Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3 September 2014 - 2 October 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

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
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: bulk
Specific details on test material used for the study:
- Name of test material (as cited in study report): 4-amino-5-ethylsulfonyl-2-methoxybenzoic acid
- Physical state: Solid
- Analytical purity: 99.81%
- Lot/batch No.: MP1032.31
- Expiration date of the lot/batch: 16 June 2017

Method

Target gene:
Histidine-requiring gene in Salmonella typhimurium and Tryptophan-requiring gene in the strain of Escherichia coli.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: rfa, uvrB
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA
Metabolic activation:
with and without
Metabolic activation system:
S9 mix from rat liver
Test concentrations with justification for top dose:
5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO.
- Justification for choice of solvent/vehicle: The solubility of the test item was examined in Distilled water, Dimethyl sulfoxide (DMSO) and Acetone as vehicles. The test item was insoluble at 100 mg/mL concentration in Distilled water. However, it was soluble at the same concentration using DMSO or Acetone as vehicles (after 5 minutes of vortex). Due to the better biocompatibility to the test system, DMSO was selected as vehicle (solvent) for the study. The behaviour of the obtained test item formulation (50 μL) with the solution of top agar and phosphate buffer was examined in a test tube without test bacterium suspension to examine the formulation compatibility (Preliminary Compatibility Test). The selected vehicle was compatible with the survival of the cells and the S9 activity.

Controls
Untreated negative controls:
yes
Remarks:
untreated control - used demonstrating that the chosen vehicle induced no deleterious or mutagenic effects
Negative solvent / vehicle controls:
yes
Remarks:
(DMSO and distilled water)
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-nitro-1,2-phenylenediamine (NPD), 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation (initial mutation test):
Molten top agar was prepared and kept at 45°C. 2 mL of top agar was aliquoted into individual test tubes (3 tubes per control or concentration level).The
equivalent number of minimal glucose agar plates was properly labelled. The test item (or controls) and other components (top agar, overnight culture of test
strain, phosphate buffer (pH 7.4) or S9 mix) were prepared freshly and added to the overlay (45°C). This solution was mixed and poured on the surface of
minimal agar plates. After preparation, the plates were incubated at 37°C for 48 hours.

Pre-incubation (confirmatory mutation test):
Before the overlaying, the test item formulation (or vehicle/solvent or reference control), the bacterial culture and the S9 mix or phosphate buffer was added into
appropriate tubes to provide direct contact between bacteria and the test item (in its vehicle/solvent). The tubes (3 tubes per control or concentration level) were
gently mixed and incubated for 20 min at 37ºC in a shaking incubator. After the incubation period, 2 mL of molten top agar was added to the tubes; the content
was mixed up and poured onto minimal glucose agar plates as described for the standard plate incorporation method. After preparation, the plates were
incubated at 37°C for 48 hours.

NUMBER OF REPLICATIONS: 3 replicates per dose and controls.

EVALUATION OF EXPERIMENTAL DATA:
The colony numbers were determined by manual counting.
Visual examination of the plates was performed, precipitation or signs of growth inhibition (if any) were recorded.
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated.
Evaluation criteria:
CRITERIA FOR A POSITIVE RESPONSE
A test item was considered mutagenic if:
- a dose–related increase in the number of revertants occurred and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurred in at least one strain with or without metabolic activation.
An increase was considered biologically relevant if:
- the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
- the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

CRITERIA FOR A NEGATIVE RESPONSE
A test article was considered non-mutagenic if it produced neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
The mean number of revertants per plate, the standard deviation and the mutation factor values were calculated for each concentration level of the test item and for the controls using Microsoft ExcelTM software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(Confirmatory Mutation Test in TA1537 strain at 5000 μg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES: The numbers of revertant colonies were mostly in the normal range (minor differences were detected in some sporadic cases, but they were without biological significance and considered as biological variability of the test system). No precipitate or signs of inhibitory, cytotoxic effect were detected on the plates in the preliminary experiment.

COMPARISON WITH HISTORICAL CONTROL DATA: The mean values of revertant colony numbers of untreated, negative (solvent) and positive control plates were within the historical control range in all strains. see below

Positive contrrol data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 331.5 1369.2 1230.9 524.3 1107.4 2458.9 2472.1 239.3 229.8 266.5
St. dev. 81.9 230.3 238.4 244.4 154.3 437.0 427.8 198.7 79.7 52.2
Range 152-1224 912-2120 208-2440 592-1708 592-1708 496-4918 1192-5240 125-2216 117-838 125-496
n 549 546 549 543 546 546 540 543 543 546

Untreated control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 23.4 105.8 9.5 6.9 31.1 30.8 116.6 11.2 8.8 39.1
St. dev. 6.2 28.2 4.5 3.5 10.0 7.2 24.8 3.8 3.7 10.2
Range 9-46 54-210 1-46 1-24 11-76 10-56 65-204 3-35 1-28 16-81
n 549 546 549 543 546 546 540 543 543 546

DMSO control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 22.1 102.4 9.8 7.1 29.8 29.9 1118.0 11.4 9.0 38.5
St. dev. 6.5 28.1 4.3 3.7 9.5 7.4 25.3 3.7 4.0 10.1
Range 9-55 40-217 1-43 1-25 7-81 11-67 62-229 3-24 2-29 18-84
n 558 555 558 552 555 554 549 551 552 555

Distilled water control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 23.8 106.9 9.7 7.8 31.9 31.5 120.3 11.1 9.4 40.5
St. dev. 6.2 30.4 4.4 3.4 9.9 7.5 27.2 3.7 3.5 9.7
Range 13-43 45-215 2-47 2-24 12-77 10-50 70-222 3-39 1-20 18-91
n 129 546 549 129 549 129 546 549 129 552

Acetone control data:
without metabolic activation (-S9 Mix) with metabolic activation (+S9 Mix)
TA98 TA100 TA1535 TA1537 E.COLI TA98 TA100 TA1535 TA1537 E.COLI
Mean 331.5 1369.2 1230.9 524.3 1107.4 2458.9 2472.1 239.3 229.8 266.5
St. dev. 81.9 230.3 238.4 244.4 154.3 437.0 427.8 198.7 79.7 52.2
Range 152-1224 912-2120 208-2440 592-1708 592-1708 496-4918 1192-5240 125-2216 117-838 125-496
n 549 546 549 543 546 546 540 543 543 546

n: number of cases

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effect of the test item (reduced background lawn and reduced revertant counts) was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 bacterial strain at 5000 μg/plate concentration without metabolic activation.


Any other information on results incl. tables

 Table No.4 Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA98

Cell count (overnight culture): 2.71 x 109 CFU/mL

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

19

33

20

38

29

40

22.7

37.0

5.51

3.61

1.01

1.16

 

1581

 

MEAN

SD

MF

23

28

28

36

28

41

26.3

35.0

2.89

6.56

1.18

1.09

 

500

 

MEAN

SD

MF

25

32

25

36

27

45

25.7

37.7

1.15

6.66

1.15

1.18

 

158.1

 

MEAN

SD

MF

18

30

19

36

20

38

19.0

34.7

1.00

4.16

0.85

1.08

 

50

 

MEAN

SD

MF

21

35

24

37

25

42

23.3

38.0

2.08

3.61

1.04

1.19

 

15.81

 

MEAN

SD

MF

22

37

23

39

29

39

24.7

38.3

3.79

1.15

1.10

1.20

 

5

 

MEAN

SD

MF

16

27

23

30

25

34

21.3

30.3

4.73

3.51

0.96

0.95

 

Untreated control

 

 

MEAN

SD

MF

27

36

30

27

20

33

25.7

32.0

5.13

4.58

1.15

1.00

 

DMSO control

 

 

MEAN

SD

MF

25

33

21

30

21

33

22.3

32.0

2.31

1.73

1.00

1.00

Positive control NPD (4ug)

 

 

 

MEAN

SD

MF

372

2488

320

2524

384

2480

358.7

2497.3

34.02

23.44

16.06

78.04

Table No.5: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA100

Cell count (overnight culture): 3.19 x 109 CFU/mL

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

83

98

117

95

107

115

102.3

102.7

17.47

10.79

0.97

0.95

 

1581

 

MEAN

SD

MF

109

115

92

101

109

111

103.3

109.0

9.81

7.21

0.98

1.01

 

500

 

MEAN

SD

MF

108

106

100

93

112

92

106.7

97.0

6.11

7.81

1.02

0.9

 

158.1

 

MEAN

SD

MF

99

99

97

89

116

100

104.0

96.0

10.44

6.08

0.99

0.89

 

50

 

MEAN

SD

MF

105

98

85

86

109

86

99.7

90.0

12.86

6.93

0.95

0.83

 

15.81

 

MEAN

SD

MF

105

101

118

104

85

97

102.7

100.7

16.62

3.51

0.98

0.93

 

5

 

MEAN

SD

MF

101

104

94

87

114

98

103.0

96.3

10.15

8.62

0.98

0.89

 

Untreated control

 

 

MEAN

SD

MF

111

102

121

102

131

121

121.0

108.3

10.00

10.97

1.15

1.00

 

DMSO control

 

 

MEAN

SD

MF

86

104

108

106

121

114

105.0

108.0

17.69

5.29

1.00

1.00

Distilled water control*

 

 

 

MEAN

SD

MF

106

103

116

113

117

115

105.0

110.3

17.69

6.43

1.00

1.02

Positive control

SAZ (2ug)

 

MEAN

SD

MF

1188                       2AA

2412

1204                       (2ug)

2424

1212

2488

1201.3

2441.3

12.22

40.86

10.63

22.60

*Distilled water control was used because of SAZ

Table No.6: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA1535

Cell count (overnight culture): 2.03 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

14

11

18

15

19

13

17.0

13.0

2.65

2.00

0.85

1.18

 

1581

 

MEAN

SD

MF

16

14

14

7

17

13

15.7

11.3

1.53

3.79

0.78

1.03

 

500

 

MEAN

SD

MF

11

16

15

9

19

11

15.00

12.0

4.00

3.61

0.75

1.09

 

158.1

 

MEAN

SD

MF

18

10

11

11

15

10

14.7

10.3

3.51

0.58

0.73

0.94

 

50

 

MEAN

SD

MF

10

11

13

8

21

10

14.7

9.7

5.69

1.53

0.73

0.88

 

15.81

 

MEAN

SD

MF

17

14

15

15

14

7

15.3

12.0

1.53

4.36

0.77

1.09

 

5

 

MEAN

SD

MF

17

6

16

14

13

11

15.3

10.3

2.08

4.04

0.77

0.94

 

Untreated control

 

 

MEAN

SD

MF

16

8

16

13

11

11

14.3

10.7

2.89

2.52

0.72

0.97

 

DMSO control

 

 

MEAN

SD

MF

20

11

19

13

21

9

20.0

11.0

1.00

2.00

1.00

1.00

Distilled water control*

 

 

 

MEAN

SD

MF

18

9

16

13

17

17

17.00

13.0

1.00

4.00

0.85

1.18

Positive control

SAZ (2ug)

 

MEAN

SD

MF

1200                   2AA

208

1100                    2ug

214

1050

101

1116.7

174.3

76.38

63.58

65.69

15.85

Table No.7: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Salmonella typhimurium TA1537

Cell count (overnight culture): 2.47 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

5

4

3

9

10

16

6.0

9.7

3.61

6.03

0.64

1.04

 

1581

 

MEAN

SD

MF

7

6

7

7

7

6

7.0

6.3

0.00

0.58

0.75

0.95

 

500

 

MEAN

SD

MF

6

10

6

8

5

9

5.7

9.0

0.58

1.00

0.61

1.35

 

158.1

 

MEAN

SD

MF

6

6

4

7

7

8

5.7

7.0

1.53

1.00

0.61

1.05

 

50

 

MEAN

SD

MF

9

7

8

6

8

5

8.3

6.00

0.58

1.00

0.89

0.90

 

15.81

 

MEAN

SD

MF

6

5

7

6

6

7

6.3

6.0

0.58

1.00

0.68

0.9

 

5

 

MEAN

SD

MF

7

10

3

11

3

6

4.3

9.0

2.31

2.65

0.46

1.35

 

Untreated control

 

 

MEAN

SD

MF

6

8

8

8

9

6

7.7

7.3

1.53

1.15

0.82

1.10

 

DMSO control

 

 

MEAN

SD

MF

8

5

6

8

14

7

9.3

6.7

4.16

1.53

1.00

1.00

Positive control

9AA (50ug)

 

 

MEAN

SD

MF

404                     2AA

201

396                      (2ug)

197

352

204

384.0

200.7

28.00

3.51

41.14

30.10

Table No.8: Initial Mutation Test (Plate Incorporation Method)

16-18 September 2014

Strain: Escherichia coli WP2 uvrA

Cell count (overnight culture): 3.03 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

35

35

48

43

31

42

38.0

40.0

8.89

4.36

0.95

1.09

 

1581

 

MEAN

SD

MF

28

49

42

45

39

37

36.3

43.7

7.37

6.11

0.91

1.19

 

500

 

MEAN

SD

MF

37

45

30

32

39

34

35.3

37.0

4.73

7.00

0.88

1.01

 

158.1

 

MEAN

SD

MF

34

32

34

41

52

53

40.0

42.0

10.39

10.54

1.00

1.15

 

50

 

MEAN

SD

MF

29

45

36

46

38

39

34.3

43.3

4.73

3.79

0.86

1.18

 

15.81

 

MEAN

SD

MF

43

38

39

51

36

43

39.3

44.0

3.51

6.56

0.98

1.20

 

5

 

MEAN

SD

MF

26

35

30

46

42

36

32.7

39.0

8.33

6.08

0.82

1.06

 

Untreated control

 

 

MEAN

SD

MF

35

31

32

39

35

35

34.0

35.0

1.73

4.00

0.85

0.95

 

DMSO control

 

 

MEAN

SD

MF

43

43

32

33

45

34

40.0

36.7

7.00

5.51

1.00

1.00

Distilled water control*

 

 

MEAN

SD

MF

38

36

36

37

35

41

36.3

38.0

1.53

2.65

0.91

1.04

Positive control

MMS (2uL)

 

MEAN

SD

MF

1140             2AA

321

1124             (50ug)

298

1240

314

1168.0

311.0

62.86

11.79

32.15

8.48

*Distilled water control was used because of MMS

Table No.9: Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimurium TA98

Cell count (overnight culture): 2.20 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

28

32

20

31

23

32

23.7

31.7

4.04

0.58

1.08

1.04

 

1581

 

MEAN

SD

MF

26

38

22

27

14

33

20.7

32.7

6.11

5.51

0.94

1.08

 

500

 

MEAN

SD

MF

19

32

17

31

23

38

19.7

33.7

3.06

3.79

0.89

1.11

 

158.1

 

MEAN

SD

MF

19

30

35

38

22

36

25.3

34.7

8.50

4.16

1.15

1.14

 

50

 

MEAN

SD

MF

27

33

22

35

22

36

23.7

34.7

2.89

1.53

1.08

1.14

 

15.81

 

MEAN

SD

MF

27

34

28

38

24

39

26.3

37.0

2.08

2.65

1.20

1.22

 

5

 

MEAN

SD

MF

26

26

22

36

22

34

23.3

32.0

2.31

5.29

1.06

1.05

 

Untreated control

 

 

MEAN

SD

MF

23

27

23

30

21

34

22.3

30.3

1.15

3.51

1.02

1.00

 

DMSO control

 

 

MEAN

SD

MF

24

25

20

35

22

31

22.0

30.3

2.00

5.03

1.00

1.00

Positive control

NPD (4ug)

 

MEAN

SD

MF

392                  2AA

2416

384                  (2ug)

2380

420

2424

398.7

2406.7

18.90

23.44

18.12

79.34

Table No.10: Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimurium TA100

Cell count (overnight culture): 1.90 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

98

130

98

98

97

109

97.7

112.3

0.58

16.26

1.09

1.14

 

1581

 

MEAN

SD

MF

129

91

123

117

106

108

119.3

105.3

11.93

13.20

1.33

1.07

 

500

 

MEAN

SD

MF

121

103

96

101

101

102

106.0

102.0

13.23

1.00

1.18

1.04

 

158.1

 

MEAN

SD

MF

103

108

126

116

111

126

113.3

116.7

11.68

9.02

1.26

1.19

 

50

 

MEAN

SD

MF

122

100

111

109

112

110

115.0

106.3

6.08

5.51

1.28

1.08

 

15.81

 

MEAN

SD

MF

96

101

99

102

111

104

102.0

102.3

7.94

1.53

1.13

1.04

 

5

 

MEAN

SD

MF

127

122

124

108

110

94

120.3

108.0

9.07

14.00

1.34

1.10

 

Untreated control

 

 

MEAN

SD

MF

121

92

113

91

113

112

115.7

98.3

4.62

11.85

1.29

1.00

 

DMSO control

 

 

MEAN

SD

MF

91

105

90

93

89

97

90.0

98.3

1.00

6.11

1.00

1.00

Distilled water control*

 

 

MEAN

SD

MF

83

92

129

104

91

130

101.0

108.7

24.58

19.43

1.12

1.11

Positive control

SAZ (2ug)

 

MEAN

SD

MF

1120          2AA

2520

1084           (2ug)

2480

1100

2452

1101.3

2484.0

18.04

34.18

10.90

25.26

*Distilled water control was used because of SAZ

Table No.11: Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimurium TA1535

Cell count (overnight culture): 3.15 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

11

9

18

10

7

11

12.0

10.00

5.57

1.00

0.77

1.25

 

1581

 

MEAN

SD

MF

17

10

19

11

12

10

16.0

10.3

3.61

0.58

1.02

1.29

 

500

 

MEAN

SD

MF

12

11

14

7

11

7

12.3

8.3

1.53

2.31

0.79

1.04

 

158.1

 

MEAN

SD

MF

13

15

16

13

9

10

12.7

12.7

3.51

2.52

0.81

1.58

 

50

 

MEAN

SD

MF

12

9

17

10

19

11

16.0

10.0

3.61

1.00

1.02

1.25

 

15.81

 

MEAN

SD

MF

15

6

13

11

19

8

15.7

8.3

3.06

2.52

1.00

1.04

 

5

 

MEAN

SD

MF

11

9

11

8

6

16

9.3

11.0

2.89

4.36

0.60

1.38

 

Untreated control

 

 

MEAN

SD

MF

16

9

14

7

17

8

15.7

8.0

1.53

1.00

1.00

1.00

 

DMSO control

 

 

MEAN

SD

MF

19

10

14

6

14

8

15.7

8.0

2.89

2.00

1.00

1.00

Distilled water control*

 

 

MEAN

SD

MF

13

8

18

12

14

6

15.0

8.7

2.65

3.06

0.96

1.08

Positive control

SAZ (2ug)

 

MEAN

SD

MF

1012              2AA

201

1140              (2ug)

204

1084

211

1078.7

205.3

64.17

5.13

71.91

25.67

*Distilled water control was used because of SAZ

Table NO.12 Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Salmonella typhimuriu

Cell count (overnight culture): 2.49 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

n.d

14

1R

15

2R

15

1.5

14.7

0.71

0.58

0.17

1.26

 

1581

 

MEAN

SD

MF

5

6

9

8

14

16

9.3

10.0

4.51

5.29

1.08

0.86

 

500

 

MEAN

SD

MF

6

11

9

14

10

16

8.3

13.7

2.08

2.52

0.96

1.17

 

158.1

 

MEAN

SD

MF

8

9

9

13

12

13

9.7

11.7

2.08

2.31

1.12

1.00

 

50

 

MEAN

SD

MF

6

13

11

14

14

15

10.3

14.0

4.04

1.00

1.19

1.20

 

15.81

 

MEAN

SD

MF

3

10

8

11

14

12

8.3

11.0

5.51

1.00

0.96

0.94

 

5

 

MEAN

SD

MF

10

11

10

13

15

14

11.7

12.7

2.89

1.53

1.35

1.09

 

Untreated control

 

 

MEAN

SD

MF

5

10

7

12

10

16

7.3

12.7

2.52

3.06

0.85

1.09

 

DMSO control

 

 

MEAN

SD

MF

5

10

9

12

12

13

8.7

11.7

3.51

1.53

1.00

1.00

Positive control

9AA

(50ug)

 

MEAN

SD

MF

404               2AA

206

412                (2ug)

208

424

216

13.3

210.0

10.07

5.29

47.69

18.00

n.d.: no data (plate was infected)

R: Reduced background

Table No.13 Confirmatory Mutation Test (Pre-Incubation Method)

30 September-02 October 2014

Strain: Escherichia coli WP2 uvrA

Cell count (overnight culture): 3.84 x 109 CFU/mL

 

                              Revertant colony number

Concentration

(μg/plate)

-S9

+S9

 

5000

 

MEAN

SD

MF

45

44

46

51

47

56

46.0

50.3

1.00

6.03

1.02

0.93

 

1581

 

MEAN

SD

MF

43

47

49

57

51

61

47.7

55.0

4.16

7.21

1.06

1.02

 

500

 

MEAN

SD

MF

41

52

41

59

50

71

44.0

60.7

5.20

9.61

0.98

1.12

 

158.1

 

MEAN

SD

MF

31

52

36

60

47

61

38.0

57.7

8.19

4.93

0.84

1.07

 

50

 

MEAN

SD

MF

39

56

48

56

57

56

48.0

56.0

9.00

0.00

1.07

1.04

 

15.81

 

MEAN

SD

MF

41

49

44

56

45

57

43.3

54.0

2.08

4.36

0.96

1.00

 

5

 

MEAN

SD

MF

36

54

49

62

68

64

51.0

60.0

16.09

5.29

1.13

1.11

 

Untreated control

 

 

MEAN

SD

MF

42

59

60

40

36

60

46.0

43.0

12.49

11.27

1.02

0.98

 

DMSO control

 

 

MEAN

SD

MF

33

52

45

50

57

60

45.0

54.0

12.00

5.29

1.00

1.00

Distilled water control*

 

 

MEAN

SD

MF

59

65

44

62

46

58

49.7

61.7

8.14

3.51

1.10

1.14

Positive control

MMS (2uL)

 

MEAN

SD

MF

1140             2AA

324

1082                (50 ug)

312

1020

296

1080.7

310.7

60.01

14.05

21.76

5.74

*Distilled water control was used because of MMS

Summary Table of the Initial Mutation Test (test item)

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

22.7

37.0

102.3

102.7

17.0

13.0

6.0

9.7

38.0

40.0

MF

1.01

1.16

0.97

0.95

0.85

1.18

0.64

1.04

0.95

1.09

1581

Mean

26.3

35.0

103.3

109.0

15.7

11.3

7.0

6.3

36.3

43.7

MF

1.18

1.09

0.98

1.01

0.78

1.03

0.75

0.95

0.91

1.19

500

Mean

25.7

37.7

106.7

97.0

15.0

12.0

5.7

9.0

35.3

37.0

MF

1.15

1.18

1.02

0.90

0.75

1.09

0.61

1.35

0.88

1.01

158.1

Mean

19.0

34.7

104.0

96.0

14.7

10.3

5.7

7.0

40.0

42.0

MF

0.85

1.08

0.99

0.89

0.73

0.94

0.61

1.05

1.00

1.15

50

Mean

23.3

38.0

99.7

90.0

14.7

9.7

8.3

6.0

34.3

43.3

MF

1.04

1.19

0.95

0.83

0.73

0.88

0.89

0.90

0.86

1.18

15.81

Mean

24.7

38.3

102.7

100.7

15.3

12.0

6.3

6.0

39.3

44.0

MF

1.10

1.20

0.98

0.93

0.77

1.09

0.68

0.90

0.98

1.20

5

Mean

21.3

30.3

103.0

96.3

15.3

10.3

4.3

9.0

32.7

39.0

MF

0.96

0.95

0.98

0.89

0.77

0.94

0.46

1.35

0.82

1.06

Summary Table of the Confirmatory Mutation Test (test item):

Concentrations

(µg/plate)

Mean values of revertants / Mutation factor (MF)

Salmonella typhimuriumtester strains

Escherichia coli

TA98

TA100

TA1535

TA1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

5000

Mean

23.7

31.7

97.7

112.3

12.0

10.0

1.5

14.7

46.0

50.3

MF

1.08

1.04

1.09

1.14

0.77

1.25

0.17

1.26

1.02

0.93

1581

Mean

20.7

32.7

119.3

105.3

16.0

10.3

9.3

10.0

47.7

55.0

MF

0.94

1.08

1.33

1.07

1.02

1.29

1.08

0.86

1.06

1.02

500

Mean

19.7

33.7

106.0

102.0

12.3

8.3

8.3

13.7

44.0

60.7

MF

0.89

1.11

1.18

1.04

0.79

1.04

0.96

1.17

0.98

1.12

158.1

Mean

25.3

34.7

113.3

116.7

12.7

12.7

9.7

11.7

38.0

57.7

MF

1.15

1.14

1.26

1.19

0.81

1.58

1.12

1.00

0.84

1.07

50

Mean

23.7

34.7

115.0

106.3

16.0

10.0

10.3

14.0

48.0

56.0

MF

1.08

1.14

1.28

1.08

1.02

1.25

1.19

1.20

1.07

1.04

15.81

Mean

26.3

37.0

102.0

102.3

15.7

8.3

8.3

11.0

43.3

54.0

MF

1.20

1.22

1.13

1.04

1.00

1.04

0.96

0.94

0.96

1.00

5

Mean

23.3

32.0

120.3

108.0

9.3

11.0

11.7

12.7

51.0

60.0

MF

1.06

1.05

1.34

1.10

0.60

1.38

1.35

1.09

1.13

1.11

Applicant's summary and conclusion

Conclusions:
The test item had no mutagenic activity in the applied bacterium tester strains under the test conditions used in this study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD Guideline 471. The experiments were carried out using Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation (S9 fraction prepared from the livers of rats). Based on the results of the Solubility Test, the test item was dissolved in DMSO. Concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate were examined in the Range Finding Test. Since the revertant colonies were in the normal range and no precipitate or cytotoxic effect were observed, the test item concentrations in the main tests were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg test item/plate. In the Initial Mutation Test and Confirmatory Mutation Test, none of the observed revertant colony numbers were above the respective biological threshold value. There were no dose-related trends and no indication of any treatment effect. In all test item treated groups, the numbers of revertant colonies did not exceed the biological relevance when compared to the solvent control and were within the normal biological variability of the test system. No precipitate was detected on the plates in the main tests in any examined bacterial strains with and without metabolic activation. Inhibitory, cytotoxic effect of the test item was observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1537 bacterial strain without metabolic activation. The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid. In conclusion, the test item had no mutagenic activity in the bacterium tester strains under the test conditions used in this study.