

(16)

7.4

15 µg

105

101

77

(94)

15.1

13

13

9

(12)

2.3

21

27

19

(22)

4.2

26

20

20

(22)

3.5

6

12

12

(10)

3.5

50 µg

107

84

102

(98)

12.1

11

17

16

(15)

3.2

19

25

30

(25)

5.5

13

19

17

(16)

3.1

9

5

6

(7)

2.1

150 µg

83

106

107

(99)

13.6

9

17

8

(11)

4.9

20

25

21

(22)

2.6

25

18

17

(20)

4.4

17

15

9

(14)

4.2

500 µg

98

99

86

(94)

7.2

0 V

0 V

0 V

(0)

0.0

15 S

10 S

12 S

(12)

2.5

17 S

9 S

6 S

(11)

5.7

5 S

5 S

5 S

(5)

0.0

1500 µg

47 S

24 S

48 S

(40)

13.6

N/T

 

0 V

0 V

0 V

(0)

0.0

0 T

0 T

0 T

(0)

0.0

N/T

 

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

440

490

520

(483)

40.4

151

171

175

(166)

12.9

87

138

247

(157)

81.7

92

111

111

(105)

11.0

128

135

110

(124)

12.9

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

2AA: 2-Aminoanthracene

N/T: Not tested at this dose level

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

Under the conditions of this study the test material was considered to be non-mutagenic in the presence and absence of S9 activation.
Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test substance in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. The test strains were treated with the test substance using both the Ames plate incorporation and pre incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). Formulated concentrations were adjusted by an appropriate factor to allow for the stated purity of the test substance. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 µg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. Eight test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item following the change in test methodology. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item induced a visible reduction in the growth of the bacterial background lawns of all of the tester strains in both the presence and absence of S9-mix. Weakened bacterial background lawns were initially noted in the absence of S9-mix from 500 µg/plate (TA1535, TA98 and TA1537) and 1500 µg/plate (TA100 and WP2uvrA). In the presence S9-mix weakened bacterial background lawns were again initially noted from 500 µg/plate (TA1535 and TA1537) and 1500 µg/plate (TA100, WP2uvrA and TA98). Consequently for the second mutation test the toxic limit was employed as the maximum dose concentration. Results from the second mutation test (pre-incubation method) confirmed the toxicity previously noted with weakened bacterial background lawns observed in the absence of S9-mix from 150 µg/plate (TA1535 and TA1537) and 500 µg/plate (WP2uvrA, TA100 and TA98). In the presence of S9-mix, weakened bacterial background lawns were noted from 500 µg/plate (TA1535, TA1537, TA98 and WP2uvrA) and 1500 µg/plate (TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9 mix and experimental methodology. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. There were no 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) in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre incubation method). It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.