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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, 2018

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:
25-07-2018 to 06-08-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH S2(R1) guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
inspected: July 2017; signature: November 2017
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine or tryptophan locus
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 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method): 0, 0.5, 1.5, 5, 15, 50, 150, 500 and/or 1500 µg/plate (depending on toxicity).
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. The dose levels were selected based on the results of Experiment 1.
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.
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 (pre-incubation) ; Experiment 2. in medium; in agar (pre-incubation)

DURATION
- Exposure duration:
Experiment 1 and 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 All of the plates were sealed in anaerobic jars or bags (one jar/bag for each concentration of test item/vehicle) during the incubation procedure (37 ± 3 ºC for approximately 48 hours) to minimize potential losses of the test item from the plates. After incubation, the plates were scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
Conditions were selected in accordance with the relevant guidelines.
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:
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
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
Remarks on result:
other: all strains/cell types tested

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)

100

122

106

(109)

11.4#

20

20

15

(18)

2.9

31

19

31

(27)

6.9

25

17

18

(20)

4.4

8

5

13

(9)

4.0

1.5 µg

125

98

117

(113)

13.9

18

17

12

(16)

3.2

29

30

27

(29)

1.5

25

25

18

(23)

4.0

16

9

15

(13)

3.8

5 µg

125

105

93

(108)

16.2

25

22

16

(21)

4.6

29

24

37

(30)

6.6

28

19

25

(24)

4.6

12

12

10

(11)

1.2

15 µg

109

90

92

(97)

10.4

16

22

18

(19)

3.1

35

27

29

(30)

4.2

30

29

35

(31)

3.2

10

15

7

(11)

4.0

50 µg

101

90

100

(97)

6.1

23

18

22

(21)

2.6

27

33

32

(31)

3.2

25

27

12

(21)

8.1

13

14

6

(11)

4.4

150 µg

89 S

82 S

105 S

(92)

11.8

18

12

20

(17)

4.2

29

34

30

(31)

2.6

23

30

26

(26)

3.5

2

13

7

(7)

5.5

500 µg

0 T

0 T

0 T

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

16 S

25 S

22 S

(21)

4.6

0 T

0 T

0 T

(0)

0.0

1500 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

20 S

21 S

24 S

(22)

2.1

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

851

813

806

(823)

24.2

1227

1387

1471

(1362)

124.0

1301

1244

1198

(1248)

51.6

241

261

262

(255)

11.8

617

368

316

(434)

160.9

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)

121

118

119

(119)

1.5#

12

13

11

(12)

1.0

37

34

56

(42)

11.9

16

23

23

(21)

4.0

8

15

16

(13)

4.4

1.5 µg

100

96

119

(105)

12.3

10

13

11

(11)

1.5

38

38

35

(37)

1.7

33

28

32

(31)

2.6

22

12

11

(15)

6.1

5 µg

131

134

120

(128)

7.4

12

15

17

(15)

2.5

35

44

40

(40)

4.5

22

33

29

(28)

5.6

16

12

19

(16)

3.5

15 µg

135

120

105

(120)

15.0

14

7

19

(13)

6.0

28

42

29

(33)

7.8

24

35

24

(28)

6.4

8

14

17

(13)

4.6

50 µg

111

116

142

(123)

16.6

12

24

8

(15)

8.3

32

40

34

(35)

4.2

18

24

34

(25)

8.1

9

10

15

(11)

3.2

150 µg

135

119

118

(124)

9.5

9

11

10

(10)

1.0

44

40

46

(43)

3.1

28

19

34

(27)

7.5

12

9

13

(11)

2.1

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

30 S

21 S

27 S

(26)

4.6

17

23

24

(21)

3.8

6 S

6 S

10 S

(7)

2.3

1500 µg

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

0 V

0 V

0 V

(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

988

1537

1823

(1449)

424.3

308

264

331

(301)

34.0

284

295

292

(290)

5.7

112

119

132

(121)

10.1

213

207

217

(212)

5.0

 

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)

90

111

104

(102)

10.7#

17

16

21

(18)

2.6

20

23

19

(21)

2.1

19

23

17

(20)

3.1

6

12

15

(11)

4.6

0.15 µg

127

117

108

(117)

9.5

33

20

25

(26)

6.6

16

16

24

(19)

4.6

N/T

15

13

19

(16)

3.1

0.5 µg

97

107

109

(104)

6.4

32

17

15

(21)

9.3

33

27

24

(28)

4.6

24

27

10

(20)

9.1

13

19

15

(16)

3.1

1.5 µg

107

112

93

(104)

9.8

11

22

22

(18)

6.4

19

14

15

(16)

2.6

25

18

16

(20)

4.7

14

16

12

(14)

2.0

5 µg

90

113

121

(108)

16.1

28

21

8

(19)

10.1

19

34

38

(30)

10.0

21

10

22

(18)

6.7

13

14

16

(14)

1.5

15 µg

126

85

82

(98)

24.6

24

18

32

(25)

7.0

29

32

24

(28)

4.0

21

24

14

(20)

5.1

21

15

10

(15)

5.5

50 µg

93

117

90

(100)

14.8

29

29

14

(24)

8.7

22

12

23

(19)

6.1

32

13

20

(22)

9.6

10

10

17

(12)

4.0

150 µg

105 S

110 S

97 S

(104)

6.6

20

24

22

(22)

2.0

25 S

26 S

20 S

(24)

3.2

15

21

14

(17)

3.8

5

6

6

(6)

0.6

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

20 S

12 S

12 S

(15)

4.6

3 S

6 S

5 S

(5)

1.5

1500 µg

N/T

N/T

N/T

0 V

0 V

0 V

(0)

0.0

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

595

617

621

(611)

14.0

232

215

219

(222)

8.9

626

597

492

(572)

70.5

213

218

209

(213)

4.5

103

86

100

(96)

9.1

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)

138

105

135

(126)

18.2#

14

11

10

(12)

2.1

36

31

32

(33)

2.6

26

24

28

(26)

2.0

16

26

21

(21)

5.0

0.15 µg

124

130

95

(116)

18.7

20

15

12

(16)

4.0

N/T

N/T

N/T

0.5 µg

119

105

121

(115)

8.7

14

21

10

(15)

5.6

34

27

29

(30)

3.6

29

37

35

(34)

4.2

21

16

14

(17)

3.6

1.5 µg

92

112

97

(100)

10.4

17

13

21

(17)

4.0

29

36

34

(33)

3.6

20

22

39

(27)

10.4

19

18

15

(17)

2.1

5 µg

124

115

126

(122)

5.9

25

18

14

(19)

5.6

37

33

31

(34)

3.1

17

21

36

(25)

10.0

15

15

12

(14)

1.7

15 µg

105

95

90

(97)

7.6

17

18

14

(16)

2.1

30

24

31

(28)

3.8

22

28

24

(25)

3.1

22

11

18

(17)

5.6

50 µg

95

118

115

(109)

12.5

13

11

9

(11)

2.0

28

23

28

(26)

2.9

23

28

21

(24)

3.6

26

17

20

(21)

4.6

150 µg

108

128

138

(125)

15.3

11

14

14

(13)

1.7

26

39

25

(30)

7.8

16

24

24

(21)

4.6

14

23

18

(18)

4.5

500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

31 S

20 S

22 S

(24)

5.9

21

25

25

(24)

2.3

6 S

7 S

8 S

(7)

1.0

1500 µg

N/T

N/T

0 V

0 V

0 V

(0)

0.0

24 S

21 S

12 S

(19)

6.2

0 V

0 V

0 V

(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

1518

1380

1502

(1467)

75.5

252

250

259

(254)

4.7

183

144

149

(159)

21.2

101

120

135

(119)

17.0

231

231

223

(228)

4.6

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

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, ICH S2(R1) and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item 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 item using both the Ames pre incubation method 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.The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was determined by the results of experiment 1 and ranged between 0.15 and 1500 µg/plate depending on strain type and/or exposure to S9 mix. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item. 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 a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 and 500 µg/plate in the absence and presence of metabolic activation (S9-mix), respectively. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).There were no biologically relevant 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. A minor statistical value was noted in Experiment 1 (TA98 at 15 µg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.No biologically relevant 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. A minor statistical value was noted in Experiment 2 (TA1535 at 5 µg/plate in the presence of S9-mix), however this response was within the in house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. It was concluded that, under the conditions of this assay, the test item 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 study available

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Key Study : OECD TG 471, 2018 : The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100, ICH S2(R1) and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item 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 item using both the Ames pre incubation method 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.The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was determined by the results of experiment 1 and ranged between 0.15 and 1500 µg/plate depending on strain type and/or exposure to S9 mix. Eight test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item. 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 a visible reduction in the growth of the bacterial background lawns of all of the tester strains initially from 150 and 500 µg/plate in the absence and presence of metabolic activation (S9-mix), respectively. No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix).There were no biologically relevant 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. A minor statistical value was noted in Experiment 1 (TA98 at 15 µg/plate in the absence of S9-mix), however this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.No biologically relevant 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. A minor statistical value was noted in Experiment 2 (TA1535 at 5 µg/plate in the presence of S9-mix), however this response was within the in house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance. It was concluded that, under the conditions of this assay, the test item 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 classification or non-classification

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