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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:
2016-10-28 to 2016-11-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Ninth Addendum to OECD Guidelines for Testing of Chemicals, Section 4, No. 471, "Bacterial Reverse Mutation Test", adopted 21st July, 1997.
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
May 30, 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
EPA 712-C-98-247, August 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

Method

Target gene:
histidine
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:
S9 mix from rat liver
Test concentrations with justification for top dose:
Pre-experiment (plate incorporation): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment I (plate incorporation test with and without rat liver S9): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate
Experiment II (pre-incubation test with and without rat liver S9): 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 μg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Aqua dest.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
S. typhimurium: TA 100, TA 1535 without metabolic activation dissolved in Aqua dest.
Positive control substance:
sodium azide
Positive controls:
yes
Remarks:
S. typhimurium: TA 98, TA 1537 without metabolic activation dissolved in DMSO
Positive control substance:
other: 4-nitro-o-phenylene-diamine (4-NOPD)
Positive controls:
yes
Remarks:
S. typhimurium: TA 102 without metabolic activation dissolved in Aqua dest.
Positive control substance:
methylmethanesulfonate
Positive controls:
yes
Remarks:
S. typhimurium: TA 98, TA 100, TA 1535, TA 1537 and TA 102 with metabolic activation dissolved in DMSO
Positive control substance:
other: 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I: in agar (plate incorporation)
Experiment II: preincubation

DURATION
Experiment I:
- Preincubation period: No
- Exposure duration: at least 48 h in the dark at 37 °C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

Experiment II:
- Preincubation period: 60 min at 37°C
- Exposure duration: 60 min at 37°C and at least 48 h in the dark at 37°C
- Expression time (cells in growth medium): Not applicable
- Selection time (if incubation with a selection agent): at least 48 h in the dark at 37°C
- Fixation time (start of exposure up to fixation or harvest of cells): Not applicable

SELECTION AGENT (mutation assays): histidine (overlay agar)

NUMBER OF REPLICATIONS: 3 (in one case only two plates were evaluated)

DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control
Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD 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.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.
Statistics:
According to OECD 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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment I
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
A mutation factor of 2.1 was observed at a concentration of 1000 µg/plate. No dose-response relationship was observed and the effect could not be reproduced in experiment II. Thus, the effect was considered as not biologicvally relevant.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experilment I
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher without metabolic activation and at concentrations of 2500 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment I
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 1000 µg/plate and higher without metabolic activation and at concentrations of 2500 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 100 µg/plate and higher without metabolic activation and at concentrations of 316 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 31.6 µg/plate without metabolic activation and at concentrations of 316 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 100 µg/plate and higher without metabolic activation and at concentrations of 316 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 31.6 µg/plate and higher without metabolic activation and at concentrations of 316 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at concentrations of 100 µg/plate and higher without metabolic activation and at concentrations of 316 µg/plate with metabolic activation
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with tester strains TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as for the main experiment I (plate incorporation test)).
The test item was tested in the pre-experiment with the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate.
For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 μL Rat Liver S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 μL Overlay agar.
For each strain and dose level, including the controls, three plates were used.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval
(e.g. 95%)
- Negative (solvent/vehicle) historical control data: The negative control plates (A. dest.) with and
without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency
are within the historical control data range (2013 -2015)):
-S9 + S9 (rat liver)
min max min max
TA 98 13 54 13 61
TA 100 49 139 67 162
TA 1535 4 39 4 32
TA 1537 2 35 3 36
TA 102 141 472 91 586

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

Any other information on results incl. tables

Table 1: Results Pre-Experiment

Substance

Dose

g/plate)

TA 98

Mutation Factor [toxicity]*

TA 100

Mutation Factor [toxicity]*

without S9

with S9

without S9

with S9

Solvent Control

(A. dest)

 

1.0

1.0

1.0

1.0

4-NOPD

10.0

11.0

-

-

-

NaN3

10.0

-

-

5.0

-

2-AA

2.50

-

66.2

-

15.0

Test Item

 

3.16

1.4

1.1

0.9

0.8

10.0

1.3

1.1

1.0

1.0

31.6

1.2

1.1

1.1

1.1

100

0.8

1.0

1.0

1.0

316

1.1

1.0

1.2

1.1

1000

0.4 [B]

0.3 [B]

1.2 [B]

2.1 [B]

2500

0.0 [B]

0.0 [B]

0.0 [B]

0.4 [B]

5000

0.1 [B]

0.0 [B]

0.0 [B]

0.0 [B]

* [toxicity parameter]: B = Background lawn reduced; N = No background lawn

 

 

Table 2: Results Experiment I (Plate-Incorporation Test)

Tester Strain : TA 98 Experiment 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

24

27

3.1

24

19

5.0

1.4

0.9

30

18

28

14

DMSO

 

19

20

1.7

27

22

4.6

1.0

1.0

22

19

19

19

Test item

3.16 µg

23

28

5.5

25

24

3.2

1.4

1.1

28

26

34

20

Test item

10.0 µg

18

25

7.0

24

23

1.7

1.3

1.1

25

21

32

24

Test item

31.6 µg

17

25

6.7

25

24

2.1

1.2

1.1

29

26

28

22

Test item

100 µg

15

17

2.9

24

21

9.8

0.8

1.0

20

10

15

29

Test item

316 µg

26

22

10.2

22

21

1.5

1.1

1.0

10

19

29

21

Test item

1000 µg

22 B

8

12.4

10 B

7

4.4

0.4

0.3

1 B

9 B

0 B

2 B

Test item

2500 µg

0 B

0

0.0

0 B

0

0.0

0.0

0.0

0 B

0 B

0 B

0 B

Test item

5000 µg

3 B

1

1.7

0 B

0

0.0

0.1

0.0

0 B

0 B

0 B

0 B

4-NOPD

10 µg

193

219

23.1

/

/

/

11.0

/

236

/

229

/

2-AA

2.5 µg

/

/

/

1030

1434

521.0

/

66.2

/

1250

/

2022

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 100 Experiment 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

124

130

4.9

142

139

6.7

1.0

1.0

132

131

133

143

DMSO

 

116

126

13.2

135

133

2.5

1.0

1.0

121

130

141

1133

Test item

3.16 µg

102

118

14.3

98

106

9.8

0.9

0.8

130

117

121

103

Test item

10.0 µg

116

123

7.5

136

130

5.3

1.0

1.0

122

126

131

128

Test item

31.6 µg

134

133

1.2

149

146

7.9

1.1

1.1

134

152

132

137

Test item

100 µg

116

128

13.7

142

133

7.8

1.0

1.0

126

131

143

127

Test item

316 µg

141

157

25.2

166

150

18.3

1.2

1.1

186

130

144

154

Test item

1000 µg

186 B

155

41.2

313 B

280

35.1

1.2

2.1

108 B

283 B

170 B

243 B

Test item

2500 µg

11 B

6

4.4

31 B

50

49.3

0.0

0.4

3 B

13 B

4 B

106 B

Test item

5000 µg

0 B

0

0.0

0 B

0

0.0

0.0

0.0

0 B

0 B

0 B

0 B

NaN3

10 µg

648

630

16.7

/

/

/

5.0

/

615

/

627

/

2-AA

2.5 µg

/

/

/

2014

1985

49.1

/

15.0

/

1928

/

2012

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 1535 Experiment 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

30

27

5.5

7

9

1.5

1.4

0.6

21

9

31

10

DMSO

 

16

19

3.1

15

15

4.5

1.0

1.0

20

11

22

20

Test item

3.16 µg

25

22

4.4

22

19

4.2

1.1

1.2

17

14

24

20

Test item

10.0 µg

23

27

7.2

14

18

3.8

1.4

1.2

22

20

35

21

Test item

31.6 µg

21

24

2.9

18

17

1.5

1.3

1.1

26

17

26

15

Test item

100 µg

26

24

6.7

15

15

1.5

1.3

1.0

30

13

17

16

Test item

316 µg

16

20

3.2

12

12

1.5

1.0

0.8

21

10

22

13

Test item

1000 µg

14 B

12

2.0

12

8

3.5

0.6

0.5

12 B

6

10 B

6

Test item

2500 µg

2 B

1

1.2

2 B

2

0.6

0.0

0.1

0 N

2 B

0 N

1 B

Test item

5000 µg

0 N

0

0.0

0 N

0

0.0

0.0

0.0

0 N

0 N

0 N

0 N

NaN3

10 µg

1028

1210

157.4

/

/

/

62.6

/

1296

/

1305

/

2-AA

2.5 µg

/

/

/

265

261

15.4

/

17.0

/

274

/

244

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 1537 Experiment 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

14

11

4.6

15

16

1.2

1.3

1.1

14

15

6

17

DMSO

 

9

9

2.0

23

15

7.2

1.0

1.0

7

10

11

11

Test item

3.16 µg

/

12

8.5

9

14

4.5

1.3

0.9

18

14

6

18

Test item

10.0 µg

16

12

4.0

13

14

1.5

1.3

1.0

12

16

8

14

Test item

31.6 µg

10

9

1.0

12

12

3.5

1.0

0.8

9

16

8

9

Test item

100 µg

17

22

4.2

12

12

1.5

2.4

0.8

25

11

23

14

Test item

316 µg

20

19

0.6

14

18

3.8

2.1

1.3

19

21

19

20

Test item

1000 µg

5 B

5

4.5

16

18

2.1

0.6

1.3

10 B

20

1 B

19

Test item

2500 µg

0 N

0

0.0

1 B

1

0.6

0.0

0.0

0 N

1 B

0 N

0 B

Test item

5000 µg

0 N

0

0.0

0 B

0

0.0

0.0

0.0

0 N

0 B

0 N

0 B

4-NOPD

40 µg

96

95

10.1

/

/

/

10.5

/

104

/

84

/

2-AA

2.5 µg

/

/

/

423

380

41.6

/

25.9

/

340

/

376

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 102 Experiment 1

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

445

424

33.0

534

479

71.1

1.1

1.2

441

399

386

505

DMSO

 

388

378

13.8

370

401

73.2

1.0

1.0

362

349

383

485

Test item

3.16 µg

373

396

23.5

471

456

15.0

1.0

1.1

395

457

420

441

Test item

10.0 µg

388

390

3.2

446

477

27.5

1.0

1.2

394

499

389

485

Test item

31.6 µg

416

406

8.9

439

455

13.9

1.1

1.1

399

463

403

463

Test item

100 µg

418

409

11.5

486

476

10.0

1.1

1.2

396

486

413

475

Test item

316 µg

383

400

25.5

503

513

60.1

1.1

1.3

429

458

387

577

Test item

1000 µg

347 B

273

166.2

514

513

14.5

0.7

1.3

390 B

527

83 B

498

Test item

2500 µg

76 B

43

28.7

165 B

161

66.1

0.1

0.4

32 B

225 B

22 B

93 B

Test item

5000 µg

0 N

0

0.0

0 N

0

0.0

0.0

0.0

0 N

0 N

0 N

0 N

MMS

1 µL

2851

2759

164.0

/

/

/

7.3

/

2570

/

2857

/

2-AA

10 µg

/

/

/

 

941

106.9

/

2.3

/

 

/

 

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

 

Table 3: Results Experiment II (Pre-incubation Test)

Tester Strain : TA 98 Experiment 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

28

23

7.6

19

27

8.5

1.0

1.1

14

36

26

26

DMSO

 

26

22

4.0

32

26

5.5

1.0

1.0

21

23

18

22

Test item

1.00 µg

14

18

4.0

20

21

2.6

0.8

0.8

22

19

17

24

Test item

3.16 µg

28

29

3.1

26

28

10.7

1.3

1.1

32

19

26

40

Test item

10.0 µg

28

24

4.0

20

18

2.1

1.1

0.7

20

17

25

16

Test item

31.6 µg

25

23

4.9

27

29

2.6

1.0

1.1

26

28

17

32

Test item

100 µg

22 B

14

11.9

34

27

6.1

0.6

1.1

19 B

26

0 B

22

Test item

316 µg

29 B

29

11.0

24 B

22

1.5

1.3

0.9

18 B

22 B

40 B

21 B

Test item

1000 µg

0 N

0

0.0

0 B

0

0.0

0.0

0.0

0 N

0 N

0 N

0 N

4-NOPD

10 µg

287

330

40.7

/

/

/

15.2

/

335

/

368

/

2-AA

2.5 µg

/

/

/

 

1715

356.8

/

66.8

/

 

/

 

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 100 Experiment 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

111

108

4.6

84

101

15.3

1.1

1.1

103

107

111

113

DMSO

 

80

97

19.3

99

93

14.4

1.0

1.0

118

77

93

104

Test item

1.00 µg

115

106

7.6

71

83

16.4

1.1

0.9

103

102

101

77

Test item

3.16 µg

96

107

11.6

102

98

9.6

1.1

1.1

105

87

119

105

Test item

10.0 µg

105

95

10.0

87

104

17.0

1.0

1.1

85

104

96

121

Test item

31.6 µg

58 B

56

8.2

96

96

8.5

0.6

1.0

63 B

88

47 B

105

Test item

100 µg

44 B

19

22.1

67

82

14.5

0.2

0.9

11 B

82

2 B

96

Test item

316 µg

0 N

0

0.0

34 B

27

23.9

0.0

0.3

0 N

46 B

0 N

0 B

Test item

1000 µg

0 N

0

0.0

0 N

0

0.0

0.0

0.0

0 N

0 N

0 N

0 N

NaN3

10 µg

772

667

90.6

/

/

/

6.9

/

614

/

616

/

2-AA

2.5 µg

/

/

/

1669

1679

117.8

/

18.0

/

1801

/

1566

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 1535 Experiment 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

20

22

1.5

14

17

3.1

1.3

2.2

23

18

22

20

DMSO

 

14

17

3.5

4

8

5.3

1.0

1.0

21

14

17

6

Test item

1.00 µg

14

12

3.2

12

10

3.5

0.7

1.3

13

12

8

6

Test item

3.16 µg

19

14

4.5

15

14

3.6

0.8

1.8

14

17

10

10

Test item

10.0 µg

17

18

6.1

14

15

5.0

1.1

1.8

25

20

13

10

Test item

31.6 µg

17

12

4.2

10

12

2.0

0.7

1.5

9

12

11

14

Test item

100 µg

14 B

11

2.5

10

10

2.5

0.7

1.2

11 B

12

9 B

7

Test item

316 µg

13 B

10

3.1

8 B

7

0.6

0.6

0.9

7 B

7 B

9 B

7 B

Test item

1000 µg

0 N

1

1.2

0 B

0

0.0

0.0

0.0

0 N

0 B

2 B

0 N

NaN3

10 µg

879

897

42.4

/

/

/

51.7

/

866

/

945

/

2-AA

2.5 µg

/

/

/

213

182

31.5

/

22.8

/

184

/

150

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 1537 Experiment 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

4

6

2.0

9

10

1.7

0.9

1.3

8

9

6

12

DMSO

 

11

7

3.8

6

8

3.8

1.0

1.0

4

12

5

5

Test item

1.00 µg

6

7

1.2

10

12

1.5

1.1

1.5

8

12

8

13

Test item

3.16 µg

9

5

3.5

13

14

3.6

0.8

1.8

3

11

3

18

Test item

10.0 µg

16

10

6.0

8

8

3.0

1.5

1.0

9

11

4

5

Test item

31.6 µg

4 B

5

0.6

3

6

4.2

0.7

0.8

5 B

5

5 B

11

Test item

100 µg

1 B

0

0.6

7

5

2.1

0.1

0.6

0 B

3

0 B

4

Test item

316 µg

3 B

1

1.7

3 B

2

2.1

0.2

0.3

0 B

4 B

0 B

0 B

Test item

1000 µg

0 N

0

0.0

0 N

0

0.0

0.0

0.0

0 N

0 N

0 N

0 N

4-NOPD

40 µg

97

88

14.5

/

/

/

13.2

/

71

/

95

/

2-AA

2.5 µg

/

/

/

99

166

76.9

/

21.7

/

149

/

250

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Tester Strain : TA 102 Experiment 2

Treatment

Dose/plate

REVERTANT COLONIES PER PLATE

MUTATION

FACTOR

Without activation (-S9)

With activation (+S9)

Counts

Mean

SD

Counts

Mean

SD

-S9

+S9

A. dest

 

318

301

14.7

341

340

4.2

1.1

1.2

296

335

290

343

DMSO

 

250

271

23.9

277

280

10.4

1.0

1.0

266

292

297

272

Test item

1.00 µg

283

273

11.2

304

292

12.0

1.0

1.0

261

291

276

280

Test item

3.16 µg

270

255

13.1

339

297

38.3

0.9

1.1

251

288

245

264

Test item

10.0 µg

268

280

12.5

259

269

12.3

1.0

1.0

293

266

280

283

Test item

31.6 µg

257

251

20.7

305

302

3.6

0.9

1.1

268

298

228

303

Test item

100 µg

114 B

128

12.1

226

268

36.4

0.5

1.0

137 B

290

132 B

288

Test item

316 µg

133 B

97

84.6

0 B

40

59.8

0.4

0.1

157 B

109 B

0 B

12 B

Test item

1000 µg

0 B

1

1.2

48 B

16

27.7

0.0

0.1

2 B

0 B

0 B

0 B

MMS

1 µL

1470

1680

200.0

/

/

/

6.2

/

1868

/

1703

/

2-AA

10 µg

/

/

/

706

720

102.2

/

2.6

/

625

/

828

SD: Standard-deviation  

P: Precipitation

B: Background lawn reduced 

 C: Contamination

N: No background lawn

Mutation factor =         mean revertants (test item)     

                               mean revertants (vehicle control)

 

Applicant's summary and conclusion

Conclusions:
During the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of the test item for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

- Experiment I: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

- Experiment II: 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate

No precipitation of the test item was noted in any tester strain used in experiment I and II (with and without metabolic activation). Toxic effects of the test item were noted in all tester strains used in experiment I and II:

- In experiment I toxic effects were observed at concentrations of 1000 µg/plate and higher (with and without metabolic activation), depending on the particular tester strain.

- In experiment II toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.

All criteria of validity were met.

In conclusion, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.