Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2016

Link to relevant study records
Reference
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:
guideline study
Remarks:
Guideline study performed under GLP. All relevant validity criteria were met.
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
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/plateExperiment 2 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plateUp 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.
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.
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, light background contamination and artefacts on the plates, 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: 3DETERMINATION 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).
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
maximum recommended dose level of 5000 µg/plate using preincubation method (EXP2) only
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:
maximum recommended dose level of 5000 µg/plate using preincubation method (EXP2) only
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'.

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)

83

78

75

(79)

4.0#

16

11

7

(11)

4.5

27

20

19

(22)

4.4

20

12

13

(15)

4.4

15

11

17

(14)

3.1

1.5 µg

95

75

91

(87)

10.6

14

11

14

(13)

1.7

17

32

28

(26)

7.8

21

21

11

(18)

5.8

16

14

10

(13)

3.1

5 µg

75

82

68

(75)

7.0

12

11

9

(11)

1.5

21

21

15

(19)

3.5

13

21

20

(18)

4.4

14

11

12

(12)

1.5

15 µg

95

100

76

(90)

12.7

17

11

13

(14)

3.1

20

21

15

(19)

3.2

20

32

27

(26)

6.0

7

9

6

(7)

1.5

50 µg

82

80

91

(84)

5.9

12

10

18

(13)

4.2

21

23

12

(19)

5.9

27

12

27

(22)

8.7

8

14

18

(13)

5.0

150 µg

94

94

98

(95)

2.3

23

13

12

(16)

6.1

20

20

23

(21)

1.7

20

24

17

(20)

3.5

6

17

7

(10)

6.1

500 µg

80

99

92

(90)

9.6

12

12

10

(11)

1.2

19

21

19

(20)

1.2

33

24

29

*

(29)

4.5

11

7

11

(10)

2.3

1500 µg

96

88

106

*

(97)

9.0

7

7

7

(7)

0.0

19

20

19

(19)

0.6

29

24

11

(21)

9.3

12

11

8

(10)

2.1

5000 µg

83

80

72

(78)

5.7

8

6

7

(7)

1.0

33

19

25

(26)

7.0

13

12

20

(15)

4.4

5

4

5

(5)

0.6

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

597

621

637

(618)

20.1

200

319

270

(263)

59.8

1200

1195

1155

(1183)

24.7

192

158

200

(183)

22.3

1219

1070

1232

(1174)

90.0

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)

102

114

114

(110)

6.9#

15

16

16

(16)

0.6

24

24

33

(27)

5.2

23

22

19

(21)

2.1

15

19

17

(17)

2.0

1.5 µg

86

91

100

(92)

7.1

13

19

11

(14)

4.2

27

20

35

(27)

7.5

23

24

24

(24)

0.6

11

16

19

(15)

4.0

5 µg

107

130

102

(113)

14.9

12

7

7

(9)

2.9

25

35

29

(30)

5.0

24

27

16

(22)

5.7

15

16

17

(16)

1.0

15 µg

120

106

94

(107)

13.0

12

10

11

(11)

1.0

27

32

29

(29)

2.5

28

21

20

(23)

4.4

15

9

12

(12)

3.0

50 µg

94

84

115

(98)

15.8

6

14

8

(9)

4.2

29

33

20

(27)

6.7

24

20

15

(20)

4.5

6

13

15

(11)

4.7

150 µg

98

90

114

(101)

12.2

8

13

7

(9)

3.2

28

36

23

(29)

6.6

18

18

25

(20)

4.0

14

17

15

(15)

1.5

500 µg

83

79

80

(81)

2.1

9

9

10

(9)

0.6

28

25

19

(24)

4.6

22

26

22

(23)

2.3

9

16

11

(12)

3.6

1500 µg

119

108

104

(110)

7.8

6

6

5

(6)

0.6

16

20

16

(17)

2.3

19

20

12

(17)

4.4

11

8

5

(8)

3.0

5000 µg

103

98

79

(93)

12.7

5

6

!

(6)

0.7

21

16

20

(19)

2.6

18

24

15

(19)

4.6

9

13

8

(10)

2.6

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

593

600

591

(595)

4.7

246

226

246

(239)

11.5

270

297

346

(304)

38.5

211

195

226

(211)

15.5

450

438

385

(424)

34.6

 

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)

99

118

131

(116)

16.1#

11

15

20

(15)

4.5

21

23

33

(26)

6.4

12

20

19

(17)

4.4

15

12

13

(13)

1.5

1.5 µg

87

132

83

(101)

27.2

13

12

19

(15)

3.8

28

32

20

(27)

6.1

15

23

20

(19)

4.0

13

17

15

(15)

2.0

5 µg

94

112

108

(105)

9.5

17

21

20

(19)

2.1

17

28

15

(20)

7.0

16

13

17

(15)

2.1

13

16

11

(13)

2.5

15 µg

104

102

74

(93)

16.8

13

23

13

(16)

5.8

23

24

27

(25)

2.1

12

25

15

(17)

6.8

12

9

12

(11)

1.7

50 µg

114

91

102

(102)

11.5

16

13

15

(15)

1.5

24

28

23

(25)

2.6

27

20

17

(21)

5.1

8

11

12

(10)

2.1

150 µg

68

100

92

(87)

16.7

16

15

13

(15)

1.5

23

23

21

(22)

1.2

25

17

20

(21)

4.0

12

12

9

(11)

1.7

500 µg

82

82

91

(85)

5.2

17

16

16

(16)

0.6

21

17

17

(18)

2.3

17

20

13

(17)

3.5

13

7

12

(11)

3.2

1500 µg

99

108

87

(98)

10.5

9

13

9

(10)

2.3

19

20

19

(19)

0.6

12

13

13

(13)

0.6

4

7

5

(5)

1.5

5000 µg

69 S

65 S

65 S

(66)

2.3

8 S

7 S

7 S

(7)

0.6

7 S

15 S

9 S

(10)

4.2

11 S

8 S

9 S

(9)

1.5

3 S

2 S

2 S

(2)

0.6

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

674

683

605

(654)

42.7

500

279

497

(425)

126.7

1026

1111

994

(1044)

60.5

293

553

338

(395)

139.0

1029

1004

1024

(1019)

13.2

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)

116

132

122

(123)

8.1#

16

19

16

(17)

1.7

32

45

24

(34)

10.6

20

23

17

(20)

3.0

11

11

16

(13)

2.9

1.5 µg

134

128

110

(124)

12.5

15

15

13

(14)

1.2

32

39

28

(33)

5.6

26

14

18

(19)

6.1

15

12

11

(13)

2.1

5 µg

130

128

136

(131)

4.2

15

12

17

(15)

2.5

32

49

44

(42)

8.7

23

15

18

(19)

4.0

11

12

13

(12)

1.0

15 µg

119

124

104

(116)

10.4

11

9

16

(12)

3.6

32

29

45

(35)

8.5

20

19

22

(20)

1.5

11

12

10

(11)

1.0

50 µg

120

112

112

(115)

4.6

21

17

16

(18)

2.6

24

32

32

(29)

4.6

17

19

18

(18)

1.0

9

11

12

(11)

1.5

150 µg

115

132

114

(120)

10.1

13

11

23

(16)

6.4

24

25

24

(24)

0.6

22

22

22

(22)

0.0

11

16

11

(13)

2.9

500 µg

120

124

106

(117)

9.5

19

13

15

(16)

3.1

28

33

44

(35)

8.2

13

15

17

(15)

2.0

7

11

16

(11)

4.5

1500 µg

111

106

112

(110)

3.2

10

8

7

(8)

1.5

25

19

28

(24)

4.6

21

20

13

(18)

4.4

7

5

9

(7)

2.0

5000 µg

88 S

79 S

64 S

(77)

12.1

6 S

5 S

6 S

(6)

0.6

16

19

11

(15)

4.0

8 S

9 S

7 S

(8)

1.0

3 S

5 S

5 S

(4)

1.2

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

436

442

418

(432)

12.5

154

163

124

(147)

20.4

235

223

253

(237)

15.1

557

446

378

(460)

90.4

218

200

186

(201)

16.0

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

4NQO : 4-Nitroquinoline-1-oxide

9AA : 9-Aminoacridine

BP : Benzo(a)pyrene

2AA : 2-Aminoanthracene

P : Test item precipitate

!: No growth at all, therefore plate considered misdosed

S: Sparse bacterial background lawn

V : Very weak bacterial background lawn

# : Standard deviation

 

Table 3. Spontaneous Mutation Rates (Concurrent Negative Controls): Experiment 1 and 2, respectively

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

68

 

16

 

25

 

25

 

12

 

79

(78)

10

(14)

21

(22)

32

(26)

5

(9)

86

 

16

 

20

 

21

 

11

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

138

 

13

 

36

 

20

 

16

 

120

(124)

16

(17)

23

(27)

20

(19)

13

(13)

114

 

23

 

21

 

17

 

11

 

Conclusions:
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). 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 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. 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), in the first mutation test (plate incorporation method), although small reductions in revertant colony frequency were noted to several of the tester strains at the upper test item dose levels. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate in the second mutation test. The test item did induce a visible reduction in the growth of the bacterial background lawns and/or small reductions in revertant colony frequency to the majority of tester strains in the second mutation test (pre-incubation method) at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). 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 toxicologically 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 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 in Experiment 2 (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1500 μg/plate (TA100) and 500 μg/plate (TA98) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls. 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.

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

Additional information

OECD TG 471, 2016 - 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). 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 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. 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), in the first mutation test (plate incorporation method), although small reductions in revertant colony frequency were noted to several of the tester strains at the upper test item dose levels. These results were not indicative of toxicity sufficiently severe enough to prevent the test item being tested up to the maximum recommended dose level of 5000 μg/plate in the second mutation test. The test item did induce a visible reduction in the growth of the bacterial background lawns and/or small reductions in revertant colony frequency to the majority of tester strains in the second mutation test (pre-incubation method) at 5000 μg/plate in both the presence and absence of metabolic activation (S9-mix). 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 toxicologically 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 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 in Experiment 2 (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first mutation test at 1500 μg/plate (TA100) and 500 μg/plate (TA98) in the absence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.9 times the concurrent vehicle controls. 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.


Justification for selection of genetic toxicity endpoint
Study selected is an in vitro study (Klimisch 1)

Short description of key information:
negative, in vitro bacterial reverse mutation (with and without S-9 activation), OECD TG 471, 2016

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The substance does not meet classification criteria under Regulation (EC) No 1272/2008 for mutagenicity