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:
09-03-2018 to 10-04-2018
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:
2018
Report Date:
2018

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
Version / remarks:
Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labor and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries (24 November 2000)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: July 2017 ; signature: November 2017
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 4ºC, in the dark, under nitrogen
- Other: clear colourless

Method

Target gene:
histidine or tryptophan locus
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 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Part of the first mutation test was repeated due to excessive toxicity (TA-strains dosed in the absence of S9-mix) employing an amended test item dose range of 0.015 to 50 µg/plate. Specifically: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and µg/plate.

Experiment 2 (pre-incubation method): 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. The dose levels were selected based on the results of Experiment 1.

Salmonella strains (All); TA98, TA100, TA1535 and TA1537 (absence of S9-mix): 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15, 50 µg/plate.
Salmonella strains TA100 and TA1537 (presence of S9-mix): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 µg/plate.
Salmonella strains TA1535 (presence of S9-mix): 0.5, 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strain TA98 (presence of S9-mix): 5, 15, 50, 150, 500, 1500, 5000 µg/plate.
E.coli strain WP2uvrA (absence and presence of S9-mix): 15, 50, 150, 500, 1500, 5000 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/increased to allow for the stated water/impurity content. See 'Test Material Information' for further details.
Controlsopen allclose all
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
Remarks:
Without metabolic activation S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
With metabolic activation S9
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).
The choice of application was due to the test item to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls.

DURATION
- Exposure duration:
Experiment 1. All of the plates were pre-incubated in sealed, small volume containers, by application of 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 to 72 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). Manual counts may be performed, where automated counting cannot be performed: e.g. colonies spreading, colonies on the edge of the plates 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.

SELECTION AGENT (mutation assays): histidine-deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Rationale for test conditions:
In accordance with the OECD TG 471 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. 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)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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).
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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
Additional information on results:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.
Remarks on result:
other: all strains/cell types tested

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)

118

115

127

(120)

6.2#

11

20

12

(14)

4.9

33

18

27

(26)

7.5

22

19

22

(21)

1.7

16

17

5

(13)

6.7

0.015 µg

129

119

125

(124)

5.0

10

14

13

(12)

2.1

N/T

24

20

17

(20)

3.5

8

7

4

(6)

2.1

0.05 µg

130

128

115

(124)

8.1

18

7

14

(13)

5.6

N/T

14

14

18

(15)

2.3

8

5

7

(7)

1.5

0.15 µg

115

109

124

(116)

7.5

15

10

14

(13)

2.6

N/T

22

21

14

(19)

4.4

8

3

4

(5)

2.6

0.5 µg

96

116

123

(112)

14.0

8

8

10

(9)

1.2

N/T

19

17

26

(21)

4.7

3

4

4

(4)

0.6

1.5 µg

114

136

131

(127)

11.5

16

8

19

(14)

5.7

30

24

38

(31)

7.0

18

13

21

(17)

4.0

7

3

4

(5)

2.1

5 µg

105

93

100

(99)

6.0

12

11

11

(11)

0.6

25

28

32

(28)

3.5

16

19

17

(17)

1.5

7

8

7

(7)

0.6

15 µg

89 S

88 S

89 S

(89)

0.6

8 S

8 S

5 S

(7)

1.7

28

40

33

(34)

6.0

17 S

14 S

11 S

(14)

3.0

2 S

3 S

4 S

(3)

1.0

50 µg

0 V

0 V

0 V

(0)

0.0

6 S

8 S

12 S

(9)

3.1

40

39

36

(38)

2.1

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

150 µg

N/T

N/T

37

34

21

(31)

8.5

N/T

N/T

500 µg

N/T

N/T

31

32

26

(30)

3.2

N/T

N/T

1500 µg

N/T

N/T

29

22

24

(25)

3.6

N/T

N/T

5000 µg

N/T

N/T

34

46

19

(33)

13.5

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

564

352

374

(430)

116.6

173

201

213

(196)

20.5

549

522

443

(505)

55.1

324

336

341

(334)

8.7

162

86

224

(157)

69.1

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)

137

136

131

(135)

3.2#

9

12

15

(12)

3.0

37

49

47

(44)

6.4

24

24

26

(25)

1.2

14

7

12

(11)

3.6

1.5 µg

131

134

128

(131)

3.0

14

8

16

(13)

4.2

49

32

34

(38)

9.3

24

19

22

(22)

2.5

7

10

12

(10)

2.5

5 µg

134

135

138

(136)

2.1

15

13

6

(11)

4.7

35

33

40

(36)

3.6

17

30

36

(28)

9.7

5

5

5

(5)

0.0

15 µg

128

129

123

(127)

3.2

11

10

15

(12)

2.6

34

39

39

(37)

2.9

32

28

27

(29)

2.6

8

13

6

(9)

3.6

50 µg

110

127

124

(120)

9.1

9

10

9

(9)

0.6

53

36

40

(43)

8.9

27

31

26

(28)

2.6

7

5

13

(8)

4.2

150 µg

74 S

93 S

77 S

(81)

10.2

15

12

12

(13)

1.7

29

50

48

(42)

11.6

31

21

28

(27)

5.1

4 S

9 S

10 S

(8)

3.2

500 µg

0 V

0 V

0 V

(0)

0.0

10 S

14 S

5 S

(10)

4.5

38

41

40

(40)

1.5

26

22

26

(25)

2.3

0 V

0 V

0 V

(0)

0.0

1500 µg

0 V

0 V

0 V

(0)

0.0

8 S

3 S

4 S

(5)

2.6

38

34

26

(33)

6.1

20

22

19

(20)

1.5

0 V

0 V

0 V

(0)

0.0

5000 µg

0 V

0 V

0 V

(0)

0.0

7 S

7 S

14 S

(9)

4.0

33

32

40

(35)

4.4

10 S

13 S

14 S

(12)

2.1

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

1855

1919

2034

(1936)

90.7

333

319

289

(314)

22.5

229

231

232

(231)

1.5

105

126

146

(126)

20.5

346

364

367

(359)

11.4

†            Experimental procedure repeated at a later date due to toxicity in the original test

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

V           Very weak bacterial background lawn

#           Standard deviation

 

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)

102

114

115

(110)

7.2#

9

9

13

(10)

2.3

34

20

21

(25)

7.8

21

17

16

(18)

2.6

10

6

7

(8)

2.1

0.015 µg

107

115

126

(116)

9.5

8

9

9

(9)

0.6

N/T

15

25

18

(19)

5.1

9

6

7

(7)

1.5

0.05 µg

108

126

116

(117)

9.0

7

10

11

(9)

2.1

N/T

21

20

15

(19)

3.2

9

5

5

(6)

2.3

0.15 µg

117

133

118

(123)

9.0

14

10

6

(10)

4.0

N/T

20

13

26

(20)

6.5

11

12

8

(10)

2.1

0.5 µg

87

111

116

(105)

15.5

10

6

9

(8)

2.1

N/T

14

18

12

(15)

3.1

9

7

3

(6)

3.1

1.5 µg

123

123

128

(125)

2.9

7

8

13

(9)

3.2

N/T

15

19

15

(16)

2.3

6

5

3

(5)

1.5

5 µg

90

100

95

(95)

5.0

11

8

9

(9)

1.5

N/T

11

21

21

(18)

5.8

12

10

5

(9)

3.6

15 µg

82 S

87 S

83 S

(84)

2.6

12 S

15 S

7 S

(11)

4.0

28

29

23

(27)

3.2

22 S

18 S

18 S

(19)

2.3

6 S

5 S

5 S

(5)

0.6

50 µg

0 V

0 V

0 V

(0)

0.0

6 S

8 S

12 S

(9)

3.1

37

29

22

(29)

7.5

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

150 µg

N/T

N/T

31

25

21

(26)

5.0

N/T

N/T

500 µg

N/T

N/T

26

28

29

(28)

1.5

N/T

N/T

1500 µg

N/T

N/T

26

33

30

(30)

3.5

N/T

N/T

5000 µg

N/T

N/T

21

30

29

(27)

4.9

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

405

464

409

(426)

33.0

1268

1403

1552

(1408)

142.1

428

444

367

(413)

40.6

351

343

348

(347)

4.0

117

123

104

(115)

9.7

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)

109

130

119

(119)

10.5#

11

13

19

(14)

4.2

41

32

35

(36)

4.6

29

23

31

(28)

4.2

10

10

15

(12)

2.9

0.15 µg

119

130

127

(125)

5.7

N/T

N/T

N/T

7

11

9

(9)

2.0

0.5 µg

112

110

113

(112)

1.5

8

10

12

(10)

2.0

N/T

N/T

12

7

8

(9)

2.6

1.5 µg

107

145

124

(125)

19.0

13

6

17

(12)

5.6

N/T

N/T

13

6

10

(10)

3.5

5 µg

120

129

131

(127)

5.9

14

10

7

(10)

3.5

N/T

27

21

24

(24)

3.0

4

13

5

(7)

4.9

15 µg

114

128

114

(119)

8.1

8

11

4

(8)

3.5

38

37

27

(34)

6.1

26

27

28

(27)

1.0

16

17

12

(15)

2.6

50 µg

104

124

106

(111)

11.0

12

12

10

(11)

1.2

41

38

33

(37)

4.0

27

19

23

(23)

4.0

12

8

11

(10)

2.1

150 µg

0 V

0 V

0 V

(0)

0.0

8

15

7

(10)

4.4

43

36

29

(36)

7.0

18

18

14

(17)

2.3

8 S

10 S

8 S

(9)

1.2

500 µg

0 V

0 V

0 V

(0)

0.0

8 S

7 S

9 S

(8)

1.0

39

29

31

(33)

5.3

13

18

27

(19)

7.1

0 V

0 V

0 V

(0)

0.0

1500 µg

N/T

0 V

0 V

0 V

(0)

0.0

30

31

41

(34)

6.1

20

10

11

(14)

5.5

N/T

5000 µg

N/T

N/T

39

26

51

(39)

12.5

10 S

17 S

27 S

(18)

8.5

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

1863

1914

1789

(1855)

62.9

312

341

327

(327)

14.5

162

177

235

(191)

38.6

104

106

132

(114)

15.6

275

233

250

(253)

21.1

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item 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 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 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. Part of the first mutation test was repeated due to excessive toxicity (all TA-strains) dosed in the absence of S9-mix employing an amended test item dose range of 0.015 to 50 µg/plate. The experiment was repeated on a separate day 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 a minimum of four non-toxic dose levels and the toxic limit of the test item. The dose range was amended following the results of Experiment 1 and ranged between 0.015 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 or the toxic limit of the test item depending on the strain type and presence of S9-mix.The test item caused a visible reduction in the growth of the bacterial background lawns of all of the Salmonella strains dosed in the absence of S9-mix from 15 μg/plate. In the presence S9-mix, weakened bacterial background lawns were notedfor all of the Salmonella strains initially from 150 μg/plate. No toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level in either the absence or presence S9-mix. In Experiment 2, both the maximum dose level (5000 μg/plate) or the toxic limit was employed as the maximum concentration in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. The test item induced an identical toxic response to the first experiment with weakened bacterial background lawns noted from 15 μg/plate to all Salmonella strains dosed in the absence and presence of S9-mix. Again, no toxicity was noted to Escherichia coli strain WP2uvrA at any test item dose level. No precipitates were observed at any dose level 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 (S9mix). 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.