Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 October - 12 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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)
Version / remarks:
21st July, 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
August 1998
Deviations:
no
Qualifier:
according to
Guideline:
other: ICH Guidance S2(R1): Guidance on Genotoxicity Testing and Data Interpretation for Pharmaceuticals Intended for Human Use, June 2012
Version / remarks:
June 2012
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
Specific details on test material used for the study:
Date of production: 26.03.2015
Expiration date: 26.03.2020

Method

Target gene:
his/trp
Genotypes of the Strains Used for Mutagenicity Testing:
In addition to histidine and tryptophan mutation, each strain has additional mutations which enhance its sensitivity to mutagens. The uvrB (uvrA) strains are defective in excision repair. It causes the strains to be more sensitive to the mutagenic and lethal effects of a wide variety of mutagens because they cannot repair DNA damages. The rfa mutation increases the permeability of the bacterial lipopolysaccharide wall for larger molecules. The plasmid pKM101 (TA98, TA100) carries the muc+ gene which participates in the error-prone "SOS" DNA repair pathway induced by DNA damage. This plasmid also carries an ampicillin resistance transfer factor (R-factor) which is used to identify its presence in the cell. The Escherichia coli strain used in this test (WP2uvrA) is also defective in DNA excision repair.

Species / strain
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and beta-naphthoflovone induce rat liver, S9-mix
Test concentrations with justification for top dose:
1600, 500, 160, 50, 16, 5, and 1.6 µg/plate
Vehicle:
Vehicle: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: DMSO was found as appropriate vehicle for preparing the test item solutions. Based on the laboratory’s historical control database, this vehicle is compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylenediamine
Remarks:
TA98, without S9 mix, 4 µg/plate
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100 and TA1535, without S9 mx, 2 µg/plate
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA1537, without S9 mix, 50 µg/plate
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E.coli WP2, without S9 mix, 2 µL/plate
Negative controls:
yes
Solvent controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All Salmonella strains tested, with S9 mix, 2 µg/plate; E.coli WP2, with S9 mix, 50 µg/plate
Details on test system and conditions:
METHOD OF APPLICATION: in agar (plate incorporation), preincubation;

DURATION
- Preincubation period:
20 min
- Exposure duration:
48 h

NUMBER OF REPLICATIONS:
3


DETERMINATION OF CYTOTOXICITY
- Method: revertant colony count, background lawn development

Rationale for test conditions:
Justification of concentrations:
Selection of the concentration range was done on the basis of a Solubility Test and a Concentration Range Finding Test.
Based on the solubility test, a stock solution (suspension) with a nominal concentration of 50 mg/mL was prepared in DMSO and diluted in 6 steps by factor of approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) was determined at the concentrations of 5000; 1600; 500; 160; 50; 16 and 5 µg/plate. In the informatory experiment the revertant colony numbers of vehicle control plates with and without S9 Mix were in line the corresponding historical control data ranges. The positive control treatments showed the expected, biological relevant increases in induced revertant colonies in both tester strains.

Evaluation criteria:
The colony numbers on the untreated, vehicle control, positive control and the test item treated plates were determined visually by manual counting, vs control

A test item is considered mutagenic if:
- a dose–related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control,
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Criteria for a Negative Response:
A test item is considered non-mutagenic if it produces 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 values and appropriate standard deviations and mutation rates were calculated by EXCEL software.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity:
no
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
Validity of the Performed Experiments
The tester strains used in this study demonstrated the specific phenotype characteristics, were in line with the corresponding historical control data ranges and showed the adequate strain culture titer. Each batch of the S9 fraction used in this test had the appropriate biological activity and was active in the applied system. Each of the investigated reference mutagens showed the expected increase (at least a 3-fold increase) in induced revertant colonies over the mean value of the respective vehicle control in both main experimental phases and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in the main experimental phases, in all tester strains.
The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates showed characteristic mean numbers agreed with the actual historical control data ranges in all strains in both main experimental phases. Seven concentration levels were investigated in the Informatory Toxicity Test and in the main mutation experiments (Initial and Confirmatory Mutation Tests). In the performed experimental phases there were at least five analyzable concentrations and a minimum of three non-toxic and non-precipitated dose levels at each tester strain. All criteria for the validity of the performed experiments have therefore been met.

Controls
In the performed Initial and Confirmatory Mutation Test multiple test items were tested with reference values from the common parallel controls. In the Initial and Confirmatory Mutation Tests the revertant colony numbers of the dimethyl sulfoxide (DMSO) vehicle control plates with and without S9 Mix were in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases in induced revertant colonies in all experimental phases, in all tester strains. The revertant colony numbers of the untreated and ultrapure water control plates in different experimental phases were slightly higher or lower than the DMSO control plates. The higher or lower revertant counts of these controls remained in the corresponding historical control data ranges. In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.

Initial and Confirmatory Mutation Tests (Plate Incorporation Test and Pre-Incubation Test)
No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with C. I. Leuco Sulfur Green 2 at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. In the performed experiments, sporadically increased revertant colony numbers were observed. These increases did not show a dose-response relationship, were of minor intensity, and all of the increases remained far below the biologically relevant thresholds for being positive. The obtained increases were therefore considered as biologically not relevant, being in the range of the biological variability of the applied test system. The highest revertant colony number increase was observed in the Initial Mutation Test (Plate Incorporation Test) in S. typhimurium TA98 strain at 50 μg/plate, in the absence of metabolic activation ( S9). This value however remained in the range of the corresponding vehicle historical control data and additional concentration related increase in revertant colony counts was not noticed. The mutation rate was 1.63, which was far below the genotoxicological threshold for being positive.
In the Initial and Confirmatory Mutation Tests, unequivocal inhibitory effect of the test item on bacterial growth was observed. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain only, in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains , in the absence of exogenous metabolic activation. The cytotoxicity was indicated by decreased revertant colony counts (some of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. For the cytotoxicity tendency (it did not show a clear dose-dependent tendency), the effect of precipitate formation was supposed. The table contains the unequivocal results where the obtained revertant colony numbers were below the vehicle (in some cases below the corresponding historical control data ranges), and/or affected background lawn development occurred.

All of the further observed lower revertant colony numbers (when compared to the revertant colony numbers of the corresponding vehicle control) remained in the range of the biological variability of the applied test system. In general, 160 µg/plate was considered as lowest concentration showing unequivocal cytotoxicity. When evaluated by naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration of 1600 µg/plate in absence and at 1600 and 500 µg/plate in the presence of S9 following the plate incorporation procedure and in the absence and presence of S9 following the pre-incubation procedure. For confirmation of manual evaluations (made by naked eye) all of the plates were checked for colony and background lawn development by microscope at 40X magnification. At this magnification further test item precipitate was not noticed at lower concentration levels.

Any other information on results incl. tables

Table 4: Summary of the Results of the Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)

 

Concentrations (µg/plate)

Salmonella typhimuriumtester strains

TA 98

TA 100

-S9

+S9

-S9

+S9

Mean values of revertants per plate and
Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

 

Untreated Control

24.3

1.28

17.7

0.83

97.3

1.15

115.3

1.07

 

DMSO Control

19.0

1.00

21.3

1.00

84.7

1.00

107.3

1.00

 

Ultrapure Water Control

100.7

1.00

 

5000

#

#

#

#

75.0

0.89

101.3

0.94

 

1600

18.3

0.96

16.7

0.78

98.0

1.16

99.3

0.93

 

500

19.7

1.04

14.7

0.69

97.7

1.15

112.3

1.05

 

160

18.7

0.98

18.7

0.88

84.7

1.00

102.7

0.96

 

50

16.0

0.84

22.7

1.06

99.3

1.17

95.7

0.89

 

16

25.0

1.32

21.0

0.98

88.7

1.05

105.0

0.98

 

5

27.7

1.46

17.7

0.83

94.7

1.12

102.7

0.96

 

NPD (4 µg/plate)

236.7

12.46

 

SAZ (2 µg/plate)

1093.3

10.86

 

2AA (2 µg/plate)

850.7

39.88

2075.3

19.34

 

MR: Mutation Rate                                   #   : the revertants could not be counted, evaluated

NPD: 4-Nitro-1,2-phenylenediamine

SAZ: Sodium azide

2AA: 2-aminoanthracene

Table 5: Summary of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (µg/plate)

Salmonella typhimuriumtester strains

Escherichiacoli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

21.7

1.33

26.0

0.88

86.0

1.01

117.3

1.27

9.3

0.88

11.0

0.94

7.3

1.29

4.7

0.64

18.3

0.83

19.3

0.94

DMSO Control

16.3

1.00

29.7

1.00

85.0

1.00

92.3

1.00

10.7

1.00

11.7

1.00

5.7

1.00

7.3

1.00

22.0

1.00

20.7

1.00

Ultrapure Water Control

102.7

1.00

12.7

1.00

19.7

1.00

1600

19.7

1.20

18.7

0.63

111.3

1.31

106.7

1.16

7.3

0.69

8.3

0.71

0.3

0.06

3.3

0.45

15.7

0.71

21.7

1.05

500

18.0

1.10

25.7

0.87

102.3

1.20

129.0

1.40

12.3

1.16

9.3

0.80

2.0

0.35

10.0

1.36

15.0

0.68

22.3

1.08

160

25.7

1.57

24.0

0.81

102.7

1.21

135.0

1.46

9.3

0.88

10.7

0.91

5.3

0.94

9.0

1.23

18.3

0.83

18.7

0.90

50

26.7

1.63

20.3

0.69

95.7

1.13

121.0

1.31

9.3

0.88

15.3

1.31

5.7

1.00

7.3

1.00

12.0

0.55

21.0

1.02

16

19.7

1.20

19.7

0.66

98.0

1.15

127.0

1.38

9.0

0.84

16.3

1.40

6.0

1.06

6.7

0.91

19.7

0.89

28.3

1.37

5

20.0

1.22

17.3

0.58

96.0

1.13

114.3

1.24

13.7

1.28

16.3

1.40

7.0

1.24

6.3

0.86

14.7

0.67

19.3

0.94

1.6

18.0

1.10

16.7

0.56

98.3

1.16

101.3

1.10

7.7

0.72

14.0

1.20

6.0

1.06

8.7

1.18

15.7

0.71

22.3

1.08

NPD (4 µg/plate)

274.7

16.82

SAZ (2 µg/plate)

1240.0

12.08

561.7

44.34

9AA (50 µg/plate)

597.3

105.41

MMS (2 µL/plate)

892.0

45.36

2AA (2 µg/plate)

2336.0

78.74

2317.3

25.10

183.7

15.74

169.7

23.14

2AA (50 µg/plate)

220.0

10.65

MR: Mutation Rate;          NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methylmethanesulfonate; 2AA: 2-aminoanthracene

Remarks: DMSO was applied as vehicle of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as vehicle for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water

Table 6: Summary of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (µg/plate)

Salmonella typhimuriumtester strains

Escherichia coli

TA 98

TA 100

TA 1535

TA 1537

WP2uvrA

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Mean values of revertants per plate Mutation rate (MR)

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Mean

MR

Untreated Control

18.0

0.95

17.7

0.98

97.3

1.20

109.7

1.02

10.7

1.23

12.0

0.97

13.7

1.00

5.7

0.81

18.0

0.93

30.0

1.22

DMSO Control

19.0

1.00

18.0

1.00

81.3

1.00

108.0

1.00

8.7

1.00

12.3

1.00

13.7

1.00

7.0

1.00

19.3

1.00

24.7

1.00

Ultrapure Water Control

90.3

1.00

14.3

1.00

22.3

1.00

1600

16.0

0.84

17.0

0.94

104.7

1.29

97.3

0.90

5.3

0.62

12.0

0.97

1.0

0.07

8.0

1.14

21.3

1.10

21.0

0.85

500

11.7

0.61

17.7

0.98

51.0

0.63

112.7

1.04

4.3

0.50

11.7

0.95

1.3

0.10

7.3

1.05

21.3

1.10

24.3

0.99

160

9.7

0.51

23.3

1.30

90.7

1.11

114.3

1.06

2.7

0.31

10.7

0.86

5.0

0.37

8.3

1.19

20.7

1.07

22.0

0.89

50

30.0

1.58

20.0

1.11

102.0

1.25

101.0

0.94

8.0

0.92

13.0

1.05

6.7

0.49

6.7

0.95

22.0

1.14

25.7

1.04

16

21.7

1.14

15.7

0.87

77.3

0.95

115.3

1.07

7.0

0.81

9.3

0.76

13.7

1.00

8.0

1.14

14.7

0.76

27.3

1.11

5

18.0

0.95

18.0

1.00

90.0

1.11

114.3

1.06

8.3

0.96

12.0

0.97

12.7

0.93

9.0

1.29

21.0

1.09

31.7

1.28

1.6

17.3

0.91

21.7

1.20

88.7

1.09

105.0

0.97

10.0

1.15

10.3

0.84

13.0

0.95

10.0

1.43

18.7

0.97

34.7

1.41

NPD (4 µg/plate)

188.7

9.93

SAZ (2 µg/plate)

1248.0

13.82

794.7

55.44

9AA (50 µg/plate)

264.0

19.32

MMS (2 µL/plate)

824.0

36.90

2AA (2 µg/plate)

684.0

38.00

984.0

9.11

160.0

12.97

93.3

13.33

2AA (50 µg/plate)

146.7

5.95

MR: Mutation Rate;          NPD: 4-Nitro-1,2-phenylenediamine; SAZ: Sodium azide; 9AA: 9-Aminoacridine; MMS: Methyl methanesulfonate; 2AA: 2-aminoanthracene

Remarks:           DMSO was applied as vehicle of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as vehicle for the SAZ and MMS. The mutation rate of the test item and the untreated control is given referring to the DMSO. The mutation rate of the NPD, 9AA and 2AA is given referring to the DMSO and the mutation rate of the SAZ and MMS positive control is given referring to the ultrapure water.

.

Table 7: Historical Control Values for Revertants/Plate (for the Period of 2008-2015)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.4

106.0

10.4

8.1

25.6

SD

3.7

27.3

1.5

2.5

5.5

Minimum

9

65

3

2

11

Maximum

39

157

23

19

45

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

28.0

117.1

11.9

9.0

34.3

SD

4.2

19.4

1.5

2.0

5.4

Minimum

12

75

4

3

18

Maximum

48

166

24

20

56

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

20.9

101.4

10.3

7.9

24.9

SD

3.5

26.2

1.4

2.5

4.9

Minimum

10

65

3

2

11

Maximum

39

150

23

20

44

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.1

114.7

12.0

8.8

34.2

SD

4.0

19.3

1.5

2.1

5.2

Minimum

15

71

4

3

16

Maximum

48

161

24

20

56

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

22.4

105.5

10.4

7.5

26.3

SD

3.6

27.6

1.6

2.3

5.9

Minimum

12

67

3

2

13

Maximum

36

156

24

15

47

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

28.0

117.4

11.5

8.7

35.2

SD

4.0

19.8

1.4

2.3

5.2

Minimum

15

83

4

4

18

Maximum

43

166

22

16

56

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

255.6

958.9

842.1

467.4

712.3

SD

30.7

149.9

134.0

105.7

57.5

Minimum

123

522

354

109

320

Maximum

647

1927

1871

1498

1283

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1224.8

1431.9

165.4

148.0

264.7

SD

293.8

339.9

35.1

21.3

74.2

Minimum

409

581

85

68

141

Maximum

2587

2923

507

407

487

Abbreviations:    TA98, TA100, TA1535, TA1537: Salmonella typhimurium TA98, TA100, TA1535, TA1537; E. coli: Escherichia coli WP2uvrA

                               SD: Standard deviation; DMSO: Dimethyl sulfoxide

Applicant's summary and conclusion

Conclusions:
In the bacterial reverse mutation assay (Ames test) according to OECD guideline 471 the test item did not induce gene mutations.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay (Ames test) according to the OECD guideline 471. The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 mix) prepared from livers of phenobarbital/beta-naphthoflavone-induced rats. The study included a Preliminary Solubility Test, a Preliminary Concentration Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Test), and a Confirmatory Mutation Test (Pre-Incubation Test). Based on the results of the Solubility Test and the Concentration Range Finding Test the test item was dissolved in dimethyl sulfoxide (DMSO). Based on the results of the preliminary Concentration Range Finding Test (Informatory Toxicity Test) the following concentrations of the test item were prepared and investigated in the Initial and Confirmatory Mutation Tests:1600; 500; 160; 50; 16; 5 and 1.6 µg/plate.

In the main experiments (when evaluated by naked eye) test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at the concentration of 1600 µg/plate in absence and at 1600 and 500 µg/plate in the presence of S9 following the plate incorporation procedure and in the absence and presence of S9 following the pre-incubation procedure. The obtained precipitate did not interfere with the scoring of the colonies in any case. In the Initial Mutation Test inhibitory effect of the test item was observed in the S. typhimurium TA1537 strain in the absence and also in the presence of exogenous metabolic activation. In the Confirmatory Mutation Test unequivocal inhibition was noticed in all examined Salmonella typhimurium strains, in the absence of exogenous metabolic activation. The inhibitory effect was indicated by absent or decreased revertant colony counts (some of them below the corresponding historical control data ranges) and affected background lawn development: reduced or slightly reduced background lawn. In general, 160 µg/plate was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges. The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (more than 3-fold increase) in induced revertant coloniesand the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive controlin all experimental phases, in all tester strains. No biologically relevant increases were observed in revertant colony numbers of any of the five test strains following treatment with at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments.

The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. In conclusion, the test item has no mutagenic activity on the applied bacterium tester strains under the test conditions used in this study.