Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2017 - 19 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives
EC Number:
288-896-1
EC Name:
Acetamide, N-(2,4-dinitrophenyl)-, reaction products with 1-methyl-2,4-dinitrobenzene and sodium sulfide (Na2(Sx)), leuco derivatives
Cas Number:
85940-08-5
Molecular formula:
not applicable
IUPAC Name:
Reaction product of N-(2,4-dinitrophenyl)acetamide with 1-methyl-2,4-dinitrobenzene and polysulfide, leuco derivatives
Test material form:
solid: particulate/powder
Details on test material:
Test item: Leuco Sulphur Brown 46
Appearance: Brown powder
CAS No: 85940-08-5

Method

Target gene:
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 (WP2 uvrA) is also defective in DNA excision repair.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/-naphthoflavone-induced rats
Test concentrations with justification for top dose:
5000; 1600; 500; 160; 50; 16 and 5 µg/plate
Vehicle / solvent:
dimethyl sulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
methylmethanesulfonate
other: 4-Nitro-1,2-phenylenediamine, 2-aminoanthracene
Details on test system and experimental conditions:
Origin of the Bacterial Strains

Tester strains: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and Escherichia coli WP2 uvrA
Supplier: Trinova Biochem GmbH; Rathenau Str. 2; D-35394 Giessen, Germany;
Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA.
Frozen stock cultures were prepared from the disc cultures.

Storage of Tester Strains

The strains are stored at -80 ± 10ºC in the Laboratory of TOXI-COOP ZRT. in the form of lyophilized discs and in frozen permanent copies. Frozen permanent cultures of the tester strains are prepared from fresh, overnight cultures to which DMSO (8 % (v/v)) is added as a cryoprotective agent.

Confirmation of Phenotypes of Tester Strains

The phenotypes of the tester strains used in the bacterial reverse mutation assays with regard to membrane permeability (rfa), UV sensitivity (uvrA and uvrB), ampicillin resistance (amp), as well as spontaneous mutation frequencies are checked regularly according to Ames et al.
Established procedures (Standard Operating Procedures) for the preparations of each batch of frozen stock culture and raw data and reports of phenotype confirmation are stored in the Laboratory of TOXI-COOP ZRT.

Spontaneous Reversion of Tester Strains

Each tester strain reverts spontaneously at a frequency that is characteristic for the strain. Spontaneous reversions of the test strains to histidine or tryptophan prototrophs are measured routinely in mutagenicity experiments and expressed as the number of spontaneous revertants per plate.

Procedure for Bacterial Cultures

The frozen bacterial cultures were thawed at room temperature and 200 µL inoculum was used to inoculate each 50 mL of Nutrient Broth No. 2 for the
overnight cultures in the assay. The cultures were incubated for approximately 10-14 hours in a 37 oC Benchtop Incubator Shaker.

Viability and the Cell Count of the Testing Bacterial Cultures

The viability of each testing culture was determined by plating 0.1 mL of the 10-5, 10-6, 10-7 and 10-8 dilutions of cultures on nutrient agar plates. The viable cell number of the cultures was determined by manual colony counting.

Metabolic Activation System

The test bacteria were also exposed to the test item in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented
post-mitochondrial fraction (S9).

Rat Liver S9 Fraction

The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF)-induced rat liver was provided by Trinova Biochem GmbH (Rathenau Str. 2; D-35394
Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).
Rationale for test conditions:
Justification of concentrations:
Selection of the concentration range for the initial mutation test was done on the basis of solubility test and concentration range finding test (informatory toxicity test). The investigated concentration range for the confirmatory mutation test was chosen based on the results of the initial mutation test.

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 (repeated plate incorporation 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 mutation test: ±S9 mix: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate.

In the initial mutation test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. The revertant colony numbers of the solvent control plates (DMSO) 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 biologically relevant increases (more than 3-fold increase) in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains.
Evaluation criteria:
Evaluation of Experimental Data

The colony numbers on the untreated, solvent control, positive control and the test item treated plates were determined visually by manual counting

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 solvent 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 solvent 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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
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
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
positive
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:
positive
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:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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 (according to the provided Certificates, Appendix VIII) 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 solvent control in all 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 the tester strains. The spontaneous revertant colony numbers of the dimethyl sulfoxide (DMSO) solvent control plates showed characteristic mean numbers agreed with the actual historical control data ranges in the examined strains in both main experimental phases. Seven concentration levels were investigated in the initial and confirmatory mutation tests. In the performed main 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) solvent 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, solvent and positive controls were in line with the criteria for validity of the assay.

Initial Mutation Test (Plate Incorporation Test)
In this test negative mutagenicity results were obtained in Salmonella typhimurium TA1537 and Escherichia coli WP2 uvrA strains in the absence and also in
the presence of an exogenous metabolic activation system (±S9 mix). Unequivocal positive results were noticed following treatment with the test item in the investigated Salmonella typhimurium TA98, TA100 and TA1535 strains (±S9 mix). The increased revertant colony numbers were above the threshold for being positive in S. typhimurium TA98 at the concentration of 5000 µg/plate (-S9 mix) and in the range of 5000-50 µg/plate (+S9 mix), in TA100 at the concentrations of 5000 and 1600 µg/plate ( S9 mix) and in the concentration range of 5000-500 µg/plate (+S9 mix); furthermore in TA1535 at 5000 µg/plate (±S9 mix). The increased revertant colony numbers were above the corresponding historical control data range, however, they were below the genotoxicological threshold for being positive in S. typhimurium TA98 at 1600, 500 and 160 µg/plate (-S9 mix). The significant revertant colony number increases showed clear dose-related tendency in S. typhimurium TA98, TA100 and TA1535 strains in the absence and presence of exogenous metabolic activation (± S9 mix). In this test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix). To confirm and to investigate the reproducibility of this positive result a confirmatory mutation test was performed with the S. typhimurium strain TA98, TA100 and TA1535 strains in the absence and presence of an exogenous metabolic activation system (±S9 mix). The strains Salmonella typhimurium
TA1537 and Escherichia coli WP2 uvrA were not further investigated.

Confirmatory Mutation Test (Plate Incorporation Test)
While the positive results already noticed in the initial mutation test in the investigated Salmonella typhimurium TA98 strain (±S9 mix), TA100 strain (±S9 mix) and TA1535 strain (+S9 mix) were successfully confirmed, the revertant colony number increases remained just below the threshold for being positive (borderline results) in TA1535 in the absence of an exogenous metabolic activation system (-S9 mix). The increased revertant colony numbers were above the threshold for being positive in S. typhimurium TA98 at the concentrations of 5000 and 1600 µg/plate (-S9 mix) and in the range of 5000-50 µg/plate (+S9 mix), in TA100 at the concentrations of 5000 and 1600 µg/plate ( S9 mix) and in the concentration range of 5000-500 µg/plate (+S9 mix); furthermore in TA1535 at 5000 µg/plate (+S9 mix). The increased revertant colony numbers were above the corresponding historical control data range; however just below the genotoxicological threshold for being positive (borderline results) in S. typhimurium TA1535 at 5000 µg/plate (-S9 mix). Similarly to the initial mutation test results the significantly increased revertant colony number showed clear dose-related tendency in absence and also in the presence of exogenous metabolic activation (± S9 mix). Non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix).

Any other information on results incl. tables

SUMMARY TABLES OF THE EXPERIMENTS

Summary Table of the Results of the Concentration Range Finding Test

Concentration Range Finding Test (Informatory Toxicity Test)

Concentrations (mg/plate)

Salmonella typhimurium tester 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

20.7

1.00

26.3

1.05

86.3

1.10

105.0

1.16

DMSO Control

20.7

1.00

25.0

1.00

78.7

1.00

90.3

1.00

Ultrapure Water Control

84.7

1.00

5000

36.0

1.74

79.7

3.19

292.3

3.72

464.0

5.14

1600

52.0

2.52

133.3

5.33

165.3

2.10

442.7

4.90

500

37.7

1.82

174.7

6.99

123.0

1.56

246.0

2.72

160

38.7

1.87

164.7

6.59

105.0

1.33

167.3

1.85

50

19.3

0.94

78.0

3.12

81.0

1.03

100.7

1.11

16

27.7

1.34

36.0

1.44

75.3

0.96

103.3

1.14

5

17.7

0.85

23.0

0.92

75.0

0.95

98.7

1.09

NPD (4mg)

223.0

10.79

SAZ (2mg)

949.3

11.21

2AA (2mg)

1421.3

56.85

2406.7

26.64

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

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

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester 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

23.7

1.37

26.7

1.36

99.7

1.17

105.7

1.17

9.3

0.82

10.0

1.03

9.3

1.22

10.7

1.14

37.0

1.10

41.0

0.95

DMSO Control

17.3

1.00

19.7

1.00

85.3

1.00

90.0

1.00

11.3

1.00

9.7

1.00

7.7

1.00

9.3

1.00

33.7

1.00

43.3

1.00

Ultrapure Water Control

85.3

1.00

10.3

1.00

37.0

1.00

5000

86.7

5.00

122.7

6.24

268.0

3.14

335.3

3.73

40.0

3.53

50.3

5.21

4.0

0.52

10.0

1.07

31.3

0.93

42.3

0.98

1600

45.0

2.60

110.7

5.63

197.7

2.32

289.3

3.21

16.0

1.41

21.7

2.24

0.7

0.09

2.0

0.21

26.7

0.79

52.0

1.20

500

41.0

2.37

190.0

9.66

127.0

1.49

248.3

2.76

12.3

1.09

12.0

1.24

7.3

0.96

14.7

1.57

32.0

0.95

54.0

1.25

160

40.0

2.31

140.0

7.12

103.3

1.21

158.0

1.76

14.7

1.29

12.3

1.28

8.7

1.13

8.0

0.86

37.3

1.11

42.3

0.98

50

21.0

1.21

78.7

4.00

83.7

0.98

100.3

1.11

10.3

0.91

8.7

0.90

8.3

1.09

9.0

0.96

36.0

1.07

36.7

0.85

16

22.7

1.31

42.7

2.17

86.3

1.01

97.7

1.09

11.0

0.97

14.3

1.48

9.0

1.17

9.7

1.04

39.3

1.17

39.3

0.91

5

19.7

1.13

31.7

1.61

77.3

0.91

81.7

0.91

9.3

0.82

9.0

0.93

5.7

0.74

9.7

1.04

31.3

0.93

44.3

1.02

NPD (4mg)

283.3

16.35

SAZ (2mg)

1397.3

16.38

1141.3

110.45

9AA (50mg)

602.7

78.61

MMS (2mL)

980.0

26.49

2AA (2mg)

2376.0

120.81

1920.0

21.33

175.7

18.17

182.7

19.57

2AA (50mg)

230.3

5.32

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 solvent of the test item and positive control substances: NPD, 9AA and 2AA and the ultrapure water was applied as solvent 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.

Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Plate Incorporation Test)

Concentrations (mg/plate)

Salmonella typhimurium tester strains

TA 98

TA 100

TA 1535

-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

Untreated Control

16.3

1.17

18.7

0.84

97.7

0.99

105.0

1.05

10.7

1.03

10.7

0.97

DMSO Control

14.0

1.00

22.3

1.00

99.0

1.00

99.7

1.00

10.3

1.00

11.0

1.00

Ultrapure Water Control

99.7

1.00

10.0

1.00

5000

46.0

3.29

107.3

4.81

215.7

2.18

297.0

2.98

30.3

2.94

43.7

3.97

1600

44.3

3.17

153.3

6.87

201.3

2.03

245.0

2.46

20.3

1.97

26.0

2.36

500

34.7

2.48

231.0

10.34

132.0

1.33

224.3

2.25

12.7

1.23

15.3

1.39

160

24.7

1.76

154.0

6.90

112.3

1.13

152.3

1.53

11.7

1.13

11.3

1.03

50

15.0

1.07

92.3

4.13

81.0

0.82

100.7

1.01

12.7

1.23

12.3

1.12

16

14.7

1.05

36.7

1.64

88.0

0.89

93.7

0.94

8.3

0.81

12.0

1.09

5

15.7

1.12

36.7

1.64

84.7

0.86

95.7

0.96

6.7

0.65

10.3

0.94

NPD (4mg)

368.7

26.33

SAZ (2mg)

1805.3

18.11

1157.3

115.73

2AA (2mg)

1240.7

55.55

1730.7

17.36

166.0

15.09

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

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

Historical Control Values for Revertants/Plate (for the Period of 2008-2016)

 

Bacterial strains

Historical control data of untreated control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.0

105.0

10.5

8.1

25.4

SD

3.7

25.7

1.4

2.3

5.2

Minimum

9

66

3

2

11

Maximum

39

155

23

19

45

n

226

236

216

214

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.5

117.1

11.8

9.0

33.9

SD

4.3

18.1

1.4

1.9

5.2

Minimum

12

75

4

2

17

Maximum

46

166

23

20

56

n

226

236

216

214

215

 

Bacterial strains

Historical control data of DMSO

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

20.4

100.1

10.3

7.9

24.7

SD

3.6

24.8

1.3

2.4

4.6

Minimum

10

64

3

2

11

Maximum

38

147

23

20

45

n

226

236

216

214

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

26.5

113.8

11.8

8.8

33.7

SD

4.1

18.3

1.5

1.9

5.0

Minimum

15

71

3

3

16

Maximum

47

162

25

20

57

n

226

236

216

214

215

 

Bacterial strains

Historical control data of Water

control

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

21.9

104.7

10.5

7.6

26.1

SD

3.7

25.9

1.5

2.2

5.5

Minimum

12

68

3

2

12

Maximum

35

154

24

16

48

n

89

236

216

89

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

27.4

117.3

11.4

8.7

34.9

SD

4.0

18.5

1.3

2.2

4.9

Minimum

15

83

4

3

18

Maximum

43

167

22

16

57

n

89

152

149

89

148

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                                               SD: Standard deviation;    DMSO: Dimethyl sulfoxide;n: number of studies

Historical Control Values for Revertants/Plate (for the Period of 2008-2016) (continued)

 

Bacterial strains

Historical control data of positive controls

‑S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

260.1

977.2

847.3

478.6

724.5

SD

31.8

150.6

126.3

104.5

65.0

Minimum

123

521

359

110

320

Maximum

664

1970

1855

1601

1313

n

226

236

216

214

215

+S9

 

TA98

TA100

TA1535

TA1537

E. coli

Average

1222.7

1436.4

164.1

147.0

257.7

SD

274.9

318.3

33.1

20.1

72.5

Minimum

386

583

85

69

140

Maximum

2676

2988

498

399

477

n

226

236

216

214

215

Abbreviations:   TA98, TA100, TA1535, TA1537: Salmonella typhimuriumTA98, TA100, TA1535,

                               TA1537;E. coli:Escherichia coliWP2uvrA

                                     SD: Standard deviation;   DMSO: Dimethyl sulfoxide;  n: number of studies

Applicant's summary and conclusion

Conclusions:
In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1535 strains carrying frameshift and base-pair substitution in the absence and presence of an exogenous metabolic activation system, under the test conditions used in this study.
Executive summary:

The test item was tested with regard to a potential mutagenic activity using the Bacterial Reverse Mutation Assay according to OECD guideline 471. The initial mutation test was 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 WP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The confirmatory mutation test was carried out using Salmonella typhimurium TA98, TA100 and TA1535. 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 (repeated plate incorporation test). Based on the results of the solubility test and the concentration range finding test the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions a correction of the concentrations for the active component content (74.99 %) was made in the experiments. 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 mutation test: ±S9 mix: 5000;1600; 500; 160; 50; 16 and 5 µg/plate. The selection of the concentration range was based on the recommendations in OECD 471 guideline. At the concentration choice the non-toxicity of the test item and the appearance of precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. To confirm and to investigate the reproducibility of the positive result from the initial mutation test in the confirmatory mutation test the same concentration levels were investigated (±S9 mix: 5000; 1600; 500; 160; 50; 16 and 5 µg/plate). When evaluated by the naked eye, non-interfering test item precipitate was noticed after about 48 hours incubation on the plates in the examined strains at 5000 and 1600 µg/plate in the absence and also in the presence of an exogenous metabolic activation system (±S9 mix). In the initial mutation test an unequivocal inhibitory, cytotoxic effect of the test item was observed in S. typhimurium TA1537 strain at 5000 and 1600 µg/plate, in the absence and also in the presence of exogenous metabolic activation (±S9 mix). The cytotoxicity was indicated by decreased revertant colony counts and/or affected background lawn development: reduced or slightly reduced background lawn. The revertant colony numbers of the solvent control plates (DMSO) 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 biologically relevant increases (more than 3-fold increase)in induced revertant colonies and the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains. Unequivocal positive results, confirmed by a repeat of the experiment, were noticed following the plate incorporation procedures (initial and confirmatory mutation test) in the investigated Salmonella typhimurium TA98, TA100 strains (±S9 mix) and in TA1535 strain (+S9 mix). The positive results obtained in the initial mutation test were not confirmed in the repeated plate incorporation procedure in case of TA1535 in the absence of exogenous metabolic activation (-S9 mix). The increased revertant colony counts remained just below the threshold for being positive (borderline results). However, the obtained increased tendencies followed a clear dose-relationship in all above listed cases. The positive mutagenicity results were obtained at higher concentration levels, mostly at precipitating concentrations (5000 and 1600 µg/plate); however unequivocal, confirmed positive results were noticed at non-precipitated concentrations in S. typhimurium TA98 at the concentrations of 500, 160 and 50 µg/plate (+S9 mix) and in TA100 at 500 µg/plate (+S9 mix). The reported data of this mutagenicity assay show that under the experimental conditions applied, the test item induced gene mutations by frameshifts and base pair substitutions in the genome of the Salmonella typhimurium TA98, TA100 and TA1535 tester strains examined. The unequivocal positive results were confirmed by a repeat of the plate incorporation procedures (initial and confirmatory mutation tests) in the absence and presence of exogenous metabolic activation (±S9 mix). In conclusion, the test item showed a mutagenic activity on Salmonella typhimurium TA98, TA100 and TA1535 strains carrying frameshift and base-pair substitution in the absence and presence of an exogenous metabolic activation system, under the test conditions used in this study.