Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this endpoint three in vitro genotoxicity assays were performed according their respective OECD TG and in compliance to GLP.

The test item did not induce an increase in mutation frequency in the Ames test, is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (Thymidine kinase locus) in mouse lymphoma L5178Y cells and did not induce structural and/or numerical chromosomal damage in human lymphocytes in the in vitro mammalian cell micronucleus test.

Link to relevant study records

Referenceopen allclose all

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
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
Target gene:
histidine
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.
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.
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.

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)

 

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-30 to 2017-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: healthy and non-smoking donor with no known recent exposure to genotoxic chemicals and radiation
Details on mammalian cell type (if applicable):
- pre-experiment and main experiment I: female donor (age between 18 and 35 years)
- experiment II: male donor (age between 18 and 35 years)
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
The selection of the concentrations used in the main experiments was based on data from the pre-experiment.
In the main experiments without and with metabolic activation 0.30 mM test item was selected as the highest concentration for microscopic evaluation based on cytotoxicity of the test item.

- Experiment I with short-term exposure (4 h):
-- without metabolic activation: 0.10, 0.25 and 0.30 mM (corresponding to 14.5, 36.3 and 43.6 µg/mL)
-- with metabolic activation: 0.10, 0.20 and 0.30 mM (corresponding to 14.5, 29.0 and 43.6 µg/mL)
- Experiment II with long-term exposure (44 h):
-- without metabolic activation: 0.10, 0.20 and 0.30 mM (corresponding to 14.5, 29.0 and 43.6 µg/mL)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
cell culture medium
Negative solvent / vehicle controls:
yes
Remarks:
cell culture medium with 1% DMSO
Positive controls:
yes
Remarks:
Clastogenic Controls
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Positive controls:
yes
Remarks:
Clastogenic Controls
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Positive controls:
yes
Remarks:
Aneugenic Control
Positive control substance:
other: Colchicine
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
CELL CULTURE CONDITIONS
- Culture medium: RPMI 1640 medium supplemented with: 15% fetal bovine serum (FBS), 100 U/100 µg/mL penicillin/streptomycin solution, 2.4 µg/mL phytohaemagglutinin (PHA)
- incubation at 37 °C, 5.5 % CO2

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 44 to 48 hours with phytohemeagglutinine (PHA)
- Exposure duration: 4 or 44 hours
- Washing: 2x
- Fixative: methanol + glacial acetic acid (3+1)

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B

STAIN (for cytogenetic assays): acridine orange solution (10 µg/mL)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION: dropping the cell suspension onto clean glass slides

NUMBER OF CELLS EVALUATED: at least 2000 binucleated cells (if possible) per concentration (1000 binucleated cells per slide) were analysed for micronuclei
- Frequency of micronucleated cells: reported as % micronucleated cells.
To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION according to the criteria of Fenech:
clearly surrounded by a nuclear membrane, having an area of less than one-third of that of the main nucleus, being located within the cytoplasm of the cell and not linked to the main nucleus via nucleoplasmic bridges.

DETERMINATION OF CYTOTOXICITY
As an assessment of the cytotoxicity, a cytokinesis block proliferation index (CBPI) was determined from 500 cells according to the following formula:
CBPI = ((c1 x 1) + (c2 x 2) + (cx x 3))/n
with: c1: mononucleate cells; c2: binucleate cells; cx: multinucleate cells; n: total number of cells

ACCEPTABILITY OF THE ASSAY:
A mutation assay is considered acceptable if it meets the following criteria:
- The concurrent negative/solvent control is considered acceptable for addition to the laboratory historical negative/solvent control database
- Concurrent positive controls should induce responses that are compatible with those generated in the laboratory’s historical positive control data base and produce a statistically significant increase compared with the concurrent negative/solvent control
- Cell proliferation criteria in the negative/solvent control should be fulfilled
- All experimental conditions are tested unless one resulted in positive results
- Adequate number of cells and concentrations are analysable
- Criteria for the selection of top concentration are fulfilled


Rationale for test conditions:
A pre-experiment was conducted under identical conditions as described for the main experiment I (4 h incubation) in order to determine the toxicity of the test item. The CBPI was used for the quantification of cytotoxicity.
The following concentrations were tested without and with S9 mix: 0.039, 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5.0, 7.5, 10 mM corresponding to 5.7, 11.3, 22.7, 45.4, 90.8, 181.5, 363, 726, 1089 and 1452 µg/mL
The concentration of 10 mM was considered to be the highest test concentration to be used in this test system following the recommendation of the corresponding OECD testing guideline 487.
Evaluation criteria:
A test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
- the increase is concentration-related in at least one experimental condition when evaluated with an appropriate trend test
- any of the results are outside the distribution of the historical negative/solvent control data (e.g. Poisson-based 95% control limits)

When all of these criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system. A test item is considered to be clearly negative if in all experimental conditions examined none of the criteria mentioned above are met.
Statistics:
The nonparametric Chi square test was performed to verify statistically significant enhancement (p<0.05) of cells with micronuclei.
The Chi square test for trend was performed to test whether there is a concentration-related increase in the micronucleated cells frequency in the experimental conditions.
Key result
Species / strain:
lymphocytes: healthy and non-smoking donor with no known recent exposure to genotoxic chemicals and radiation
Remarks:
4h treatment
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: healthy and non-smoking donor with no known recent exposure to genotoxic chemicals and radiation
Remarks:
44 h treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No precipitate of the test item was noted in any concentration group evaluated in the main experiments.

Ethylmethanesulfonate (EMS, 600 µg/mL and 1400 µg/mL) and cyclophosphamide (CPA, 15 µg/mL) were used as clastogenic controls. Colchicine (Colc, 0.02 µg/mL and 0.8 µg/mL) was used as aneugenic control. All induced distinct and statistically significant increases of the micronucleus frequency.
This demonstrates the validity of the assay.
Remarks on result:
other: no biologically relevant increase of the micronucleus frequency

Summary: Experiment I and II, without metabolic activation

  Dose group Concentration (mM) Cytostasis (%) Relative Cell Growth (%) Micronucleated Frequency (%)
Exp. I, 4 h treatment, 44 h fixation interval C 0 1.2 1.2 0.85
S 0 0 100 1.2
3 0.1 23 77 1.2
5 0.25 37 63 1.38
6 0.3 52 48 2.03
EMS 1400µg/mL 44 56 3.65
Colc 0.8 µg/mL 46 54 2.84
Exp. II, 44 h treatment, 44 h fixation interval C 0 0* 111 0.55
S 0 0 100 0.55
7 0.1 5 95 0.75
8 0.2 37 63 0.8
9 0.3 66 34 0.5
EMS 600 µg/mL 47 53 3.1
Colc 0.02 µg/mL 42 58 1.55

C: Negative Control (Culture medium)

S: Solvent Control (DMSO 1% v/v in culture medium)

P: Precipitation (+: precipitation, -: no precipitation)

EMS: Ethylmethanesulfonate, Positive Control (without metabolic activation) [600 µg/mL and 1400 µg/mL]

Colc: Positive Control (without metabolic activation) [0.02 µg/mL and 0.8 µg/mL]

Relative Cell Growth: 100 x ((CBPITest conc– 1) / (CBPIcontrol -1))

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

Summary: Experiment I, with metabolic activation

  Dose group Concentration (mM) Cytostasis (%) Relative Cell Growth (%) Micronucleated Frequency (%)
Exp. I, 4 h treatment, 44 h fixation interval C 0 1 99 0.9
S 0 0 100 1.4
2 0.1 25 75 1.63
3 0.2 29 71 1.1
4 0.3 53 47 1.8
CPA 15µg/mL 26 74 5.35

C: Negative Control (Culture medium)

S: Solvent Control (DMSO 1% v/v in culture medium)

P: Precipitation (+: precipitation, -: no precipitation)

EMS: Ethylmethanesulfonate, Positive Control (without metabolic activation) [600 µg/mL and 1400 µg/mL]

Colc: Positive Control (without metabolic activation) [0.02 µg/mL and 0.8 µg/mL]

Relative Cell Growth:100 x ((CBPITest conc– 1) / (CBPIcontrol -1))

Cytostasis [%] = 100- Relative Cell Growth [%]

*: the cytostasis is defined 0, when the relative cell growth exceeds 100%

Conclusions:
It can be concluded that the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro mammalian cell micronucleus test.

Executive summary:

In the current study the potential of the test item to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487 and according to GLP regulations.

The selection of the concentrations used in the main experiments was based on data from the pre-experiment. In the main experiments without and with metabolic activation 0.30 mM test item was selected as the highest concentration for microscopic evaluation based on cytotoxicity of the test item.

The following concentrations were evaluated for micronuclei frequencies:

- Experiment I with short-term exposure (4 h):

without metabolic activation: 0.10, 0.25 and 0.30 mM (corresponding to 14.5, 36.3 and 43.6 µg/mL)

with metabolic activation: 0.10, 0.20 and 0.30 mM (corresponding to 14.5, 29.0 and 43.6 µg/mL)

- Experiment II with long-term exposure (44 h):

without metabolic activation: 0.10, 0.20 and 0.30 mM (corresponding to 14.5, 29.0 and 43.6 µg/mL)

 

No precipitate of the test item was noted in any concentration group evaluated in the main experiments.

In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.10 mM. At a concentration of 0.25 mM a cytostasis of 37% and at a concentration of 0.30 mM a cytostasis of 52% was noted.

In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.20 mM. At a concentration of 0.30 mM a cytostasis of 53% was noted.

In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.10 mM. At a concentration of 0.20 mM a cytostasis of 37% and at a concentration of 0.30 mM a cytostasis of 66% was noted.

In experiment I without and with metabolic activation and in experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

It can be concluded that the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro mammalian cell micronucleus test.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-06-19 to 2017-07-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
- Principle of test: This in vitro experiment is carried out to assess the potential of the test item to induce gene mutations by means of a Thymidine Kinase assay using the mouse lymphoma cell line L5178Y. The Thymidine Kinase (TK) system detects base pair mutations, frameshift mutations, small deletions as well as large, non-lethal deletions and rearrangements of the relevant chromosomes.

- Short description of test conditions: the experiment with and without metabolic activation was performed as 4 hours short-term exposure assay

- Parameters analysed / observed: potential mutagenicity and/or clastogencity
GLP compliance:
yes (incl. certificate)
Type of assay:
other: In vitro Mammalian Cell Gene Mutation Assay in Mouse Lymphoma L5178Y Cells
Target gene:
Thymidine Kinase Locus/TK+/-)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: successfully in in vitro experiments for many years.
- Cell cycle length, doubling time or proliferation index: 10-12 h doubling time


- Methods for maintenance in cell culture if applicable: Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase). This allows the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection.
- Modal number of chromosomes:
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 supplemented with hypoxanthine, thymidine, glycine, ethotrexate. The cells are resuspended in medium without methotrexate but thymidine, hypoxanthine and glycine for 1-3 days.
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Homogenate
Test concentrations with justification for top dose:
The toxicity of the test item was determined in a pre-experiment up to a maximum concentration of 10 mM. Six concentrations [0.2, 0.5, 2.5, 5.0, 7.5 and 10 mM] (correspond to 0.03, 0.07, 0.36, 0.73, 1.09 and 1.45 mg/mL) were tested without and with metabolic activation.
Dose groups that were not evaluable due to very high toxicity and precipitation of the test item are not reported.

The test item was investigated at the following concentrations:
- without metabolic activation: 0.0075, 0.01, 0.02, 0.04, 0.06 and 0.08 mM (0.0011, 0.0015, 0.003, 0.006, 0.009 and 0.012 mg/mL)
- with metabolic activation: 0.0075, 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 mM (0.0011, 0.0015, 0.003, 0.006, 0.009, 0.012 and 0.015 mg/mL)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Based on the results of the solubility test (performed in Ames Test Eurofins Munich # 167244) DMSO was used as solvent (1% DMSO). Different test item stock solutions were prepared and added to the samples. The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
other: methylmethanesulfonate (MMS)
Remarks:
The dilutions of the stock solutions of the positive controls were prepared on the day of the experiment and used immediately.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; 11 mL RPMI medium with 5% horse serum (25 cm2 flasks)
- Cell density at seeding (if applicable): 1 x 10E7 cells

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 2 days
- Density check: each days and adjusted to 3x10E5 cells/mL. (total volume 20mL)


NUMBER OF REPLICATIONS: 4

NUMBER OF CELLS EVALUATED: the plates were scored after incubation period of 12 days

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (RSG)

Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
- the induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 10E6 cells and
- a dose-dependent increase in mutant frequency is detected

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥ 40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal abberations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation of the results.

A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend of the test is negative.
Statistics:
For the microwell method used here the Poisson distribution was used to calculate the plating efficiencies for cells cloned without and with TFT selection.
The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the solvent/negative controls were used as reference.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Please see table 4 and 7 under section "other information on results" section
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Without metabolic activation: Relative Total Growth decreased starting at 0.04 mM (39.5%), 0.06 mM (36.3%) and 0.08 mM (14.4%) With metabolic activation: Relative Total Growth decreased starting at 0.08 mM (19.6%) and 0.1 mM (10.3%)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Please see the table under the section "Any other information on results".
The acceptance criteria as recommended by IWGT were fulfilled

1. Pre-experiment for Toxicity, without metabolic activation:

Test Group Concentration (mM) Number of Cells 4 h after treatment Number of cells 24h after treatment Number of cells 48h after treatment SG RSG (%)
C1 0 331000 1170000 1580000 18.5 110.3
C2 335000 1190000 1520000 18.1 107.9
S1 0 303000 1030000 1540000 15.9 100.0
S2 381000 1170000 1510000 17.7
1 0.2 246000 185000 162000 0.5 2.9
2 0.5 261000 167000 170000 0.5 3.0
3 2.5 235000 205000 124000 0.4 2.2
4 5 221000 196000 155000 0.5 2.8

C: Negative Control

S: Solvent Control

SG: Suspension Growth

RSG: Relative Suspension Growth

2. Pre-experiment for Toxicity, with metabolic activation:

Test Group Concentration (mM) Number of Cells 4 h after treatment Number of cells 24h after treatment Number of cells 48h after treatment SG RSG (%)
C1 0 525000 1000000 1490000 14.9 91.6
C2 297000 1040000 1440000 15.0 92.1
S1 0 284000 967000 1670000 16.1 100.0
S2 312000 1050000 1560000 16.4
1 0.2 237000 233000 211000 0.6 3.9
2 0.5 276000 237000 206000 0.6 3.8
3 2.5 212000 181000 144000 0.4 2.7
4 5 238000 228000 153000 0.5 2.8

C: Negative Control

S: Solvent Control

SG: Suspension Growth

RSG: Relative Suspension Growth

3. Main Experiment - Toxicity Data, without metabolic activation:

Test Group Concentration (mM) Number of Cells 4h after Treatment Number of cells 24h after Treatment Number of cells 48 h after treatment SG RSG (%) RCE (%- RTG (%)
C1 0 341000 944000 1520000 14.3 100.8 102.6 103.4
C2 0 311000 1050000 1470000 15.4 108.5 96.0 104.1
S1 0 298000 789000 1590000 12.5 100.0 100.0 100.0
S2 0 359000 1090000 1460000 15.9 100.0 100.0 100.0
4 0.0075 356000 1000000 1470000 14.7 103.3 79.7 82.3
5 0.01 386000 900000 1490000 13.4 94.2 88.6 83.5
6 0.02 281000 828000 1430000 11.8 83.2 104.3 86.8
7 0.04 269000 582000 1230000 7.2 50.3 78.5 39.5
8 0.06 310000 540000 1200000 6.5 45.5 79.7 36.3
9 0.08 304000 334000 987000 3.3 23.2 62.4 14.4
EMS 300µg/mL 309000 763000 1430000 10.9 76.7 80.9 62.0
MMS 10µg/mL 321000 592000 1460000 8.6 60.7 75.1 45.6

C: Negative Control

S: Solvent Control

SG: Suspension Growth

RSG: Relative Suspension Growth

RCE: Relative Cloning Efficiency

RTG: Relative Total Growth

EMS: Ethylmethane sulfonate

MMS: Methylmethanesulfonate

4. Main Experiment - Mutagenicity Data, without metabolic activation:

Cloning Efficiency (CE) Mutagenicity Data
Test group Concentration (mM) Plate 1e Plate 2

CE (%)

MF:

Number of cultures / 96 wells MF (mutant / 106 cells)

IMF (mutants/

106 cells)

Plate 1 Plate 2 Plate 3 Plate 4 Mean
C1 0 76 82 108.2 10 14 12 14 12.5 64.6 /
C2

0

82 72 101.2 13 11 12 14 12.5 68.9 /
S1 0 85 79 120.3 10 16 20 16 15.5 73.6 /
S2 0 73 74 90.7 12 8 8 9 9.3 56.0 /
4 0.0075 69 73 84.1 19 24 20 19 20.5 143.1 78.3
5 0.01 78 71 93.5 25 20 14 22 20.3 127.4 62.6
6 0.02 80 79 110.1 18 24 22 19 20.8 110.9 46.1
7 0.04 78 63 82.9 23 23 28 34 27.0 200.6 135.9
8 0.06 71 71 84.1 38 30 45 39 38.0 302.1 237.3
9 0.08 65 60 65.8 48 45 42 51 46.5 505.1 440.3
EMS 300µg/mL 76 67 85.4 74 67 60 65 66.5 700.2 635.5
MMS 10µg/mL 71 67 79.3 56 59 47 54 54.0 524.7 460.0

C: Negative Control

S: Solvent Control

e: Number of culture with cell growth

CE: Cloning efficiency

MF: Mutant Frequency

IMF: Induced Mutant Frequency

EMS: Ethylmethane sulfonate

MMS: Methylmethanesulfonate

5. Main Experiment - Colony Sizing, without metabolic activation:

Test Group Concentration (mM) Wells with at least 1 colony Large colonies Small colonies % small colonies
C1 0 50 39 11 22.0
C2 50 37 13 26.0
S1 0 62 40 22 35.5
S2 37 25 12 32.4
7 0.04 108 87 21 19.4
8 0.06 152 102 50 32.9
9 0.08 186 111 75 40.3
MMS 10µg/mL 216 98 118 54.6

C: Negative Control

S: Solvent Control

MMS: Methylmethanesulfonate

6. Main Experiment - Toxicity Data, with metabolic activation:

Test Group Concentration (mM) Number of Cells 4h after Treatment Number of cells 24h after Treatment Number of cells 48 h after treatment SG RSG (%) RCE (%) RTG (%)
C1 0 253000 833000 1310000 10.9 87.5 92.1 80.6
C2 318000 928000 1310000 12.2 97.5 90.8 88.5
S1 0 351000 822000 1420000 11.7 100.0 100.0 100.0
S2 319000 922000 1440000 13.3 100.0 100.0 100.0
4 0.0075 262000 894000 1490000 13.3 106.8 97.7 104.3
5 0.01 268000 820000 1500000 12.3 98.6 110.3 108.7
6 0.02 313000 994000 1230000 12.2 98.0 99.2 97.2
7 0.04 265000 596000 1240000 7.4 56.2 97.7 57.9
8 0.06 288000 480000 1120000 5.4 43.1 88.2 38.0
9 0.08 257000 398000 1010000 4.0 32.2 60.9 19.6
10 0.1 278000 377000 744000 2.8 22.5 45.9 10.3
B(a)P 1.5µg/mL 268000 576000 1420000 8.2 65.6 85.7 56.2

C: Negative Control

S: Solvent Control

SG: Suspension Growth

RSG: Relative Suspension Growth

RCE: Relative Cloning Efficiency

RTG: Relative Total Growth

B(a)P: Benzo(a)pyrene

7. Main Experiment - Mutagenicity Data, with metabolic activation:

Cloning Efficiency (CE) Mutagenicity Data
Test group Concentration (mM) Plate 1 Plate 2 CE Number of cultures / 96 wells MF (mutant / 106 cells)

IMF (mutants/

106 cells)

Plate 1 Plate 2 Plate 3 Plate 4 Mean
C1 0 69 68 78.1 7 8 9 16 10.0 70.9 /
C2 63 73 77.0 7 10 8 14 9.8 69.9 /
S1 0 69 71 81.6 14 11 12 16 13.3 91.1 /
S2 68 77 88.0 10 10 11 14 11.3 71.0 /
4 0.0075 69 72 82.9 12 14 13 9 12.0 80.7 -0.3
5 0.01 73 76 93.5 12 14 21 11 14.5 88.2 7.1
6 0.02 72 70 84.1 20 13 11 11 13.8 92.5 11.5
7 0.04 71 70 82.9 16 14 19 18 16.8 115.9 34.8
8 0.06 69 65 74.8 25 16 19 25 21.3 168.1 87.1
9 0.08 54 54 51.7 29 35 33 35 33.0 408.3 327.3
10 0.1 47 42 38.9 40 38 41 33 38.0 649.1 568.0
B(a)P 1.5µg/mL 63 69 72.7 49 50 42 48 47.3 467.4 386.4

C: Negative Control

S: Solvent Control

e: Number of culture with cell growth

CE: Cloning efficiency

MF: Mutant Frequency

IMF: Induced Mutant Frequency

B(a)P: Benzo(a)pyrene

8. Main Experiment - Colony Sizing, with metabolic activation:

Test Group Concentration (mM) Wells with at least 1 colony Large colonies Small colonies % small colonies
C1 0 40 31 9 22.5
C2 39 37 2 5.1
S1 0 53 44 9 17.0
S2 45 42 3 6.7
8 0.04 85 70 15 17.6
9 0.06 132 88 44 33.3
10 0.08 152 80 72 47.4
B(a)P 0.1 189 93 96 50.8

C: Negative Control

S: Solvent Control

B(a)P: Benzo(a)pyrene

9.Biometry - Main Experiment, without metabolic activation

Test Group Concentration (mM) Mean Mutant Frequency Mean Induced Mutant Frequency p-value Statistical significance
C1 0 64.6 / / /
C2 0 68.9 / / /
S1 0 73.6 / / /
S2 0 56.0 / / /
4 0.0075 143.1 78.3 0.004 +
5 0.01 127.4 62.6 0.008 +
6 0.02 110.9 46.1 0.008 +
7 0.04 200.6 135.9 0.004 +
8 0.06 302.1 237.3 0.004 +
9 0.08 505.1 440.3 0.004 +
EMS 300µg/mL 700.2 635.5 0.004 +
MMS 10µg/mL 524.7 460.0 0.004 +

C: Negative Control

S: Solvent Control

EMS: Ethylmethane sulfonate

MMS: Methylmethanesulfonate

+: significant

-: not significant

10. Biometry - Main Experiment, with metabolic activation:

Test Group Concentration (mM) Mean Mutant Frequency Mean Induced Mutant Frequency p-value Statistical significance
C1 0 70.9 / / /
C2 69.9 / / /
S1 0 91.1 / / /
S2 71.0 / / /
4 0.0075 80.7 -0.3 0.897 -
5 0.01 88.2 7.1 0.772 -
6 0.02 92.5 11.5 0.651 -
7 0.04 115.9 34.8 0.026 +
8 0.06 168.1 87.1 0.004 +
9 0.08 408.3 327.3 0.004 +
10 0.1 649.1 568.0 0.004 +
B(a)P 1.5µg/mL 467.4 386.4 0.004 +

C: Negative Control

S: Solvent Control

B(a)P: Benzo(a)pyrene

+: significant

-: not significant

Conclusions:
The test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay in mouse lymphoma L5178Y cells.
Executive summary:

In this in vitro mammalian cell gene mutation assay, the mutagen potential of the test item was assessed according to the OECD 490 and under GLP without significant deviations. The test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The mutant selection cloning was performed by using liquid medium in 96 -microwell plates. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

A pre-experiment was performed in order to select the concentrations of the test item that were used in the main experiment. The test item was investigated at the following concentrations:

- Without metabolic activation: 0.0075, 0.01, 0.02, 0.04, 0.06 and 0.08 mM corresponding to the final formulation of the test item to 0.0011, 0.0015, 0.003, 0.006, 0.009 and 0.012 mg/mL of the test item.

- With metabolic activation: 0.0075, 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 mM corresponding to the final formulation of the test item to 0.0011, 0.0015, 0.003, 0.006, 0.009, 0.012 and 0.015 mg/mL of the test item.

No precipitation of the test item was observed.

Growth inhibition was observed in the main experiment without and with metabolic activation (indication of cytotoxicity). The relative total growth (RTG) was 14.4% (without metabolic activation) and 10.3% (with metabolic activation) for the highest concentration evaluated.

Biological relevant increase of mutant was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF) was exceeded by the induced mutant frequency at concentration 0.04 mM and higher (without metabolic activation) and at concentration of 0.08 mM and higher (with metabolic activation). Moreover, a dose-response relationship was observed.

Additionaly, colony sizing showed a clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

In conclusion the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (Thymidine kinase locus) in mouse lymphoma L5178Y cells.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test

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.

The test was performed according to the OECD TG 471 and under GLP.

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).

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.

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.

In vitro mouse lymphoma assay

In this in vitro mammalian cell gene mutation assay, the mutagen potential of the test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

The test was performed according to the OECD TG 490 and under GLP.

The test item was investigated at the following concentrations:

-Without metabolic activation: 0.0075, 0.01, 0.02, 0.04, 0.06 and 0.08 mM corresponding to the final formulation of the test item to 0.0011, 0.0015, 0.003, 0.006, 0.009 and 0.012 mg/mL of the test item.

-With metabolic activation: 0.0075, 0.01, 0.02, 0.04, 0.06, 0.08 and 0.1 mM corresponding to the final formulation of the test item to 0.0011, 0.0015, 0.003, 0.006, 0.009, 0.012 and 0.015 mg/mL of the test item.

Growth inhibition was observed in the main experiment without and with metabolic activation (indication of cytotoxicity). The relative total growth (RTG) was 14.4% (without metabolic activation) and 10.3% (with metabolic activation) for the highest concentration evaluated.

Biological relevant increase of mutant was found after treatment with the test item (without and with metabolic activation). The Global Evaluation Factor (GEF) was exceeded by the induced mutant frequency at concentration 0.04 mM and higher (without metabolic activation) and at concentration of 0.08 mM and higher (with metabolic activation). Moreover, a dose-response relationship was observed.

Additionaly, colony sizing showed a clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

In conclusion the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (Thymidine kinase locus) in mouse lymphoma L5178Y cells.

In vitro micronucleus assay

In the current study the potential of the test item to induce micronuclei in human lymphocytes in vitro was assessed according to OECD 487 and according to GLP regulations.

In the main experiments without and with metabolic activation 0.30 mM test item was selected as the highest concentration for microscopic evaluation based on cytotoxicity of the test item.The following concentrations were evaluated for micronuclei frequencies:

- Experiment I with short-term exposure (4 h):

without metabolic activation: 0.10, 0.25 and 0.30 mM (corresponding to 14.5, 36.3 and 43.6 µg/mL)

with metabolic activation: 0.10, 0.20 and 0.30 mM (corresponding to 14.5, 29.0 and 43.6 µg/mL)

- Experiment II with long-term exposure (44 h):

without metabolic activation: 0.10, 0.20 and 0.30 mM (corresponding to 14.5, 29.0 and 43.6 µg/mL)

 In experiment I without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.10 mM. At a concentration of 0.25 mM a cytostasis of 37% and at a concentration of 0.30 mM a cytostasis of 52% was noted.

In experiment I with metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.20 mM. At a concentration of 0.30 mM a cytostasis of 53% was noted.

In experiment II without metabolic activation no increase of the cytostasis above 30% was noted up to a concentration of 0.10 mM. At a concentration of 0.20 mM a cytostasis of 37% and at a concentration of 0.30 mM a cytostasis of 66% was noted.

In experiment I without and with metabolic activation and in experiment II without metabolic activation no biologically relevant increase of the micronucleus frequency was noted after treatment with the test item.

In conclusion, the test item did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro mammalian cell micronucleus test.

 

Justification for classification or non-classification

3 studies are available in which genetic toxicity is addressed:

Ames test

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

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 conclusion, it can be stated that 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.

In vitro mouse lymphoma assay

In this in vitro mammalian cell gene mutation assay, the mutagen potential of the test item was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Growth inhibition was observed in the main experiment without and with metabolic activation. Biological relevant increase of mutants was found after treatment with the test item (without and with metabolic activation). Moreover a dose-response relationship was observed. Colony sizing showed a clastogenic effect induced by the test item.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item is considered to be mutagenic in the in vitro mammalian cell gene mutation assay (Thymidine kinase locus) in mouse lymphoma L5178Y cells.

In vitro micronucleus assay

The potential of the test item for induction of micronuclei in human lymphocytes was tested in an in vitro micronucleus assay at concentrations up to 0.3 mM with (4h exposure) and without metabolic activation.

The test item did not induce structural and/or numerical chromosomal damage in human lymphocytes. Therefore, the substance is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the in vitro mammalian cell micronucleus test.

 

Conclusion

The test substance is considered to be non-mutagenic in the Ames test, it was mutagenic in the micronuclei in human lymphocytes and non-mutagenic with respect to clastogenicity and/or aneugenicity in the mammalian cell micronucleus test.

The results of the available data are inconclusive and do not allow for a final assessment for classification or non-classification of the test substance in regards to potential genotoxicity according to the EU Regulation No. 1272/2008, section 3.5. Additional in vivo testing is recommended.