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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2017 - 12 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
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: particulate/powder
Details on test material:
Test item: Blue Sema
Appearance: Black to brownish black, solid
CAS No: 1040873-93-5
EC No: 600-519-8

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. 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 / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
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)
Test concentrations with justification for top dose:
5000, 1600, 500, 160, 50, 16 and 5 µg/plate.
Vehicle / solvent:
dimethyl sulfoxide
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 were obtained from:
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..

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 (Section: 5.4.2) for the overnight cultures in the assay. The cultures were incubated for approximately 11-14 hours in a 37oC 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 was done on the basis of solubility tests and concentration range finding tests (informatory toxicity tests). In the solubility tests the test item behavior was investigated in the applied test system when formulated in ultrapure water or DMSO. In the preliminary phase of this study two pre-experiments were performed to find the most appropriate solvent for the main experiments.

Based on the solubility tests, stock suspensions with a concentration of 50 mg/mL were prepared in ultrapure water and 25 mg/mL in dimethyl sulfoxide (DMSO), respectively and diluted accordingly. In the informatory toxicity tests any correction factor, based on the active component of the test item (87 %) was not taken into consideration; therefore the 50 and 25 mg/mL stock suspension concentrations corresponded to 43.5 and 21.8 mg active component/mL. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined in both tests.
Evaluation criteria:
The colony numbers on the controls (untreated, vehicle, positive) and the test plates were determined (counted manually), the mean values and appropriate standard deviations and mutation rates were calculated.

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.

Results and discussion

Test resultsopen allclose all
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:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
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:
not examined
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:
not examined
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:
not examined
Positive controls validity:
valid

Any other information on results incl. tables

Summary Table of the Results of the Initial Mutation Test

Initial Mutation Test (Plate Incorporation Test)

Concentrations (mg/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

24.0

1.57

26.7

1.51

96.3

1.11

101.3

1.06

12.7

1.27

11.0

1.10

7.0

0.91

8.0

1.14

22.7

0.89

32.3

1.31

DMSO Control (100 µL)

15.3

1.00

21.3

1.00

87.0

1.00

95.0

1.00

11.7

1.00

12.3

1.00

7.0

1.00

8.3

1.00

22.0

1.00

37.0

1.00

DMSO Control (200µL)

15.3

1.00

17.7

1.00

86.7

1.00

95.7

1.00

10.0

1.00

10.0

1.00

7.7

1.00

7.0

1.00

25.3

1.00

24.7

1.00

Ultrapure Water Control

85.3

1.00

9.7

1.00

25.0

1.00

5000

10.3

0.67

13.3

0.75

85.0

0.98

78.7

0.82

7.7

0.77

9.3

0.93

0.0

0.00

0.3

0.05

21.0

0.83

27.3

1.11

1600

11.7

0.76

18.0

1.02

79.0

0.91

100.3

1.05

7.7

0.77

9.3

0.93

0.0

0.00

12.7

1.81

21.3

0.84

25.0

1.01

500

19.3

1.26

21.0

1.19

98.0

1.13

94.7

0.99

10.3

1.03

13.0

1.30

5.7

0.74

20.7

2.95

23.0

0.91

24.0

0.97

160

16.7

1.09

27.0

1.53

86.3

1.00

104.7

1.09

9.7

0.97

9.7

0.97

9.0

1.17

12.3

1.76

23.0

0.91

19.3

0.78

50

10.7

0.70

23.3

1.32

94.7

1.09

86.3

0.90

11.3

1.13

10.7

1.07

7.3

0.96

13.0

1.86

19.7

0.78

26.0

1.05

16

17.3

1.13

27.7

1.57

87.0

1.00

91.0

0.95

7.7

0.77

6.3

0.63

9.0

1.17

6.7

0.95

22.7

0.89

25.7

1.04

5

20.7

1.35

27.0

1.53

82.7

0.95

100.7

1.05

8.7

0.87

11.7

1.17

8.7

1.13

7.3

1.05

21.3

0.84

30.0

1.22

NPD (4mg)

240.3

15.67

SAZ (2mg)

1154.7

13.53

962.7

99.59

9AA (50mg)

318.0

45.43

MMS (2mL)

644.0

25.76

2AA (2mg)

1325.3

62.13

1429.3

15.05

146.7

11.89

95.7

11.48

2AA (50mg)

204.0

5.51

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

Remarks:           DMSO (200 µL) was applied as solvent of the test item and DMSO (100 µL) was applied as solvent of the positive control substances 9AA, NPD and 2AA. The ultrapure water (100 µL) was applied as solvent of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (200 µL); the mutation rate of the 9AA, NPD and 2AA refers to the DMSO sample (100 µL). The mutation rate of MMS and SAZ refers to ultrapure water (100 µL).


Summary Table of the Results of the Confirmatory Mutation Test

Confirmatory Mutation Test (Pre-Incubation Test)

Concentrations (mg/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

16.7

1.14

24.0

1.16

99.3

1.27

115.0

1.58

9.3

1.04

9.7

1.07

8.0

1.33

9.0

1.08

34.3

1.37

37.3

1.12

DMSO Control (100 µL)

21.7

1.00

18.0

1.00

98.7

1.00

90.7

1.00

9.3

1.00

10.0

1.00

7.3

1.00

7.7

1.00

27.7

1.00

35.7

1.00

DMSO Control (200µL)

14.7

1.00

20.7

1.00

78.3

1.00

73.0

1.00

9.0

1.00

9.0

1.00

6.0

1.00

8.3

1.00

25.0

1.00

33.3

1.00

Ultrapure Water Control

100.7

1.00

11.3

1.00

29.7

1.00

5000

4.3

0.30

11.7

0.56

25.0

0.32

53.7

0.74

3.3

0.37

7.0

0.78

0.0

0.00

4.3

0.52

25.0

1.00

32.7

0.98

1600

7.3

0.50

15.3

0.74

45.3

0.58

103.0

1.41

5.7

0.63

5.7

0.63

0.0

0.00

4.3

0.52

28.7

1.15

27.7

0.83

500

9.0

0.61

17.3

0.84

72.7

0.93

106.7

1.46

7.3

0.81

11.3

1.26

1.3

0.22

16.7

2.00

34.7

1.39

30.0

0.90

160

16.7

1.14

20.7

1.00

79.7

1.02

84.7

1.16

10.0

1.11

8.0

0.89

6.7

1.11

15.7

1.88

32.7

1.31

31.0

0.93

50

13.7

0.93

20.3

0.98

100.3

1.28

93.3

1.28

7.7

0.85

10.0

1.11

5.0

0.83

18.0

2.16

23.7

0.95

38.7

1.16

16

14.0

0.95

23.3

1.13

83.3

1.06

87.7

1.20

9.3

1.04

10.0

1.11

7.7

1.28

12.7

1.52

20.3

0.81

32.0

0.96

5

10.7

0.73

23.3

1.13

77.3

0.99

86.0

1.18

9.7

1.07

12.3

1.37

8.7

1.44

6.7

0.80

21.0

0.84

31.7

0.95

NPD (4mg)

226.0

10.43

SAZ (2mg)

1829.3

18.17

525.3

46.35

9AA (50mg)

273.7

37.32

MMS (2mL)

1344.0

45.30

2AA (2mg)

1213.3

67.41

1626.7

17.94

148.3

14.83

155.3

20.26

2AA (50mg)

162.0

4.54

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

Remarks:           DMSO (200 µL) was applied as solvent of the test item and DMSO (100 µL) was applied as solvent of the positive control substances 9AA, NPD and 2AA. The ultrapure water (100 µL) was applied as solvent of the positive control substances MMS and SAZ. The mutation rate of the test item and the untreated control refers to the DMSO sample (200 µL); the mutation rate of the 9AA, NPD and 2AA refers to the DMSO sample (100 µL). The mutation rate of MMS and SAZ refers to ultrapure water (100 µL).

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

Applicant's summary and conclusion

Conclusions:
The test item has no mutagenic activity on the applied bacterium tester strains 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 experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimuriumTA98, TA100, TA1535 and TA1537), and the tryptophan-requiring auxotroph strain ofEscherichia coli(Escherichia coliWP2uvrA) in the presence and absence of a post mitochondrial supernatant (S9) prepared from livers of Phenobarbital/b-naphthoflavone-induced rats. The study included preliminary solubility tests, preliminary concentration range finding tests (informatory toxicity tests), an initial mutation test (plate incorporation test), and a confirmatory mutation test (pre-incubation test). Based on the results of the solubility tests and the concentration range finding tests the test item was dissolved in dimethyl sulfoxide (DMSO). At the formulation of test item solutions correction of concentrations for active component content (86.65 % with rounding 87 %) was made in the main experiments. Based on the results of the preliminary concentration range finding tests (informatory toxicity tests) the following concentrations of the test item were prepared and investigated in the initial and confirmatory mutation tests: 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 precipitation of the test item in the final treatment mixture were taken into consideration. The observations were made by naked eye. 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 range of 5000-500 µg/plate in the absence and at 5000 and 1600 µg/plate in presence of S9 following the plate incorporation and pre-incubation procedures. An inhibitory effect of the test item was observed in the initial mutation test in the S. typhimurium TA1537 strain, in the confirmatory mutation test in the S. typhimurium TA98, TA100 and TA1537 strains in the absence and also presence of exogenous metabolic activation (slight inhibition was noticed in TA1535, in absence of S9). The inhibitory effect was indicated by absent or decreased revertant colony counts (most of them below the corresponding historical control data ranges) and/or affected background lawn development: reduced or slightly reduced background lawn. In general, 500 µg/plate (noticed in S. typhimurium TA98 and TA1537) was considered as lowest concentration showing cytotoxicity. The revertant colony numbers of solvent 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 colonies and the number of revertants mostly fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in 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 the test item 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.