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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 July - 25 August 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 471 without any deviation

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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
dated 21 July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
dated 30 May 2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
dated August 1998
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
28 October 2016
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate
EC Number:
264-935-8
EC Name:
[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate
Cas Number:
64519-44-4
Molecular formula:
C15H25NO3
IUPAC Name:
[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate
Constituent 2
Chemical structure
Reference substance name:
[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-D-prolinate
EC Number:
268-568-4
EC Name:
[1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-D-prolinate
Cas Number:
68127-22-0
Molecular formula:
C15H25NO3
IUPAC Name:
(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl (2R)-5-oxopyrrolidine-2-carboxylate
Constituent 3
Chemical structure
Reference substance name:
L-menthol
EC Number:
218-690-9
EC Name:
L-menthol
Cas Number:
2216-51-5
Molecular formula:
C10H20O
IUPAC Name:
(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexan-1-ol
Test material form:
liquid: viscous
Remarks:
Extreme high viscous hardly flowable, very sticky, clear yellow liquid, with a ropy consistency
Details on test material:
Name: QUESTICE L
Batch no.: G16 330-1
Appearance Extreme high viscous hardly flowable, very sticky, clear yellow liquid, with a ropy consistency
Composition: [1R-(1α,2β,5α)]-2-isopropyl-5-methylcyclohexyl 5-oxo-L-prolinate : 93.5%
CAS No.: 64519-44-4
EINECS-No.: 264-935-8
Molecular formula: C15H25NO3
Molecular weight: 267.37 g/mol
Purity: 93.5% (Isomer 1)
Homogeneity: homogeneous
Production date: Nov. .2016
Expiry date: Nov. 2018
Storage: Room temp. (20+-5°C), Keep away from light and humidity, keep under inert gas;

Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer and sonication for 10 minutes at approximately 40 °C on the day of each experiment. No correction was required for purity. Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino silicate pellets with a nominal pore diameter of 4 x 10^-4 microns. All formulations were used within four hours of preparation and were assumed to be stable for this period.

Method

Target gene:
histidine locus for Salmonella strains and tryptophan for E. coli strain
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix
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 in house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
untreated
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
Untreated negative controls:
yes
Remarks:
untreated
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
Details on test system and experimental conditions:
SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from:
- University of California, Berkeley, on culture discs, on 04 August 1995
- British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation

DURATION
- Preincubation period: 20 minutes in Experiment 2.
- Exposure duration: ca. 48 hours

CONTROLS:
- Vehicle/solvent control: DMSO
- Negative (untreated) controls were performed to assess the spontaneous revertant colony rate.
- Positive control items used demonstrated a direct and indirect acting mutagenic effect depending on the presence or absence of metabolic activation.
- Sterility controls were performed in triplicate as follows:
Top agar and histidine/biotin or tryptophan in the absence of S9-mix;
Top agar and histidine/biotin or tryptophan in the presence of S9-mix; and
The maximum dosing solution of the test item in the absence of S9-mix only (test in singular only).

NUMBER OF REPLICATIONS: Triplicate

- OTHER: All of the plates were incubated at 37 ± 3 °C 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). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.
Rationale for test conditions:
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 dose range was the same as Experiment 1 (15 to 5000 µg/plate). Eight test item concentrations were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxic limit of the test item following the change in test methodology.
Evaluation criteria:
Criteria for determining a positive result:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- 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 will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
None

Results and discussion

Test results
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
refer tables 7.6.1/2 to 7.6.1/5
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

MUTAGENICITY
- 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.
- In the first experiment (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains without metabolic activation (S9), initially from 1500 µg/plate (TA100, TA98 and TA1537) and at 5000 µg/plate (TA1535 and WP2uvrA). In the presence of S9, toxicity was observed at and above 1500 µg/plate to TA98 and TA1537 and at 5000 µg/plate to strains TA100, TA1535 and WP2uvrA. Additionally, a substantial decrease in the frequency of revertant colonies was observed to TA100 at 1500 µg/plate.
In the second experiment (pre-incubation method), the test item caused a slightly stronger toxic response with visible reductions in the growth of the bacterial background lawn noted in the absence of S9 from 500 µg/plate (TA100, TA1535, WP2uvrA, and TA1537) and from 1500 µg/plate (TA98). In the presence of S9-mix, toxicity to the bacterial background lawns of all the tester strains was observed at 1500 µg/plate. Again, a substantial decrease in the frequency of revertant colonies was observed to TA100 at 500 µg/plate (presence of S9).
- 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 1 (plate incorporation method) and Experiment 2 (pre incubation method).
- Refer Tables 7.6.1/1 to 7.6.1/5 for more details.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Refer Table 7.6.1/6
- Negative (solvent/vehicle) historical control data: Refer Table 7.6.1/6

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile.
- Results for the negative controls (spontaneous mutation rates) are presented in 7.6.1/1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

Any other information on results incl. tables

Table 7.6.1/1:Spontaneous Mutation Rates (Concurrent Negative Controls)

 

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

Experiment 1

TA100

TA1535

WP2uvrA

TA98

TA1537

74

 

14

 

15

 

25

 

12

 

71

(71)

21

(16)

12

(13)

22

(23)

6

(9)

69

 

13

 

13

 

23

 

8

 

Experiment 2

TA100

TA1535

WP2uvrA

TA98

TA1537

75

 

20

 

16

 

28

 

14

 

66

(73)

19

(20)

21

(19)

16

(20)

12

(11)

78

 

21

 

20

 

17

 

8

 

 

Table 7.6.1/2:Test Results: Experiment 1 – Without Metabolic Activation

 

Test Period

From: 08 August 2017

To: 11 August 2017

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)

63

68

80

(70)

8.7#

16

18

18

(17)

1.2

22

22

19

(21)

1.7

35

29

35

(33)

3.5

8

13

12

(11)

2.6

1.5 µg

60

68

70

(66)

5.3

16

22

23

(20)

3.8

17

19

20

(19)

1.5

27

26

31

(28)

2.6

9

16

9

(11)

4.0

5 µg

72

73

94

(80)

12.4

24

22

18

(21)

3.1

20

13

16

(16)

3.5

27

27

25

(26)

1.2

23

14

8

(15)

7.5

15 µg

83

61

80

(75)

11.9

19

21

17

(19)

2.0

20

21

15

(19)

3.2

25

22

22

(23)

1.7

12

19

8

(13)

5.6

50 µg

76

80

75

(77)

2.6

12

8

14

(11)

3.1

13

14

17

(15)

2.1

25

25

17

(22)

4.6

13

13

10

(12)

1.7

150 µg

84

69

88

(80)

10.0

14

25

14

(18)

6.4

11

16

15

(14)

2.6

18

19

17

(18)

1.0

10

12

18

(13)

4.2

500 µg

72

73

62

(69)

6.1

21

14

14

(16)

4.0

11

15

19

(15)

4.0

20

22

14

(19)

4.2

7

22

10

(13)

7.9

1500 µg

54 S

61 S

47 S

(54)

7.0

20

18

22

(20)

2.0

17

16

17

(17)

0.6

12 S

12 S

21 S

(15)

5.2

0 V

0 V

0 V

(0)

0.0

5000 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

7 S

5 S

5 S

(6)

1.2

0 V

0 V

0 V

(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

547

472

(489)

51.6

317

374

477

(389)

81.1

531

543

570

(548)

20.0

161

176

178

(172)

9.3

158

273

236

(222)

58.7

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

S:       Sparse bacterial background lawn

V:       Very weak bacterial background lawn

T:       Toxic, no bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/3:Test Results: Experiment 1 – With Metabolic Activation

 

Test Period

From: 08 August 2017

To: 11 August 2017

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)

65

90

88

(81)

13.9#

11

24

13

(16)

7.0

27

27

15

(23)

6.9

26

28

27

(27)

1.0

10

6

15

(10)

4.5

1.5 µg

82

97

77

(85)

10.4

27

26

10

(21)

9.5

25

19

19

(21)

3.5

29

26

29

(28)

1.7

11

17

12

(13)

3.2

5 µg

68

81

72

(74)

6.7

22

20

16

(19)

3.1

12

19

28

(20)

8.0

26

27

28

(27)

1.0

14

15

2

(10)

7.2

15 µg

84

109

70

(88)

19.8

12

17

28

(19)

8.2

25

22

19

(22)

3.0

21

27

27

(25)

3.5

4

12

15

(10)

5.7

50 µg

74

82

84

(80)

5.3

23

15

20

(19)

4.0

20

22

13

(18)

4.7

34

12

18

(21)

11.4

16

20

6

(14)

7.2

150 µg

66

72

66

(68)

3.5

12

19

19

(17)

4.0

17

18

20

(18)

1.5

24

25

13

(21)

6.7

11

8

10

(10)

1.5

500 µg

62

61

64

(62)

1.5

14

11

14

(13)

1.7

18

22

21

(20)

2.1

24

24

23

(24)

0.6

16

8

10

(11)

4.2

1500 µg

45

59

47

(50)

7.6

16

16

19

(17)

1.7

25

16

23

(21)

4.7

18 S

11 S

12 S

(14)

3.8

8 S

8 S

8 S

(8)

0.0

5000 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

17 S

22 S

16 S

(18)

3.2

0 V

0 V

0 V

(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

1129

1130

1390

(1216)

150.4

234

244

202

(227)

21.9

111

81

87

(93)

15.9

188

155

175

(173)

16.6

338

308

265

(304)

36.7

2AA:   2-Aminoanthracene

BP:     Benzo(a)pyrene

S:       Sparse bacterial background lawn

V:       Very weak bacterial background lawn

T:        Toxic, no bacterial background lawn

#:       Standard deviation

 

 

Table 7.6.1/4:Test Results: Experiment 2 – Without Metabolic Activation

 

Test Period

From: 22 August 2017

To: 25 August 2017

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)

77

70

66

(71)

5.6#

23

15

21

(20)

4.2

20

19

21

(20)

1.0

15

13

20

(16)

3.6

11

14

16

(14)

2.5

1.5 µg

79

68

67

(71)

6.7

23

18

16

(19)

3.6

12

18

25

(18)

6.5

12

26

20

(19)

7.0

13

8

9

(10)

2.6

5 µg

64

71

76

(70)

6.0

18

18

20

(19)

1.2

14

13

18

(15)

2.6

22

22

17

(20)

2.9

13

12

9

(11)

2.1

15 µg

78

64

64

(69)

8.1

14

22

24

(20)

5.3

21

28

21

(23)

4.0

17

21

14

(17)

3.5

13

8

19

(13)

5.5

50 µg

76

61

68

(68)

7.5

26

24

25

(25)

1.0

17

21

17

(18)

2.3

24

17

23

(21)

3.8

13

10

15

(13)

2.5

150 µg

61

72

69

(67)

5.7

11

22

17

(17)

5.5

26

20

19

(22)

3.8

24

21

22

(22)

1.5

10

6

18

(11)

6.1

500 µg

61 S

50 S

47 S

(53)

7.4

16 S

17 S

17 S

(17)

0.6

20 S

9 S

11 S

(13)

5.9

14

17

17

(16)

1.7

4 S

5 S

3 S

(4)

1.0

1500 µg

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

11 S

14 S

25 S

(17)

7.4

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(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

686

770

706

(721)

43.9

353

416

455

(408)

51.5

760

695

863

(773)

84.7

235

280

250

(255)

22.9

246

308

332

(295)

44.4

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

4NQO:4-Nitroquinoline-1-oxide

9AA:   9-Aminoacridine

S:       Sparse bacterial background lawn

V:       Very weak bacterial background lawn

T:       Toxic, no bacterial background lawn

#:       Standard deviation

 

Table 7.6.1/5:Test Results: Experiment 2 – With Metabolic Activation

 

Test Period

From: 22 August 2017

To: 25 August 2017

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)

71

70

69

(70)

1.0#

12

17

17

(15)

2.9

25

24

28

(26)

2.1

29

22

25

(25)

3.5

17

14

14

(15)

1.7

1.5 µg

67

66

63

(65)

2.1

21

17

18

(19)

2.1

22

38

22

(27)

9.2

20

24

25

(23)

2.6

17

9

11

(12)

4.2

5 µg

64

74

64

(67)

5.8

25

23

17

(22)

4.2

28

37

28

(31)

5.2

18

15

27

(20)

6.2

17

5

9

(10)

6.1

15 µg

63

69

68

(67)

3.2

22

22

22

(22)

0.0

28

21

36

(28)

7.5

18

12

25

(18)

6.5

10

18

19

(16)

4.9

50 µg

68

68

64

(67)

2.3

22

24

12

(19)

6.4

29

18

22

(23)

5.6

25

32

29

(29)

3.5

10

8

6

(8)

2.0

150 µg

73

64

70

(69)

4.6

13

18

24

(18)

5.5

20

21

20

(20)

0.6

24

20

24

(23)

2.3

13

25

13

(17)

6.9

500 µg

73

50

38

(54)

17.8

26

19

26

(24)

4.0

23

21

23

(22)

1.2

19

23

16

(19)

3.5

16

8

12

(12)

4.0

1500 µg

50 V

0 V

0 V

(17)

28.9

11 S

12 S

11 S

(11)

0.6

16 S

10 S

18 S

(15)

4.2

0 V

0 V

0 V

(0)

0.0

0 V

0 V

0 V

(0)

0.0

5000 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

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

970

923

1076

(990)

78.4

231

279

241

(250)

25.3

197

140

246

(194)

53.1

137

128

163

(143)

18.2

470

395

425

(430)

37.7

2AA:   2-Aminoanthracene

BP:      Benzo(a)pyrene

S:       Sparse bacterial background lawn

V:       Very weak bacterial background lawn

T:       Toxic, no bacterial background lawn

#:       Standard deviation

 

 

Table 7.6.1/6:History Profile of Vehicle and Positive Control Values

 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2015

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Values†

274

278

504

285

26

13

461

229

526

299

506

282

42

51

39

49

Min

60

61

7

7

222

278

10

12

11

10

4

6

87

98

89

93

Max

166

175

31

29

376

388

58

43

45

46

27

27

237

254

174

177

Mean

91

95

16

14

286

333

24

27

21

24

12

13

156

164

123

137

SD

19.3

19.1

4.5

4.0

48.7

37.6

5.6

5.9

6.2

6.1

3.8

3.4

42.2

35.6

23.1

21.2

POSITIVE CONTROL VALUES 2015

 

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

Values

276

280

252

264

13

13

231

227

262

276

253

261

20

35

20

35

 

Min

222

250

79

118

953

673

116

103

100

78

164

97

430

494

745

325

 

Max

2266

2402

2779

457

3140

1655

2769

550

502

705

2318

823

1696

2264

3662

1174

 

Mean

614

927

472

246

2303

1093

792

266

222

218

911

336

761

1461

2257

569

 

SD

260.6

452.5

434.8

55.7

815.2

376.5

342.1

97.7

70.2

107.6

412.4

135.7

350.0

382.0

790.7

220.3

 

COMBINED VEHICLE AND UNTREATED CONTROL VALUES 2016

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Values

399

401

758

393

60

30

690

345

788

415

762

398

32

32

16

24

Min

63

66

8

8

216

221

10

13

8

12

3

4

97

104

78

52

Max

154

156

34

39

340

375

53

53

49

51

24

23

268

243

148

166

Mean

90

93

15

15

268

310

22

27

21

25

12

13

161

159

118

110

SD

14.5

14.3

4.5

5.2

26.4

31.1

5.8

6.3

4.8

5.7

3.5

3.5

39.2

32.3

17.0

29.3

POSITIVE CONTROL VALUES 2016

 

Strain

S9-Mix

TA100

TA1535

TA102

WP2uvrA

TA98

TA1537

WP2uvrA

pKM101

WP2pKM101

 

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

 

Values

409

406

381

386

30

28

341

335

388

385

379

381

14

24

8

16

 

Min

221

284

84

92

897

629

107

102

100

96

95

101

445

574

1674

372

 

Max

2222

2863

2994

879

2326

2140

1611

637

449

4357

1413

639

1117

1855

2823

945

 

Mean

724

1264

854

240

1633

950

718

240

186

188

406

290

743

1271

2379

535

 

SD

320.4

562.9

664.9

62.1

564.5

382.7

338.6

98.2

49.8

230.8

227.0

92.7

214.6

326.5

426.2

143.3

 

SD: Standard deviation

Min: Minimum value

Max: Maximum value

†: Number of mean values used to create dataset

Applicant's summary and conclusion

Conclusions:
Under the test conditions, the test item is not considered as mutagenic in Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:

- Experiment 1 - Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

- Experiment 2 - Pre-Incubation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate, with and without S9-mix

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

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.

In the first experiment (plate incorporation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains both with and without metabolic activation (S9), initially from 1500 µg/plate.

In the second experiment (pre-incubation), the test item caused a visible reduction in the growth of the bacterial background lawn in all of the tester strains, initially from 500 and 1500 µg/plate in the absence and presence of 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 S9-mix.

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 1 (plate incorporation method) and Experiment 2 (pre incubation method).

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.