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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:
From 18 March 2009 to 16 July 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

Method

Target gene:
The histidine gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: histidine deficient (His G 46), rfa and uvrb mutations
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: histidine deficient (His G 46), rfa and uvrb mutations, addition of the plasmid pKM101
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: histidine deficient (His G 428 (pAQ1), rfa mutation, addition of the plasmid pKM101
Species / strain / cell type:
S. typhimurium TA 1537
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: histidine deficient (His C 3076), rfa and uvrb mutations
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: histidine deficient (His D 3052), rfa and uvrb mutations, addition of the plasmid pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254
Test concentrations with justification for top dose:
- Preliminary toxicity test: 10, 100, 500, 1000, 2500 or 5000 µg/plate
- Mutagenicity experiments: Since the test item was toxic in the preliminary test, the choice of the highest dose-level retained for the main experiments was based on the level of toxicity. See "Any other information" for details.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: DMSO (Batch number K36449950707 and K36994550731, supplier: Merck KGaA, Darmstadt, Germany)
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Without S9 mix: sodium azide (1 µg/plate) for TA 1535 and TA 100; 9-Aminoacridine (50 µg/plate) for TA 1537; 2-Nitrofluorene (0.5 µg/plate) for TA 98: Mitomycin C (0.5 µg/plate) for TA 102.
Remarks:
In the absence of S9 Mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9 mix: 2-Anthramine (2 µg/plate) for TA 1535, TA 1537 and TA 98; Benzo(a)pyrene (5 µg/plate) for TA 100 and 2-Anthramine (10 µg/plate) for TA 102.
Remarks:
In the presence of S9 Mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) except for the second test with S9 mix, which was performed according to the preincubation method

DURATION
- Preincubation period:
Plate incorporation method: not applicable
Preincubation method: 60 minutes at 37 °C
- Exposure duration: 48 to 72 hours at 37°C

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

SELECTION AGENT (mutation assays):
- histidine production
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive
result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Statistics:
No data

Results and discussion

Test resultsopen allclose all
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
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no precipitate was observed in the Petri plates when scoring the revertants at any dose-level.
- Other confounding effects: no data

PRELIMINARY TOXICITY TEST: A moderate to strong toxicity (decrease in the number of revertants and thinning of the bacterial lawn) was noted at dose-levels ≥ 1000 μg/mL in the TA 100 strain and at dose-levels ≥ 2500 μg/mL in the TA 98 and TA 102 strains, either with or without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA:
- Without S9 mix: A slight increase in the number of revertants was noted in the TA 1537 strain in the second experiment (2.2-fold the vehicle control value). Since this increase did not reach the threshold of 3-fold the vehicle control value and since it was due to only one out of the three plates used, and was neither dose-related nor observed in the first experiment, it was not considered as biologically relevant. A slight increase in the number of revertants was noted in the TA 98 strain in the second experiment (2-fold the vehicle control value). This increase reached the threshold of 2- fold the vehicle control value but it was neither dose-related, nor observed in the first and third experiment. Therefore, it was considered as non-biologically relevant.
- With S9 mix: In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 1250 μg/plate in the TA 1535 and TA 100 strains and at dose-levels ≥ 2500 μg/plate in the TA 1537, TA 98 and TA 102 strains. In the second experiment, performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at dose-levels ≥ 625 μg/plate towards the TA 102 strain.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Without S9 mix: A moderate to strong toxicity was noted at dose-levels ≥ 625 μg/plate in the TA 1537, TA 98, TA 100 and TA 102 strains and at dose-levels ≥ 1250 μg/plate in the TA 1535 strain.
- With S9 mix: In the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 1250 μg/plate in the TA 1535 and TA 100 strains and at dose-levels ≥ 2500 μg/plate in the TA 1537, TA 98 and TA 102 strains. In the second experiment, performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5 μg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at dose-levels ≥ 625 μg/plate towards the TA 102 strain.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 7.6.1/1: Preliminary toxicity test – Direct plate incorporation method

Strain

Compound

Dose level per plate

S-9 mix

Mean revertant colony count

SD

Ratio treated / solvent

Individual revertant colony count

TA 98

DMSO

TEST ITEM

 

 

 

 

 

 

DMSO

TEST ITEM

 

10 µg

100 µg

500 µg

1000 µg

2500 µg

5000 µg

 

 

10 µg

100 µg

500 µg

1000 µg

2500 µg

5000 µg

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

20

47

25

32

22

5

0

 

41

30

44

48

37

14

0

-

-

-

-

-

-

-

 

-

-

-

-

-

-

-

 

2.4

1.3

1.6

1.1

0.3

0.0

 

 

0.7

1.1

1.2

0.9

0.3

0.0

20

47

25

32

22

5ST

0ST

 

41

30

44

48

37

14MT

0ST

TA 100

DMSO

TEST ITEM

 

 

 

 

 

 

DMSO

TEST ITEM

 

10 µg

100 µg

500 µg

1000 µg

2500 µg

5000 µg

 

 

10 µg

100 µg

500 µg

1000 µg

2500 µg

5000 µg

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

145

139

187

145

109

36

0

 

147

159

141

164

132

5

0

-

-

-

-

-

-

-

 

-

-

-

-

-

-

-

 

1.0

1.3

1.0

0.8

0.2

0.0

 

 

1.1

1.0

1.1

0.9

0.0

0.0

145

139

187

145

109MT

36ST

0ST

 

147

159

141

164

132

5 St

0 St

TA 102

DMSO

TEST ITEM

 

 

 

 

 

 

DMSO

TEST ITEM

 

10 µg

100 µg

500 µg

1000 µg

2500 µg

5000 µg

 

 

10 µg

100 µg

500 µg

1000 µg

2500 µg

5000 µg

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

396

405

432

322

325

85

0

 

517

526

578

519

395

157

0

-

-

-

-

-

-

-

 

-

-

-

-

-

-

-

 

1.0

1.1

0.8

0.8

0.2

0.0

 

 

1.0

1.1

1.0

0.8

0.3

0.0

396

405

432

322

325

85ST

0ST

 

517

526

578

519

395

157MT

0ST

SD: Standard Deviation

MT: Moderate toxicity

ST: Strong toxicity

- Absence of S9

+ Presence of S9

 

Table 7.6.1/2: First experiment – Direct plate incorporation method

Strain

Compound

Dose level per plate

S-9 mix

Mean revertant colony count

SD

Ratio treated / solvent

Individual revertant colony count

TA 1535

DMSO

TEST ITEM

 

 

 

 

NAN3

 

DMSO

TEST ITEM

 

 

 

 

2 AM

 

78.1 µg

156.3 µg

312.5 µg

625 µg

1250 µg

1 µg

 

 

156.3 µg

312.5 µg

625 µg

1250 µg

2500 µg

2 µg

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

26

21

33

25

32

20

681

 

21

22

20

17

15

6

175

8

8

8

9

3

3

18

 

3

2

2

7

3

5

23

 

0.8

1.3

1.0

1.2

0.8

25.8

 

 

1.0

1.0

0.8

0.7

0.3

8.3

22, 35, 22

30, 16,16

41, 26, 32

31, 30, 15

34, 34, 28

18, 18, 24

687, 695, 660

 

18, 23, 22

23, 22, 20

19, 20, 22

23, 19, 10

14MT, 18MT, 13MT

11ST, 2ST, 5ST

183, 150, 193

TA 1537

DMSO

TEST ITEM

 

 

 

 

 

9AA

 

DMSO

TEST ITEM

 

 

 

 

2 AM

 

39.1 µg

78.1 µg

156.3 µg

312.5 µg

625 µg

1250 µg

50 µg

 

 

156.3 µg

312.5 µg

625 µg

1250 µg

2500 µg

2 µg

-

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

10

7

13

14

12

15

8

1036

 

7

13

9

14

12

7

177

8

1

3

3

5

1

5

273

 

3

1

5

3

2

3

8

 

0.7

1.4

1.4

1.2

1.5

0.9

107.1

 

 

1.7

1.3

1.9

1.7

1.0

24.1

6, 9, 14

7, 8, 6

11, 16, 13

13, 11, 17

17, 11, 8

14, 16, 14

6MT, 5MT, 14MT

911, 847, 1359

 

10, 5, 7

14, 12, 12

13, 4, 11

12, 12, 18

13, 14, 10

11ST, 5ST, 5ST

181, 181, 168

TA 98

DMSO

TEST ITEM

 

 

 

 

2NF

 

DMSO

TEST ITEM

 

 

 

 

2 AM

 

78.1 µg

156.3 µg

312.5 µg

625 µg

1250 µg

0.5 µg

 

 

156.3 µg

312.5 µg

625 µg

1250 µg

2500 µg

2 µg

-

-

-

-

-

-

-

 

 

+

+

+

+

+

+

31

36

23

25

28

25

201

 

41

34

42

34

23

7

1606

10

3

9

7

3

5

12

 

1

7

7

8

8

2

174

 

1.2

0.7

0.8

0.9

0.8

6.5

 

 

0.8

1.0

0.8

0.6

0.2

38.9

38, 20, 35

40, 34, 34

32, 23, 14

32, 26, 18

29, 25, 30

20, 25, 30

190, 198, 214

 

41, 41, 42

32, 29, 42

35, 49, 42

32, 43, 28

20, 17, 32

8ST, 7ST, 5ST

1637, 1419, 1762

TA 100

DMSO

TEST ITEM

 

 

 

 

 

NAN3

 

DMSO

TEST ITEM

 

 

 

 

 

BAP

 

39.1 µg

78.1 µg

156.3 µg

312.5 µg

625 µg

1250 µg

1 µg

 

 

78.1 µg

156.3 µg

312.5 µg

625 µg

1250 µg

2500 µg

5 µg

-

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

+

157

132

153

145

153

104

21

1027

 

128

112

110

110

86

27

5

391

3

11

22

20

8

4

20

28

 

12

13

14

9

14

3

5

32

 

0.8

1.0

0.9

1.0

0.7

0.1

6.5

 

 

0.9

0.9

0.9

0.7

0.2

0.0

3.1

156, 155, 160

126, 144, 125

153, 175, 132

146, 125, 164

162, 152, 146

108MT, 102MT, 101MT

44ST, 11ST, 7ST

1035, 996, 1050

 

137, 114, 133

119, 97, 121

108, 125, 97

103, 120, 107

97, 71, 91

30MT, 26MT, 24MT

1ST, 10ST, 5ST

422, 358, 392

TA 102

DMSO

TEST ITEM

 

 

 

 

 

MMC

 

DMSO

TEST ITEM

 

 

 

 

2 AM

 

19.5 µg

39.1 µg

78.1 µg

156.3 µg

312.5 µg

625 µg

0.5 µg

 

 

156.3 µg

312.5 µg

625 µg

1250 µg

2500 µg

10 µg

-

-

-

-

-

-

-

-

 

+

+

+

+

+

+

+

383

330

398

395

425

399

224

3227

 

439

316

596

554

321

139

3992

22

48

32

23

25

58

57

77

 

15

90

17

32

51

15

96

 

0.9

1.0

1.0

1.1

1.0

0.6

8.4

 

 

0.7

1.4

1.3

0.7

0.3

9.1

392, 400, 358

381, 323, 286

361, 413, 419

418, 396, 372

454, 411, 410

333, 441, 424

159MT, 249MT, 165MT

3286, 3140, 3256

 

544, 438, 425

365, 372, 212

611, 578, 598

543, 590, 528

262, 344, 356

126MT, 156MT, 135MT

4079, 4009, 3889

SD: Standard Deviation

MT: Moderate toxicity

ST: Strong toxicity

- Absence of S9

+ Presence of S9

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA1535, TA1537, TA98, TA100 and TA102 of S. typhimurium were exposed to the substance (> 98%).

A preliminary toxicity test was performed to define the dose-levels of the substance to be used for the mutagenicity study. The test item was then tested in three independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post-mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254. All experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method.

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered as valid.

Since the test item was toxic in the preliminary test, the choice of the highest dose-level retained for the main experiments was based on the level of toxicity, according to the criteria specified inthe international guidelines.

No precipitate was observed in the Petri plates when scoring the revertants at any of thedose-levels tested.

In the absence of S9 mix, a moderate to strong toxicity was noted at dose-levels ≥ 625μg/plate in the TA 1537, TA 98, TA 100 and TA 102 strains and at dose-levels ≥ 1250μg/plate in the TA 1535 strain. A slight increase in the number of revertants was noted in the TA 1537 strain in the second experiment (2.2-fold the vehicle control value). Since this increase did not reach the threshold of 3-fold the vehicle control value and since it was due to only one out of the three plates used, and was neither dose-related nor observed in the first experiment, it was not considered as biologically relevant. A slight increase in the number of revertants was noted in the TA 98 strain in the second experiment (2-fold the vehicle control value). This increase reached the threshold of 2- fold the vehicle control value but it was neither dose-related, nor observed in the first andthird experiment. Therefore, it was considered as non-biologically relevant.

In the presence of S9 mix, in the first experiment, using the direct plate incorporation method, a moderate to strong toxicity was noted at dose-levels ≥ 1250μg/plate in the TA 1535 and TA 100 strains and at dose-levels ≥ 2500μg/plate in the TA 1537, TA 98 and TA 102 strains. In the second experiment, performed using the preincubation method, a moderate to strong toxicity was noted at dose-levels ≥ 312.5μg/plate in the TA 1535, TA 1537, TA 98 and TA 100 strains and at dose-levels ≥ 625μg/plate towards the TA 102 strain.

The test item did not induce any noteworthy increase in the number of revertants, in any of thefive tester strains.

Under the experimental conditions, the test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

This study is classified as acceptable. This study satisfies the requirement of OECD 471 guideline for in vitro mutagenicity (bacterial reverse gene mutation).