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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:
2005-06-20 to 2006-04-24
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

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:
2000
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: V3186/11
- Date of manufacture: December 7, 2004

Letter of Confirmation: CAS No 90530-15-7 and 93940-97-7 refer to the same substance and substance composition.

Method

Target gene:
his; trp
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of Aroclor induced rat liver
Test concentrations with justification for top dose:
both experiments:
0; 20; 100; 500; 2500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in water, DMSO was selected as the vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylendiamine
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine
Untreated negative controls:
yes
Remarks:
sterility control
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 - 72 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants; clearing or diminution of the background lawn (= reduced his- or trp- background growth); reduction in the titer

Rationale for test conditions:
according to guidelines
Evaluation criteria:
The test chemical is considered positive in this assay if the following criteria are met:
A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.

A test substance is generally considered nonmutagenic in this test if:
The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

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:
valid
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:
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
- Effects of pH: no
- Effects of osmolality: no
- Evaporation from medium: no
- Water solubility: not soluble in water
- Precipitation: no
- Other confounding effects: none

HISTORICAL CONTROL DATA
please refer to "any other informations on results"

Any other information on results incl. tables

 Experiment 1 Standard plate test

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli

 

TA 100

TA 98

TA 1535

TA 1537

WP2 uvrA

 

 

 

Results without S9

DMSO

111 ± 8

31 ± 5

16 ± 3

8 ± 2

31 ± 4

20

108 ± 4

24 ± 6

17 ± 1

8 ± 1

27 ± 5

100

108 ± 6

28 ± 1

19 ± 3

8 ± 2

34 ± 3

500

102 ± 4

24 ± 4

15 ± 2

9 ± 2

29 ± 1

2500

102 ± 11

26 ± 1

16 ± 1

7 ± 1

31 ± 2

5000

107 ± 7

17 ± 4

16 ± 2

6 ± 1

32 ± 5

MNNG (5)

648 ± 36

-

634 ± 43

-

-

NOPD (10)

-

504 ± 26

-

-

-

AAC (100)

-

-

-

429 ± 20

-

4-NQO (5)

-

-

-

-

556 ± 24

 

 

 

Results with S9 (1:9)

DMSO

114 ± 9

35 ± 3

16 ± 3

13 ± 2

35 ± 5

20

117 ± 12

30 ± 3

16 ± 2

11 ± 3

32 ± 6

100

116 ± 11

35 ± 11

15 ± 2

10 ± 2

35 ± 3

500

105 ± 4

33 ± 3

18 ± 3

8 ± 1

38 ± 5

2500

101 ± 4

30 ± 5

13 ± 3

9 ± 3

40 ± 6

5000

114 ± 18

31 ± 3

14 ± 3

7 ± 1

33 ± 2

2- AA (2.5; 60)

828 ± 27

593 ± 39

130 ± 21

133 ± 8

231 ± 18

 

Experiment 2 Preincubation test

Dose (µg/plate)

Mean number of revertant colonies of 3 replicates (± S.D.) for different strains of S. typhimurium and E. coli

 

TA 100

TA 98

TA 1535

TA 1537

WP2 uvrA

 

 

 

Results without S9

DMSO

110 ± 4

30 ± 1

17 ± 2

8 ± 1

31 ± 5

20

108 ± 6

27 ± 3

15 ± 1

8 ± 3

29 ± 6

100

101 ± 10

28 ± 3

17 ± 2

8 ± 2

29 ± 3

500

103 ± 2

28 ± 4

16 ± 1

9 ± 5

27 ± 6

2500

101 ± 7

23 ± 4

15 ± 1

8 ± 2

25 ± 4

5000

99 ± 3

14 ± 2

15 ± 1

5 ± 3

21 ± 0

MMNG (5)

674 ± 50

-

553 ± 24

-

-

NOPD (10)

-

422 ± 29

-

-

-

AAC (100)

-

-

-

397 ± 25

-

4-NQO (5)

-

-

-

-

620 ± 19

 

 

 

Results with S9 (1:9)

DMSO

107 ± 6

31 ± 2

17 ± 3

9 ± 2

35 ± 6

20

104 ± 12

32 ± 3

15 ± 2

11 ± 3

30 ± 6

100

104 ± 1

31 ± 3

17 ± 2

11 ± 4

30 ± 6

500

101 ± 5

25 ± 3

15 ± 3

7 ± 2

27 ± 2

2500

99 ± 6

22 ± 3

14 ± 4

7 ± 1

27 ± 6

5000

96 ± 11

13 ± 2

12 ± 1

5 ± 3

24 ± 4

2- AA (2.5)

631 ± 21

598 ± 78

141 ± 19

120 ± 20

225 ± 22

 

Table 3 Historical control values for revertants/plate: Standard Plate Test

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

29

108

18

10

32

SD

4

8

2

2

5

Minimum

19

87

11

5

25

Maximum

48

140

25

16

53

+S9 (2 -AA)

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

36

110

17

11

35

SD

5

9

2

2

5

Minimum

21

91

10

5

25

Maximum

50

154

24

18

58

Historical control data of positive

control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

750

953

853

440

671

SD

125

258

205

94

159

Minimum

332

506

502

300

502

Maximum

1200

1795

1582

1042

1478

+S9 (2-AA

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

786

957

139

136

323

SD

157

225

29

26

28

Minimum

504

573

85

200

153

Maximum

1745

1878

254

136

324

 

Table 4 Historical control values for revertants/plat: Preincubation test

 

 

Bacterial strains

Historical control data of DMSO control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

28

109

17

10

32

SD

4

9

2

2

5

Minimum

17

84

12

5

25

Maximum

40

148

23

17

51

+S9 (2 -AA)

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

32

110

17

10

33

SD

5

9

2

2

5

Minimum

20

147

10

5

25

Maximum

49

91

24

16

50

Historical control data of positive

control

-S9

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

739

842

747

420

599

SD

123

170

145

72

77

Minimum

388

511

502

278

500

Maximum

1129

1583

1830

842

1150

+S9 (2-AA)

 

TA 98

TA 100

TA 1535

TA 1537

E. coli

Average

665

740

122

115

230

SD

103

143

21

20

27

Minimum

501

500

80

80

150

Maximum

1120

1380

258

200

307

Applicant's summary and conclusion