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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:
07 - 24 May 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted in 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
adopted in 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
adopted in 1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
his operon
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/beta-naphthoflavone
Test concentrations with justification for top dose:
Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation

5000 µg/plate was chosen as maximal concentration since minor toxic effects were observed in the pre-test/experiment I.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The solvent DMSO was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (4-NOPD), 2-aminoanthracene (2-AA)
Remarks:
+ S9: 2-AA (2.5 µg/plate in TA 1535, TA 1537, TA 98, TA 100; 10 µg/plate in TA 102) - S9: sodium azide (10 µg/plate in TA 1535, TA 100), 4-NOPD (10 µg/plate in TA 98; 50 µg/plate in TA 1537), methylmethanesulfonate (2.0 µL/plate in TA 102)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replications each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: other: number of revertants
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration. An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
Mean values and standard deviation were calculated.

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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
2500 and 5000 µg/plate
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, but tested up to precipitating concentrations
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, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the pre-experiment/experiment 1 the test item precipitated in the overlay agar in the test tubes and on the incubated agar plates from 1000 to 5000 μg/plate. In experiment II precipitation of the test item was observed in the overlay agar in the test tubes and on the incubated agar plates from 2500 to 5000 μg/plate. The undissolved particles had no influence on the data recording.

RANGE-FINDING/SCREENING STUDIES: A pre-test (experiment I, plate incorporation method) was performed with all strains used to evaluate the toxicity of the test item and to choose a concentration range for experiment II. The test substance was tested with and without metabolic activation at eight concentrations ranging from 3 - 5000 µg/plate. A reduction in the number was observed in one strain only (TA 1537) at 2500 and 5000 µg/plate (precipitating concentrations) in the absence of metabolic activation. Thus, the same concentrations with the exception of 3 µg/plate were used in experiment II.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The historical control data represent approximately 550 experiments for TA 1535, TA 1537, TA 98 and TA 100 and approximately 200 experiments for TA 102 (from 2011; see table 1).
- Negative (solvent/vehicle) historical control data: The historical control data represent approximately 550 experiments for TA 1535, TA 1537, TA 98 and TA 100 and approximately 200 experiments for TA 102 (from 2011; see table 1).

Any other information on results incl. tables

Table 1 Historical control data

Strain

 

without S9 mix

with S9 mix

Mean

SD

Min

Max

Mean

SD

Min

Max

TA 1535

Solvent control

14

2.37

9

23

20

3.75

11

35

Untreated control

14

2.87

7

25

20

3.82

10

32

Positive control

1751

226.44

710

2385

367

93.12

126

703

TA 1537

Solvent control

12

3.31

5

26

16

4.34

7

30

Untreated control

12

4.03

5

29

18

4.92

6

33

Positive control

88

38.92

61

448

342

144.31

77

809

TA 98

Solvent control

30

5.60

17

47

40

6.08

21

58

Untreated control

31

6.36

17

55

42

6.83

24

68

Positive control

372

78.05

158

595

2167

717.60

249

4089

TA 100

Solvent control

142

29.42

86

243

156

29.4

99

249

Untreated control

450

28.19

86

248

163

31.26

94

281

Positive control

1741

488.75

569

3082

2642

796.59

825

4503

TA 102

Solvent control

380

43.64

305

510

502

89.88

321

677

Untreated control

372

41.71

300

479

511

87.27

315

659

Positive control

2499

898.06

1080

4678

2329

598.00

1091

3972

Mean = mean value of revertants/plate; SD = standard deviation; Min = minimal value; Max = maximal value

Table 2. Test results of the pre-experiment/experiment I (plate incorporation)

With or without S9-Mix

Test substance concentration [µg/plate]

Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)

TA 1535

TA 1537

TA 98

TA 100

TA 102

-

0 (DMSO)

13 ± 3

10 ± 4

33 ± 3

88 ± 4

355 ± 22

-

0

14 ± 1

11 ± 2

37 ± 6

98 ± 6

329 ± 24

-

3

15 ± 2

10 ± 5

34 ± 6

83 ± 2

325 ± 49

-

10

11 ± 2

7 ± 2

30 ± 6

85 ± 4

366 ± 29

-

33

12 ± 4

9 ± 1

24 ± 3

90 ± 6

366 ± 19

-

100

16 ± 5

8 ± 0

35 ± 9

89 ± 23

311 ± 29

-

333

13 ± 3

7 ± 2

30 ± 1

87 ± 12

317 ± 9

-

1000

13 ± 8P

8 ± 2P

27 ± 6P

90 ± 23P

316 ± 2P

-

2500

11 ± 2P,M

4 ± 2P,M

28 ± 4P,M

94 ± 9P,M

224 ± 27P,M

-

5000

9 ± 2P,M

3 ± 1P,M

21 ± 1P,M

92 ± 12P,M

172 ± 9P,M

Positive controls,

- S9

Na-azide,

10 µg/plate

1913 ± 54

 

 

1949 ± 122

 

4-NOPD,

10 µg/plate

 

 

254 ± 69

 

 

4-NOPD,

50 µg/plate

 

72 ± 6

 

 

 

MMS,

2.0 µL/plate

 

 

 

 

3681 ± 508

+

0 (DMSO)

20 ± 3

18 ± 4

29 ± 6B,M

137 ± 10

484 ± 36

+

0

16 ± 5

16 ± 1

36 ± 6B,M

147 ± 10

533 ± 17

+

3

19 ± 6

18 ± 3

27 ± 5B,M

131 ± 10

460 ± 34

+

10

16 ± 4

16 ± 4

26 ± 6B,M

137 ± 11

494 ± 26

+

33

21 ± 4

17 ± 5

23 ± 3B,M

126 ± 8

512 ± 65

+

100

18 ± 7

15 ± 2

28 ± 4B,M

140 ± 3

459 ± 47

+

333

17 ± 4

21 ± 2

29 ± 2B,M

132 ± 11

439 ± 9

+

1000

18 ± 4

12 ± 3

31 ± 7B,M

111 ± 8

485 ± 39

+

2500

16 ± 3P

10 ± 1P,M

31 ± 4B,P,M

115 ± 11P

514 ± 11P

+

5000

12 ± 2P,M

10 ±3P,M

33 ± 8B,P,M

119 ± 6P,M

410 ± 1P,M

Positive controls,

+ S9

2-AA,

2.5 µg/plate

414 ± 23

373 ± 35

1905 ± 100B,M

3106 ± 300

 

2-AA,

10 µg/plate

 

 

 

 

3158 ± 70

Na-azide: sodium azide; 4-NOPD: 4-nitro-o-phenylene diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene

P = precipitate; M = manual count, B = extensive bacterial growth

Table 3. Test results of the experiment II (preincubation)

With or without S9-Mix

Test substance concentration [µg/plate]

Mean number of revertant colonies per plate (average of 3 plates ± standard deviation)

TA 1535

TA 1537

TA 98

TA 100

TA 102

-

0 (DMSO)

14 ± 5

9 ± 0

25 ± 9

90 ± 3

363 ± 4

-

0

15 ± 4

8 ± 4

32 ± 12

98 ± 5

384 ± 19

-

10

12 ± 6

9 ± 2

25 ± 7

101 ± 15

391 ± 18

-

33

13 ± 3

8 ± 0

25 ± 4

81 ± 3

381 ± 20

-

100

11 ± 3

9 ± 1

25 ± 3

98 ± 12

359 ± 27

-

333

13 ± 5

9 ± 3

28 ± 2

85 ± 7

368 ± 34

-

1000

12 ± 2

8 ± 1

22 ± 7

98 ± 19

398 ± 22

-

2500

14 ± 1P

8 ± 2P,M

24 ± 6P

87 ± 11P

399 ± 10P

-

5000

11 ± 3P,M

5 ± 3P,M

20 ± 2P,M

89 ± 11P,M

330 ± 42P,M

Positive controls,

- S9

Na-azide,

10 µg/plate

1939 ± 64

 

 

2144 ± 26

 

4-NOPD,

10 µg/plate

 

 

342 ± 28

 

 

4-NOPD,

50 µg/plate

 

68 ± 7

 

 

 

MMS,

2.0 µL/plate

 

 

 

 

3761 ± 15

+

0 (DMSO)

20 ± 3

18 ± 4

44 ± 8

129 ± 13

522 ± 27

+

0

18 ± 3

20 ± 5

45 ± 3

152 ± 10

493 ± 6

+

10

20 ± 4

17 ± 3

42 ± 5

133 ± 17

522 ± 25

+

33

22 ± 9

15 ± 7

37 ± 3

137 ± 21

527 ± 37

+

100

18 ± 4

19 ± 6

47 ± 15

137 ± 11

546 ± 63

+

333

19 ± 6

21 ± 2

41 ± 6

142 ± 14

476 ± 29

+

1000

23 ± 3

17 ± 2

50 ± 2

127 ± 9

509 ± 19

+

2500

20 ± 3P

15 ± 5P

61 ± 4P

124 ± 4P

557 ± 22P

+

5000

13 ± 5P,M

12 ± 4P,M

46 ± 8P,M

127 ± 9P,M

534 ± 23P,M

Positive controls,

+ S9

2-AA,

2.5 µg/plate

415 ± 40

269 ± 8

2314 ± 458

3638 ± 20

 

2-AA,

10 µg/plate

 

 

 

 

2217 ± 259

Na-azide: sodium azide; 4-NOPD: 4-nitro-o-phenylene diamine; MMS: methylmethanesulfonate; 2-AA: 2-aminoanthracene

P = precipitate; M = manual count

Applicant's summary and conclusion