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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP.

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
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: March 2013; signature: May 2013
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Physical state: liquid
- Storage condition of test material: In refrigerator (2-8°C) in the dark
- Other: Clear colourless

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Dose range finding study: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 ug/plateExperiment 1: TA1535, TA1537 and TA98: 0.5, 1.7, 5.4, 52, 164, 512 ug/plate
Experiment 2: TA1535, TA1537 and TA100 and TA98 strains: 9,16, 30, 51, 92, 164 ug/plate; WP2uvrA strain: 492, 1568, 2800, 5000 ug/plate
Experiment 3: TA1535 and TA98 strains only: 92, 164, 492 and 878 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble in dimethyl sulfoxide (DMSO).
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter.

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (bacterial background lawn) and reduction in the number of revertants

OTHER:
- Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix.
- To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test substance was tested up to the dose level of 164 μg/plate in strains TA1535, TA1537, TA98, TA100 and 5000 μg/plate in strain WP2uvrA.- Since no toxicity and no precipitate on the plates was observed in tester strains TA1535 and TA98 in the presence of S9-mix in experiment 2, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The following dose range was selected for the third mutation assay: 92, 164, 492 and 878 μg/plate.
Evaluation criteria:
See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.
Statistics:
No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test resultsopen allclose all
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity was observed at the top two dose levels dose of 5000 μg/plate in tester strain TA1535 and TA100 (absence and presence of S9-mix) and TA1535 and TA98 (with and without S9-mix)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of test substance on the plates was not observed at the start or at the end of the incubation period in any tester strain.

RANGE-FINDING/SCREENING STUDIES:
Test substance was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. The highest concentration the test substance used in the subsequent mutation assay was 512 μg/plate (experiment 1) based on the results of this assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Dose range finding test: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain

 

 

TA100   

 

WP2uvrA

 

 

Without S9-mix

 

 

 

 

Positive control

853

±

1221

± 76

Solvent control

117

±

30

± 1

1.7

108

±

33

± 5

5.4

123

±

36

± 4

17

121

±

33

± 8

52

111

±

33

± 4

164

 

e MC

27

± 4

512

0

± 0 a

32

± 9

1600

0

± 0 a

24

± 5 n

5000

0

± 0 a NP

15

± 5 s NP

With S9-mix #1

 

 

 

 

Positive control

1249

± 53

141

± 27

Solvent control

116

± 21

46

± 10

1.7

94

± 6

43

± 9

5.4

115

± 9

39

± 9

17

117

± 20

37

± 7

52

114

± 16 n

42

± 5

164

61

± 8 m

37

± 3

512

 

e MC

33

± 9

1600

0

± 15 n

25

± 7

5000

0

± 12 s NP

2

± 2 n NP

#1          Plate incorporation assay (5% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 2 Experiment 1: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium

 

 

TA1535

 

TA1537

 

 

TA98

 

Without S9-mix

 

 

 

 

 

 

Positive control

641

± 35

310

± 11

716

± 33

Solvent control

24

± 2

8

± 2

17

± 4

0.5

22

± 8

7

± 4

21

± 14

1.7

30

± 6

11

± 4

15

± 1

5.4

28

± 2

9

± 7

10

± 2

17

26

± 9 n

8

± 3 n

21

± 8 n

52

13

± 3 s

5

± 3 m

13

± 6 m

164

 

e MC

0

± 0 a

0

± 0 a

512

0

± 0 a NP

0

± 0 a NP

0

± 0 a NP

With S9-mix #1

 

 

 

 

 

 

Positive control

289

± 9

351

±

769

± 59

Solvent control

20

± 9

12

±

25

± 4

0.5

20

± 6

13

±

26

± 4

1.7

13

± 5

11

±

21

± 8

5.4

22

± 9

12

±

24

± 7

17

19

± 1

7

±

24

± 5

52

11

± 3

9

±

20

± 4 n

164

 

e MC

 

e MC

 

e MC

512

 

e NP MC

0

± 0 NP

 

e MC

#1          Plate incorporation assay (5% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

 

Table 3 Experiment 2: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 

 

TA1535

 

TA1537

 

 

TA98

 

TA100

 

WP2uvrA

 

Without S9-mix

 

 

 

 

 

 

 

 

 

 

Positive control

713

± 21

445

± 23

823

± 60

981

± 46

1168

± 355

Solvent control

19

± 4

7

± 6

18

± 5

115

± 5

18

± 6

9

22

± 3

7

± 4

19

± 2

114

± 15

-

-

16

27

± 4

9

± 3

22

± 3

114

± 4

-

-

30

23

± 3 n

5

± 1 n

19

± 4 n

123

± 22 n

-

-

51

15

± 5 m

7

± 2 m

17

± 2 s

103

± 5 s

-

-

92

 

e MC

 

e MC

 

e MC

 

e MC

-

-

164

 

e NP MC

0

± 0 a NP

0

± 0 a NP

0

± 0 a NP

-

-

492

-

-

-

-

-

-

-

-

20

± 2

878

-

-

-

-

-

-

-

-

17

± 4

1568

-

-

-

-

-

-

-

-

21

± 9 n

2800

-

-

-

-

-

-

-

-

21

± 3 s

5000

-

-

-

-

-

-

-

-

10

± 3 m NP

With S9-mix #1

 

 

 

 

 

 

 

 

 

 

Positive control

169

± 5

473

± 10

409

± 27

1279

± 57

163

± 10

Solvent control

12

± 6

15

± 6

27

± 4

114

± 23

33

± 5

9

16

± 8

9

± 3

27

± 4

102

± 14

-

-

16

14

± 2

13

± 2

29

± 2

108

± 10

-

-

30

20

± 2

15

± 2

25

± 7

106

± 14

-

-

51

11

± 6

12

± 5

27

± 8

107

± 10

-

-

92

16

± 3

9

± 2 n

17

± 6

115

± 8 n

-

-

164

7

± 4 n NP

0

± 0 a NP

19

± 5 n NP

81

± 5 m NP

-

-

492

-

-

-

-

-

-

-

-

20

± 5

878

-

-

-

-

-

-

-

-

26

± 5

1568

-

-

-

-

-

-

-

-

22

± 8

2800

-

-

-

-

-

-

-

-

20

± 5

5000

-

-

-

-

-

-

-

-

27

± 11 n NP

#1          Plate incorporation assay (10% S9)

MC        Microcolonies

NP         No precipitate

a            Bacterial lawn absent

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

s            Bacterial background lawn slightly reduced

-             Not tested

 

Table 4 Experiment 3: Mutagenic response of test substance in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)

Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium.

 

TA1535

 

TA98

 

 

With S9-mix #1

 

 

 

 

Positive control

176

± 15

472

± 74

Solvent control

16

± 8

22

± 10

92

15

± 7 n

14

± 3

164

10

± 2 m

27

± 4 n

492

 

e MC

 

e MC

878

 

e NP MC

 

e NP MC

 

 

 

 

 

#1          Plate incorporation assay (10% S9)

MC        Microcolonies

NP         No precipitate

e            Bacterial lawn extremely reduced

m           Bacterial lawn moderately reduced

n            Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:
Interpretation of results:
negative
Under the conditions of this study the test item was considered to be non-mutagenic in the presence and absence of S9 activation. Negative and strain specific positive control values were within laboratory historical control data.
Executive summary:

The study was performed to OECD 471, EU Method B.13/14, EPA OPPTS 870.5100 and the Japan Guidelines for Screening Mutagenicity of Chemicals in accordance with GLP; to evaluate the mutagenic activity of the test substance in the Salmonella typhimurium and the Escherichia coli in a reverse mutation assay (with independent repeat). In the dose range finding test, the test substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test substance did not precipitate on the plates at this dose level. In tester strain TA100, toxicity was observed at dose levels of 164 μg/plate and above in the absence and presence of S9-mix. In tester strain WP2uvrA, toxicity was observed at the dose level of 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test the test substance was tested in the first mutation assay at a concentration range of 0.5 to 512 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. Toxicity was observed in all three tester strains. In an independent repeat of the assay with additional parameters, the test substance was tested at a concentration range of 9 to 164 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strain WP2uvrA. In the second mutation assay, in the absence of S9-mix, toxicity was observed in all tester strains. In the presence of S9-mix, toxicity was only observed in tester strains TA1537 and TA100. Since in the second experiment in the presence of S9-mix, insufficient toxicity without precipitate on the plates was observed in tester strains TA1535 and TA98, an additional mutation experiment was performed in the presence of 10% (v/v) S9-mix. The test substance was tested up to the dose level of 878 μg/plate. In the third mutation assay, toxicity was observed in both tester strains. The test substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the conditions of this study it is concluded that that the test substance was not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.