Registration Dossier

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:
2015
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:
2016
Report Date:
2016

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 2016
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
gas under pressure: refrigerated liquefied gas
Details on test material:
- Physical state: liquid
- Storage condition of test material: Approximately 4 °C in the dark.
- Other: Clear colourless liquid

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 (pre-incubation method): 0, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Experiment 2 (pre-incubation method):
Details are provided in the following tables given in 'any other information on results incl. tables'.
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 limit of the test item.
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. Dimethyl sulphoxide was selected as the vehicle. No correction was made for purity.
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 and 2. in medium; in agar (plate incorporation)

DURATION
- Exposure duration:
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 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 interference in the base agar e.g. minor precipitation of salts/dust particles and marks on the base of the plates slightly distorting the counts

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:
and or tested up to guideline limit concentrations
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:
and or tested up to guideline limit concentrations
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

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

4NQO: 4-Nitroquinoline-1-oxide

9AA: 9-Aminoacridine

BP: Benzo(a)pyrene

N/T : Not tested at this dose level

2AA: 2-Aminoanthracene

S: Sparse bacterial background lawn

V: Very weak bacterial background lawn

**: p < or = 0.01

#: Standard deviation

 

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)

69

75

67

(70)

4.2#

10

14

9

(11)

2.6

19

15

11

(15)

4.0

13

17

13

(14)

2.3

5

5

15

(8)

5.8

1.5 µg

80

65

70

(72)

7.6

13

9

13

(12)

2.3

12

17

9

(13)

4.0

19

11

17

(16)

4.2

9

13

12

(11)

2.1

5 µg

66

61

65

(64)

2.6

13

15

16

(15)

1.5

9

16

12

(12)

3.5

21

8

20

(16)

7.2

7

7

12

(9)

2.9

15 µg

63

69

60

(64)

4.6

9

16

15

(13)

3.8

19

25

15

(20)

5.0

13

20

23

(19)

5.1

8

11

5

(8)

3.0

50 µg

61

61

60

(61)

0.6

13

9

11

(11)

2.0

20

17

21

(19)

2.1

19

13

13

(15)

3.5

4

4

4

(4)

0.0

150 µg

64

66

62

(64)

2.0

43 S

67 S

14 S

**

(41)

26.5

15

12

15

(14)

1.7

19

16

8

(14)

5.7

3

3

5

(4)

1.2

500 µg

62

64

62

(63)

1.2

10 S

7 S

5 S

(7)

2.5

20

11

15

(15)

4.5

15

13

5

(11)

5.3

3 S

7 S

3 S

(4)

2.3

1500 µg

65 S

66 S

61 S

(64)

2.6

0 V

0 V

0 V

(0)

0.0

16

12

15

(14)

2.1

8 S

12 S

5 S

(8)

3.5

1 S

3 S

4 S

(3)

1.5

5000 µg

43 S

49 S

65 S

(52)

11.4

0 V

0 V

0 V

(0)

0.0

13 S

20 S

20 S

(18)

4.0

12 S

15 S

13 S

(13)

1.5

0 V

0 V

0 V

(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

438

314

330

(361)

67.4

519

445

652

(539)

104.9

337

326

310

(324)

13.6

103

86

126

(105)

20.1

231

184

335

(250)

77.3

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Solvent Control

(DMSO)

80

72

61

(71)

9.5#

14

10

11

(12)

2.1

27

15

27

(23)

6.9

11

10

14

(12)

2.1

8

9

7

(8)

1.0

1.5 µg

71

74

60

(68)

7.4

11

11

8

(10)

1.7

31

35

27

(31)

4.0

12

13

14

(13)

1.0

8

13

5

(9)

4.0

5 µg

61

65

65

(64)

2.3

13

12

8

(11)

2.6

24

21

15

(20)

4.6

11

19

21

(17)

5.3

12

4

3

(6)

4.9

15 µg

71

69

63

(68)

4.2

10

11

9

(10)

1.0

20

28

25

(24)

4.0

20

11

20

(17)

5.2

5

9

3

(6)

3.1

50 µg

69

69

66

(68)

1.7

12

15

9

(12)

3.0

12

24

29

(22)

8.7

15

13

10

(13)

2.5

5

7

17

(10)

6.4

150 µg

71

73

61

(68)

6.4

16

10

17

(14)

3.8

12

25

19

(19)

6.5

13

20

12

(15)

4.4

4

7

5

(5)

1.5

500 µg

65

60

61

(62)

2.6

12 S

11 S

13 S

(12)

1.0

23

23

17

(21)

3.5

25 S

17 S

16 S

*

(19)

4.9

7

3

11

(7)

4.0

1500 µg

60 S

50 S

50 S

(53)

5.8

0 V

0 V

0 V

(0)

0.0

12 S

15 S

23 S

(17)

5.7

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

5000 µg

44 S

50 S

45 S

(46)

3.2

0 V

0 V

0 V

(0)

0.0

15 S

5 S

8 S

(9)

5.1

0 V

0 V

0 V

(0)

0.0

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

235

237

278

(250)

24.3

152

134

214

(167)

42.0

195

265

315

(258)

60.3

98

79

96

(91)

10.4

204

159

168

(177)

23.8

 

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)

94

76

89

(86)

9.3#

7

12

8

(9)

2.6

27

35

19

(27)

8.0

12

11

12

(12)

0.6

7

6

9

(7)

1.5

0.15 µg

N/T

11

9

15

(12)

3.1

N/T

N/T

N/T

0.5 µg

N/T

9

12

7

(9)

2.5

N/T

N/T

9

6

8

(8)

1.5

1.5 µg

74

76

84

(78)

5.3

7

17

11

(12)

5.0

23

28

20

(24)

4.0

11

16

20

(16)

4.5

9

7

12

(9)

2.5

5 µg

68

72

72

(71)

2.3

7

7

11

(8)

2.3

16

24

19

(20)

4.0

17

21

9

(16)

6.1

9

8

6

(8)

1.5

15 µg

59

77

66

(67)

9.1

14

14

16

*

(15)

1.2

20

19

19

(19)

0.6

16

14

10

(13)

3.1

3

4

8

(5)

2.6

50 µg

61

65

67

(64)

3.1

17 S

15 S

12 S

(15)

2.5

21

9

17

(16)

6.1

8

9

9

(9)

0.6

5

3

6

(5)

1.5

150 µg

48

59

58

(55)

6.1

11 S

12 S

14 S

(12)

1.5

20

13

19

(17)

3.8

9

8

11

(9)

1.5

3 S

2 S

0 S

(2)

1.5

500 µg

39

48

49

(45)

5.5

11 V

13 V

12 V

(12)

1.0

11

21

13

(15)

5.3

11 S

12 S

8 S

(10)

2.1

2 S

3 S

4 S

(3)

1.0

1500 µg

33 S

56 S

48 S

(46)

11.7

N/T

11

23

7

(14)

8.3

11 S

9 S

8 S

(9)

1.5

2 S

3 S

5 S

(3)

1.5

5000 µg

42 S

27 S

40 S

(36)

8.1

N/T

16 S

13 S

20 S

(16)

3.5

6 S

10 S

10 S

(9)

2.3

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

428

470

404

(434)

33.4

600

521

577

(566)

40.6

358

319

382

(353)

31.8

262

204

261

(242)

33.2

299

564

500

(454)

138.3

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Solvent Control

(DMSO)

90

91

89

(90)

1.0#

9

9

7

(8)

1.2

41

39

48

(43)

4.7

16

19

12

(16)

3.5

11

6

13

(10)

3.6

0.5 µg

N/T

6

8

10

(8)

2.0

N/T

21

14

16

(17)

3.6

10

12

8

(10)

2.0

1.5 µg

83

91

96

(90)

6.6

6

6

8

(7)

1.2

20

32

32

(28)

6.9

18

22

24

(21)

3.1

6

10

7

(8)

2.1

5 µg

87

84

90

(87)

3.0

8

13

8

(10)

2.9

32

36

19

(29)

8.9

14

17

19

(17)

2.5

7

11

8

(9)

2.1

15 µg

85

67

80

(77)

9.3

13

6

8

(9)

3.6

19

36

27

(27)

8.5

14

18

19

(17)

2.6

9

15

6

(10)

4.6

50 µg

84

83

69

(79)

8.4

7

14

7

(9)

4.0

35

20

31

(29)

7.8

14

17

12

(14)

2.5

8

6

5

(6)

1.5

150 µg

84

82

79

(82)

2.5

10

11

6

(9)

2.6

39

20

28

(29)

9.5

13

20

25

(19)

6.0

10

7

6

(8)

2.1

500 µg

65 S

55 S

63 S

(61)

5.3

15 S

7 S

12 S

(11)

4.0

15

21

13

(16)

4.2

16 S

10 S

13 S

(13)

3.0

3

4

4

(4)

0.6

1500 µg

35 S

38 S

45 S

(39)

5.1

0 V

0 V

0 V

(0)

0.0

12 S

8 S

5 S

(8)

3.5

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

5000 µg

51 S

51 S

51 S

(51)

0.0

N/T

21 S

11 S

12 S

(15)

5.5

N/T

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

331

382

343

(352)

26.7

156

139

146

(147)

8.5

210

182

214

(202)

17.4

96

104

110

(103)

7.0

174

211

212

(199)

21.7

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
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 item at 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). Since the test item had unknown volatility all testing was performed using the pre-incubation modification. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended slightly, based on these results. Eight test item dose levels were again selected in Experiment 2 in order to achieve both a minimum of four non-toxic dose levels. 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. 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, the test item induced avisible reduction in the growth of the bacterial background lawns and/or a substantial reduction in revertant colony frequency to all of the tester strains, initially from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence of S9-mix. Consequently the same maximum dose level (5000 μg/plate) or the toxic limit was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA1535), 150 μg/plate (TA1537), 500 μg/plate (TA98), 1500 μg/plate (TA100) and at 5000 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns and/or substantial reductions in revertant colony frequency were noted to all of the tester strains from 500 μg/plate. 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. Small, statistically significant increase in TA1535 revertant colony frequency were noted in the absence of S9-mix at 150 μg/plate and to TA98 at 500 μg/plate in the presence of S9-mix. These increases were considered to have no biological relevance because weakened bacterial background lawns were also noted at the same concentrations. Similarly, no toxicologically 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. A small, statistically significant increase in TA1535 revertant colony frequency was also observed in the absence of S9-mix at 15 μg/plate in this test. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. the individual revertant colony counts at 15 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.6 times the concurrent vehicle control. Under the conditions of this study, the test substance was considered non-mutagenic.