Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity (OECD 471), Ames: negative with and without metabolic activation

Mammalian mutageniticty (OECD 490), in vitro mammalian cell gene mutation using the thymidine kinase gene: negative with and without metabolic activation

Mammalian cytogenicity (OECD 473), chromosome aberration: negative with and without metabolic activation

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:
25 July - 11 Aug 2017
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:
21 July 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
his operon
Species / strain / cell type:
S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
Metabolic activation:
with and without
Metabolic activation system:
Cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from livers treated with Aroclor 1254 (500 mg/kg bw)
Test concentrations with justification for top dose:
Experiment 1a, plate incorporation method
S. typhimurium TA 97a, TA 98, TA 100, TA 102 and TA 1535: 5000, 1500, 500, 150 and 50 μg/plate with and without metabolic activation

Experiment 1b, plate incorporation method
S. typhimurium TA102: 5000 μg/plate, 1500, 500, 150, 50, 15 and 5 μg/plate with and without metabolic activation

Experiment 2, pre-incubation method
S. typhimurium TA 97a, TA 98, TA 100 and TA 1535): 5000, 2500, 1250, 625, 313, 156 and 78 μg/plate with and without metabolic activation
S. typhimurium TA 102: 500, 250, 125, 62.5, 31.3, 15.6 and 7.8 μg/plate with and without metabolic activation
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was not sufficiently soluble in demineralized water, dimethyl sulfoxide (DMSO), ethanol and tetrahydrofurane. Based on these results, ethanol was used as solvent in all experiments, because the test item showed in ethanol the best result for handling in the experiments. The test suspension was homogeneous and pipettable. In addition, ethanol does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 2-Amino-anthracene (2-AA): +S9: 1 µg/plate (in DMSO) for TA 97a, TA 100, TA 102, TA 1535; 4-Nitro-1,2-phenylenediamine (NPD): -S9: 20 µg/plate (in DMSO) for TA 97a, TA 98, TA 102
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation, experiments 1a and 1b) and pre-incubation (experiment 2)

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

NUMBER OF REPLICATIONS: triplicates each in two independent experiments (experiments 1a/1b and 2)

DETERMINATION OF CYTOTOXICITY
- Method: Inspection of the bacterial background lawn and of the number of revertants
Evaluation criteria:
The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants minus mean spontaneous revertants) was given.

A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.
Statistics:
The colonies were counted visually and the numbers were recorded. A validated spreadsheet was used to calculate mean values and standard deviations of each treatment, solvent control and positive control.
Species / strain:
S. typhimurium, other: TA 97a, TA 98, TA 100 and TA 1535
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
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
-/+S9: 5000 and 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed in any experiment at any concentration

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : data is summarised in "Any other information on results incl. tables"

Table 1: Mean Revertants Experiment 1a (all strains tested)

 

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

85

80

29

34

73

93

307

377

27

26

sd

6.9

12.7

6.4

9.8

5.9

13.6

48.4

61.1

1.5

7.0

DMSO

Mean

98

97

35

38

78

78

320

388

22

24

sd

19.6

4.0

5.9

6.7

5.5

3.5

31.2

28.8

3.2

4.0

Ethanol

Mean

76

83

24

26

72

72

295

313

22

22

sd

11.4

7.0

6.4

6.0

10.6

8.6

96.4

37.2

3.8

5.9

Positive Controls*

Mean

637

781

383

86

451

1528

1411

1472

323

183

sd

99.4

64.2

27.2

8.1

22.7

424.2

100.0

36.7

56.6

28.4

f(I)

6.50

8.05

10.94

2.26

6.18

19.59

4.41

3.79

11.96

7.63

5000

µg/plate

Mean

99

93

22

26

76

82

79

48

20

19

sd

18.9

15.1

3.1

5.9

4.0

18.9

12.6

14.5

2.1

1.5

f(I)

1.30

1.12

0.92

1.00

1.06

1.14

0.27

0.15

0.91

0.86

1500

µg/plate

Mean

79

98

24

21

75

74

66

95

17

19

sd

16.2

9.0

4.0

6.4

11.5

8.0

19.9

23.1

1.5

2.5

f(I)

1.04

1.18

1.00

0.81

1.04

1.03

0.22

0.30

0.77

0.86

500

µg/plate

Mean

90

94

26

24

82

77

285

331

23

19

sd

22.0

15.9

5.5

2.9

6.0

13.6

60.2

10.1

4.5

3.2

f(I)

1.18

1.13

1.08

0.92

1.14

1.07

0.97

1.06

1.05

0.86

150

µg/plate

Mean

79

84

26

20

66

76

281

384

18

23

sd

15.0

20.3

6.9

2.6

5.5

10.7

89.3

10.6

3.6

0.6

f(I)

1.04

1.01

1.08

0.77

0.92

1.06

0.95

1.23

0.82

1.05

 

50 µg/plate

Mean

88

89

25

32

83

64

291

355

22

24

sd

5.2

6.4

4.0

1.2

14.5

6.2

63.8

64.7

4.6

4.2

f(I)

1.16

1.07

1.04

1.23

1.15

0.89

0.99

1.13

1.00

1.09

f(I) = increase factor

* Different positive controls were used

Table 2: Mean Revertants Experiment 1b (strain TA 102)

 

Strain

TA102

Induction

-S9

+S9

Demin. water

Mean

356

345

sd

62.9

72.6

DMSO

Mean

257

324

sd

26.6

20.8

Ethanol

Mean

292

258

sd

58.9

24.0

Positive Controls*

Mean

689

1083

sd

60.2

400.0

f(I)

2.68

3.34

5000

µg/plate

Mean

12

4

sd

1.0

1.5

f(I)

0.04

0.02

1500

µg/plate

Mean

80

56

sd

10.0

9.0

f(I)

0.27

0.22

500

µg/plate

Mean

315

268

sd

46.4

58.9

f(I)

1.08

1.04

150

µg/plate

Mean

284

284

sd

52.3

52.0

f(I)

0.97

1.10

 

50 µg/plate

Mean

355

292

sd

50.0

34.6

f(I)

1.22

1.13

 

15 µg/plate

Mean

339

349

sd

47.7

120.2

f(I)

1.16

1.35

 

5 µg/plate

Mean

304

287

sd

65.8

33.5

f(I)

1.04

1.11

f(I) = increase factor

* Different positive controls were used

Table 3: Mean Revertants Experiment 2 (strains TA 97a, TA 98, TA 100 and TA 1535)

 

Strain

TA97a

TA98

TA100

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

64

81

24

28

51

64

22

23

sd

4.0

17.9

4.7

2.1

4.0

6.7

4.2

6.1

DMSO

Mean

82

67

28

34

44

62

24

24

sd

13.7

11.0

8.2

5.6

3.6

7.5

4.4

4.2

Ethanol

Mean

61

87

32

35

40

48

21

23

sd

8.5

14.2

4.0

4.2

1.0

1.5

5.0

4.9

Positive Controls*

Mean

631

237

459

165

429

491

280

141

sd

83.2

59.8

88.1

8.1

113.5

132.8

59.7

9.2

f(I)

7.70

3.54

16.39

4.85

8.41

7.92

12.73

5.88

5000

µg/plate

Mean

71

80

22

29

54

64

15

17

sd

5.7

1.2

7.2

3.0

6.1

8.5

1.7

2.6

f(I)

1.16

0.92

0.69

0.83

1.35

1.33

0.71

0.74

2500

µg/plate

Mean

69

76

25

29

46

73

18

26

sd

2.5

5.0

4.9

1.5

9.1

6.1

2.0

2.5

f(I)

1.13

0.87

0.78

0.83

1.15

1.52

0.86

1.13

1250

µg/plate

Mean

86

103

21

31

52

63

18

23

sd

14.7

19.3

2.1

10.1

7.0

14.5

2.0

5.5

f(I)

1.41

1.18

0.66

0.89

1.30

1.31

0.86

1.00

625

µg/plate

Mean

71

85

26

28

57

68

18

28

sd

12.7

17.2

9.6

4.7

4.6

9.0

1.5

3.5

f(I)

1.16

0.98

0.81

0.80

1.43

1.42

0.86

1.22

313

µg/plate

Mean

66

72

28

25

42

56

17

23

sd

0.0

4.2

5.2

4.5

7.0

5.6

2.3

2.1

f(I)

1.08

0.83

0.88

0.71

1.05

1.17

0.81

1.00

156

µg/plate

Mean

74

95

30

35

55

69

21

26

sd

2.9

4.0

7.2

7.9

8.7

8.1

6.6

4.0

f(I)

1.21

1.09

0.94

1.00

1.38

1.44

1.00

1.13

 

78 µg/plate

Mean

72

74

27

28

37

45

16

23

sd

4.6

6.4

8.0

9.0

7.9

11.4

3.1

2.9

f(I)

1.18

0.85

0.84

0.80

0.93

0.94

0.76

1.00

f(I) = increase factor

* Different positive controls were used

Table 4: Mean Revertants Experiment 2 (strain TA 102)

 

Strain

TA102

Induction

-S9

+S9

Demin. water

Mean

287

283

sd

19.7

28.1

DMSO

Mean

287

268

sd

42.0

38.2

Ethanol

Mean

253

335

sd

42.4

43.9

Positive Controls*

Mean

952

1325

sd

298.2

51.4

f(I)

3.32

4.94

500

µg/plate

Mean

320

332

sd

39.4

35.6

f(I)

1.26

0.99

250

µg/plate

Mean

292

292

sd

13.9

10.6

f(I)

1.15

0.87

125

µg/plate

Mean

301

287

sd

58.0

26.6

f(I)

1.19

0.86

62.5

µg/plate

Mean

271

269

sd

24.1

64.8

f(I)

1.07

0.80

31.3

µg/plate

Mean

276

283

sd

52.9

42.0

f(I)

1.09

0.84

15.6

µg/plate

Mean

289

315

sd

42.8

77.7

f(I)

1.14

0.94

7.8

µg/plate

Mean

280

275

sd

17.4

15.1

f(I)

1.11

0.82

f(I) = increase factor

* Different positive controls were used

Table 5: Historical Data of Spontaneous Revertants and Comparison with Experiments 1a, 1b and 2

 

Strain

 

TA97a

TA98

TA100

TA102

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

 

 

H2O de-min.

Mean

92

97

15

18

93

97

279

296

17

17

Min

60

63

6

8

62

66

85

67

6

7

Max

144

138

37

41

141

141

425

511

31

33

SD

19

16

6

6

15

15

65

75

6

6

Exp 1a

85

80

29

34

73

93

307

377

27

26

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

356

345

n.r.

n.r.

Exp 2

64

81

24

28

51

64

287

283

22

23

 

 

DMSO

Mean

91

100

15

16

90

93

279

289

17

16

Min

58

70

7

8

60

63

79

80

8

6

Max

135

144

46

36

136

199

393

459

33

31

SD

18

16

7

6

16

19

61

65

6

6

Exp 1a

98

97

35

38

78

78

320

388

22

24

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

257

324

n.r.

n.r.

Exp 2

82

67

28

34

44

62

287

268

24

24

 

 

Ethanol

Mean

89

99

16

17

86

90

284

275

16

16

Min

57

65

8

9

61

69

111

141

9

9

Max

181

205

35

40

116

129

368

339

28

29

SD

24

29

7

8

13

16

52

47

6

6

Exp 1a

76

83

24

26

72

72

295

313

22

22

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

292

258

n.r.

n.r.

Exp 2

61

87

32

35

40

48

253

335

21

23

 

 

Positive Controls*

Mean

552

501

394

83

513

716

1160

1237

254

115

Min

264

241

100

39

223

273

491

408

55

45

Max

1152

1181

793

333

984

1912

2331

6083

484

712

SD

169

144

143

49

153

290

452

659

89

79

Exp 1a

637

781

383

86

451

1528

1411

1472

323

183

Exp 1b

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

689

1083

n.r.

n.r.

Exp 2

631

237

459

165

429

491

952

1325

280

141

* Different positive controls were used

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 Aug - 9 Oct 2017
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)
Version / remarks:
adopted 29 July 2016
Deviations:
yes
Remarks:
Some of the total suspension growth (TSG) values of the solvent controls were slightly below the range specified in the guideline. In experiment II only 3 test item concentrations were evaluated.
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
adopted 30 May 2008
Deviations:
yes
Remarks:
Some of the total suspension growth (TSG) values of the solvent controls were slightly below the range specified in the guideline. In experiment II only 3 test item concentrations were evaluated.
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of assay:
other: in vitro gene mutation study in mammalian cells
Target gene:
TK-locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC (Wesel, Germany)
- Cell doubling time (h): 10 - 12

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with horse serum, sodium pyruvate and penicillin/streptomycin, 5.0 ±
0.5% CO2
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254 for 5 days
Test concentrations with justification for top dose:
Pre-test: 663.0, 331.5, 165.8, 82.9, 41.4, 20.7, and 10.4 µg/mL
Experiment I (+S9): 665.0, 332.5, 166.3, 83.1, 41.6, and 20.8 µg/mL
Experiment I (-S9): 332.5, 166.3, 83.1, 41.6, 20.8, and 10.4 µg/mL
Experiment II (-S9): 166.4, 83.2, 41.6, 20.8, 10.4, and 5.2 µg/mL

Precipitation was noted at the highest test item concentration (665.0 μg/mL). Turbidity occurred down to a concentration of 166.3 μg/mL. Relevant cytotoxic effects were determined at 665.0 μg/mL following 4 h-treatment with metabolic activation and at 665.0 μg/mL and 332.5 μg/mL following 4 h-treatment without metabolic activation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: The test item was not soluble in any of the solvents usually employed. The best result was obtained with ethanol.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: approx. 1 x 10E7

SELECTION AGENT: trifluorothymidine (TFT), 5 µg/mL

NUMBER OF REPLICATIONS: two

EXPERIMENTAL PROCEDURE AND DETAILS:
Pre-Test for Cytotoxicity (with and without S9-mix, two replicates per culture, 7 concentrations):
- Treatment for 4 h in the presence and in the absence of S9
- Remove treatment medium (with/without S9-mix) and test item
- Add fresh medium after washing
- Transfer of 2 cells per well into 96-well microtiter plates
- Incubation for 10-12 days
- Evaluation of relative cloning efficiency

Experiment I with S9-mix (two replicates per culture, 6 concentrations):
- Treatment for 4 h in the presence of S9-mix
- Remove treatment medium, S9-mix and test item
- Add fresh medium after washing
- Measure cell density directly after treatment as well as 24 ± 4 h and 48 ± 4 h after treatment
- Evaluation of % relative suspension growth (RSG)
- Transfer of cells into 96-well microtiter plates (2 cells per well for viability and 4000 cells per well for mutagenicity (with TFT))
- Incubation for 10-12 days
- Evaluation of cytotoxicity and mutagenicity

Experiment I without S9-mix (two replicates per culture, 6 concentrations):
- Treatment for 4 h in the absence of S9-mix
- Remove treatment medium and test item
- Add fresh medium after washing
- Measure cell density directly after treatment as well as 24 ± 4 h and 48 ± 4 h after treatment
- Evaluation of % relative suspension growth (RSG)
- Transfer of cells into 96 well microtiter plates (2 cells per well for viability and 4000 cells per well for mutagenicity (with TFT))
- Incubation for 10-12 days
- Evaluation of cytotoxicity and mutagenicity

Experiment II without S9, extended exposure (two replicates per culture, 6 concentrations):
- Treatment for 24 h in the absence of S9
- Remove treatment medium and test item
- Add fresh medium after washing
- Measure cell density directly after treatment as well as 24 ± 4 h and 48 ± 4 h after treatment
- Evaluation of toxicity and % relative suspension growth (RSG)
- Transfer of cells into 96-well microtiter plates (2 cells per well for viability 4000 cells per well for mutagenicity (with TFT))
- Incubation for 10-12 days
- Evaluation of cytotoxicity and mutagenicity

DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency
Evaluation criteria:
The test item is considered to have mutagenic effects if:
- the induced mutant frequency reproducibly exceeds a threshold of 126 colonies per 1 x 10E6 cells above the corresponding solvent control.
- the relative increase of the mutant frequency shows a dose relationship.

The test item is considered to have no mutagenic effects if:
- the induced mutation frequency does not exceeds a threshold of 126 colonies per 1 x 10E6 cells above the corresponding solvent control.
- the relative increase of the mutation frequency does not show a does relationship.

A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. Results of test groups are generally rejected if the relative total growth is less than 10% of the solvent control. The biological relevance of the results was always considered first. Appropriate statistical methods were used as an aid in evaluating the test results. However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data were also taken into consideration.

Reference: Moore MM, Honma M, Clements J, et al. (2007): Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment. Mutat Res.627(1):36-40.
Statistics:
Statistical significance was confirmed by means of the non-parametric χ2 test. However, both biological and statistical significance were considered together.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: 665.0, 332.5 μg/mL (+S9); 332.5, 166.3 µg/mL (-S9) Experiment II: 166.3, 83.1, 41.6 µg/mL (-S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Potential effects have been assessed in a preparative experiment. No effects were observed.
- Effects of osmolality: Potential effects have been assessed in a preparative experiment. No effects were observed.
- Water solubility: Test item was not soluble in water
- Precipitation: Observed at 665.0 μg/mL and turbidity occurred down to a concentration of 166.3 μg/mL.

VALIDATION AND COMPARISON WITH HISTORICAL CONTROL DATA.
- positive controls showed a distinct increase in induced total mutant colonies and exceeded the number of mutant colonies of more than 300 in comparison to the corresponding solvent control.
- In both experiments (with and without S9-mix) the range of the solvent controls was in the range of the historical data as well as in the range of the spontaneous mutant frequency of the L5178Y cells.

Table 2: Final results from experiment I and II (mean values of both cultures)

 

Content

Relative Total Growth [%]

Mutants per 106Cells

 

Exp I

Exp II

Exp I

Exp II

 

+S9

-S9

-S9

+S9

-S9

-S9

Threshold

-

-

-

182

178

176

Solvent Control of Test Item

-

-

-

56

52

50

Solvent Control of Positive Control

-

-

-

63

52

53

Positive Control

54

54

32

378

358

441

Test Item

665.0 µg/mL

0

 

 

*

 

 

Test Item

332.5 µg/mL

10

0

 

*

*

 

Test Item

166.3 µg/mL

32

15

0

76

*

*

Test Item

83.1 µg/mL

69

56

1

62

61

*

Test Item

41.6 µg/mL

82

93

10

56

55

*

Test Item

20.8 µg/mL

84

97

34

57

68

64

Test Item

10.4 µg/mL

 

99

55

 

64

68

Test Item

5.2 µg/mL

 

 

84

 

 

60

* Cultures were not evaluated because of cytotoxicity.

Note: The threshold is defined as the number of mutant colonies per 1 x 10E6 cells of the respective solvent control plus 126 and was defined by Moore et al., 2007

Moore MM, Honma M, Clements J, et al. (2007): Mouse lymphoma thymidine kinase gene mutation assay: meeting of the International Workshop on Genotoxicity Testing, San Francisco, 2005, recommendations for 24-h treatment. MutatRes.627(1):36-40.

Conclusions:
Interpretation of results: negative
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Aug - 09 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 2016
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted in 2017
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: cultured peripheral lymphocytes treated with anti-coagulant (heparin)
Details on mammalian cell type (if applicable):
CELLS USED
- Cell cycle length, doubling time or proliferation index: not specified
- Sex, age and number of blood donors if applicable: male and female healthy volunteers (approximately 18 - 35 years of age), non-smokers, with no known illness or recent exposure to genotoxic agents
- Whether whole blood or separated lymphocytes were used: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration: RPMI 1640 medium (CO2 concentration 5 ± 0.5%) supplemented with
- 15%(v/v) fetal calf serum (FCS)
- 1% (v/v) penicillin/streptomycin (per mL: 10,000 IU Pen / 10 mg Strep)
- 2% (v/v) phytohaemagglutinin solution
Metabolic activation:
with and without
Metabolic activation system:
cofactor supplemented post-mitochondrial fraction (S9-mix), prepared from the livers of rats treated with Aroclor 1254
Test concentrations with justification for top dose:
4 h-treatment with and without metabolic activation: 20.8, 41.6, 83.1, 166.3, 332.5 and 665.0 µg/mL
24 h-treatment without metabolic activation: 20.8, 41.6, 83.1, 166.3, 332.5 and 665.0 µg/mL

As the highest test item concentration (665.0 µg/mL) showed severe cytotoxicity, the 4 subsequent lower concentrations were selected for the evaluation of structural chromosome aberrations.
Vehicle / solvent:
- Vehicles/solvents used: ethanol (0.5%)
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined in demineralized water, ethanol, tetrahydrofuran, acetone and dimethyl sulfoxide (DMSO). The test item was not soluble in any of the solvents. The best results were obtained with ethanol. At a concentration of 133 mg/mL and after 45 minutes ultrasonic treatment, a homogeneous and pipettable suspension could be achieved. However, even at this concentration a separation in two phases was visible after some hours.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h with and without metabolic activation and 24 h without metabolic activation

SPINDLE INHIBITOR: 0.1 μg/mL colchicine (3 h before harvest)

STAIN: 10% solution of Giemsa

NUMBER OF REPLICATIONS: duplicates in two valid independent experiments (first approach without metabolic activation in experiment I was invalid (technical error) and had to be repeated)

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Each cell culture was harvested and processed separately. The cells were spun down by gentle centrifugation (10 min, 500 g). The supernatant was discarded and the cells were re-suspended in 12 mL hypotonic KCl solution. Then the cell suspension was allowed to stand at room temperature for 20 min. After removal of the hypotonic solution by centrifugation (10 min, 500 g), the cell pellet was exposed to fixative (3:1 mixture of methanol and glacial acetic acid). After fixation at 2 - 8 °C, minimum 30 min, the cell suspension was spun down by gentle centrifugation (10 min, 500 g), the supernatant was discarded and the cell pellet was re-suspended in fixative again. The washing procedures were repeated until the cell pellet was white. The slides were prepared by dropping the cell suspension onto clean microscope slides. The slides were then stained with a 10% solution of Giemsa. All slides were independently coded before microscopic analysis.

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: At least 300 well-spread metaphases
per concentration – 150 metaphases per replicate, if applicable - were scored for cytogenetic damage.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Only metaphases with 46 ± 2 centromeric regions were included in the analysis.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
Evaluation criteria:
A test item is classified as non-mutagenic if:
• Neither a statistically significant nor a concentration-related increase of the number of cells with structural chromosomal aberrations in the evaluated test concentrations is observed.
• The obtained results lie within the range of the historical laboratory control data for solvent controls.
A test item is classified as mutagenic if in any of the experimental conditions:
• The number of induced structural chromosome aberrations (excluding gaps) lies above the range of the historical laboratory control data for solvent controls, at least at one concentration of the test item.
• A dose-related increase in the number of cells with structural chromosome aberrations is observed.
• A statistically significant increase of structural chromosomal aberrations is found, at least at one concentration of the test item.
Statistics:
The number of metaphases with structural aberrations in each treatment group was compared with the solvent control value. Statistical significance was tested using Fisher’s exact test at the 5% level (p < 0.05).
Key result
Species / strain:
lymphocytes: cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In both valid experiments without metabolic activation, cytotoxicity in varying degrees was detected. In the approach with metabolic activation, no cytotoxic effect was detected.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: None of the tested positive and solvent controls or the test item provoked a critical change of the pH value in comparison to the solvent controls.
- Effects of osmolality: None of the tested positive and solvent controls or the test item provoked a critical change of the osmolarity in comparison to the solvent controls.
- Precipitation: Precipitation of the test substance was not observed.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
see Tables 1 and 2

Table 3. Results of Valid Cytotoxicity Experiment I without Metabolic Activation

 Treatment

 Mitotic Index in %

Mitotic Index in % of solvent control

  Cytotoxicity in %

Solvent control MCM

26.5

100

-

Solvent control ethanol 0.5% v/v

20.1

100

-

Positive control EMS 300 µg/mL

19.4

97

3

Positive control EMS 600 µg/mL

15.9

79

21

Test item 665 µg/mL

18.0

68

32

Test item 332.5 µg/mL

11.9

45

55

Test item 166.3 µg/mL

15.9

60

40

Test item 83.1 µg/mL

21.5

81

19

Test item 41.6 µg/mL

24.4

92

8

Test item 20.8 µg/mL

27.4

104

-4

Generally, cytotoxicity was not a limiting factor for the evaluation of chromosomal aberrations, but the highest test item concentration did not show the highest cytotoxicity. Therefore, the 4 highest test item concentrations (including the concentration with the highest cytotoxicity of 55% and concentrations with moderate/low cytotoxicity) were scored for structural chromosomal aberrations.

Table 4. Results of Valid Cytotoxicity Experiment I with Metabolic Activation

 Treatment

  Mitotic Index in %

Mitotic Index in % of solvent control

  Cytotoxicity in %

Solvent control ethanol 0.5% v/v

12.1

100

-

Solvent control 0.9% NaCl 0.5% v/v

11.8

100

-

Positive control CPA 30 µg/mL

11.2

94

6

Positive control CPA 20 µg/mL

10.7

90

10

Test item 665 µg/mL

8.3

68

32

Test item 332.5 µg/mL

9.2

76

24

Test item 166.3 µg/mL

12.9

107

-7

Test item 83.1 µg/mL

13.6

113

-13

Test item 41.6 µg/mL

14.6

121

-21

Test item 20.8 µg/mL

13.7

114

-14

No limiting cytotoxicity was observed. Therefore, the 3 highest test item concentrations were scored for structural chromosomal aberrations.

Table 5. Results Chromosomal Aberration Assay Experiment I - without Metabolic Activation

Treatment

Aberrant cells in %

Including gaps*

Excluding gaps*

with exchanges

Without metabolic activation

Solvent control MCM

0.7

0.7

0.0

Solvent control ethanol 0.5% v/v

6.7

2.7

0.0

Positive control EMS 600 µg/ml

21.0

16.7S

4.0

Test item 665 µg/mL

4.7

1.7

0.0

Test item 332.5 µg/mL

3.6

1.6

0.0

Test item 166.3 µg/mL

4.5

1.9

0.0

Test item 83.1 µg/mL

3.3

1.0

0.0

With metabolic activation

Solvent control ethanol 0.5% v/v

6.0

2.7

0.7

Solvent control 0.9% NaCl 0.5% v/v

4.7

2.7

0.3

Positive control CPA 30 µg/ml

20.7

14.0S

2.0

Test item 665 µg/mL

5.7

1.3

0.0

Test item 332.5 µg/mL

7.0

2.0

0.5

Test item 166.3 µg/mL

3.0

1.7

0.0

* including cells carrying exchanges

S aberration frequency statistically significantly higher than corresponding control values

Table 6. Results Cytotoxicity Experiment II without Metabolic Activation

 Treatment

  Mitotic Index in %

Mitotic Index in % of solvent control

  Cytotoxicity in %

Solvent control MCM

9.2

100

--

Solvent control ethanol 0.5% v/v

9.5

100

--

Positive control EMS 300 µg/mL

4.4

46

54

Positive control EMS 600 µg/mL

1.6

16

84

Test item 665 µg/mL

2.6

28

72

Test item 332.5 µg/mL

8.1

89

11

Test item 166.3 µg/mL

9.0

98

2

Test item 83.1 µg/mL

10.1

110

-10

Test item 41.6 µg/mL

9.4

102

-2

Test item 20.8 µg/mL

9.6

104

-4

Table 7. Results Chromosomal Aberration Assay Experiment II

Treatment

Aberrant cells in %

Including gaps*

Excluding gaps*

with exchanges

Solvent control MCM

8.3

3.3

0

Solvent control ethanol 0.5% v/v

6.3

2.0

0.3

Positive control EMS 300 mg/mL

29.3

16.3S

3.3

Test item 332.5 µg/mL

6.0

2.0

0

Test item 166.3 µg/mL

4.7

1.7

0

Test item 83.1 µg/mL

7.0

2.3

0

Test item 41.6 µg/mL

6.3

2.3

0

* including cells carrying exchanges

S aberration frequency statistically significantly higher than corresponding control values

Conclusions:
Interpretation of results: negative
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro gene mutation in bacteria and mammalian cells as well as chromosome aberration in mammalian cells were tested with 4-(octadecylamino)-4-oxoisocrotonic acid.

Genetic toxicity (mutagenicity) in bacteria in vitro

A bacterial gene mutation assay (Ames test) was performed with 4-(octadecylamino)-4-oxoisocrotonic acid according to OECD 471 (LAUS, 2017c). S. typhimurium strains TA 97a, TA 98, TA 100 and TA 102 and TA1535 were tested conducting both the plate incorporation and the pre-incubation method in the absence and presence of a metabolic activation (Aroclor 1254-induced rat liver S9-mix). The experiment was conducted in 3 replicates in two independent experiments each up to the limit concentration of 5000 µg/plate. Cytotoxicity was observed for S. typhimurium strain TA102 at 5000 and 1500 µg/plate (with and without metabolic activation) in the plate incorporation test, therefore the dose used for the pre-incubation method was reduced to a maximum of 500 µg/plate. No increase in the number of revertant colonies was noted in any of the bacterial strains, with and without metabolic activation system. Appropriate positive and solvent controls were included in the test and showed the expected results. Under the conditions of the study, 4-(octadecylamino)-4-oxoisocrotonic acid did not induce mutations in the bacterial mutation assay in the absence and presence of a metabolic activation system in any of the strains tested.

Genetic toxicity (cytogenicity) in mammalian cells in vitro

An in vitro mammalian chromosome aberration test was conducted with 4-(octadecylamino)-4-oxoisocrotonic acid in accordance with OECD 473 under GLP conditions (LAUS, 2018b). The induction of structural chromosome aberrations was evaluated in vitro in lymphocytes of cultured heparinised human whole blood, incubated for 4 h or 24 h with and without metabolic activation (S9-mix from rats treated with Arocolor 1254). Test substance concentrations of 20.8 - 665 µg/mL with and without metabolic activation were applied. The negative as well as the positive controls showed the expected results. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in this chromosomal aberration test with and without metabolic activation performed in peripheral human lymphocytes in vitro.

Genetic toxicity (mutagenicity) in mammalian cells in vitro

The in vitro mammalian cell gene mutation study of 4-(octadecylamino)-4-oxoisocrotonic acid was carried out according to OECD 490 under GLP conditions (LAUS, 2018c). Gene mutations in the thymidine kinase locus were investigated in L5178Y mouse lymphoma cells in the presence and absence of a metabolic activation system (Arocolor 1254-induced rat liver, S9-mix). In the first experiment, cells were exposed to six test substance concentrations between 20.8 - 665.0 µg/mL with metabolic activation and six concentrations between 10.4 - 332.5 µg/mL without metabolic activation for 4 h. In the second experiment, cells were exposed to six test substance concentrations between 5.2 -166.4 µg/mL without metabolic activation for 24 h. The vehicle and positive controls in the study showed the expected results and were within the range of historical control data of the laboratory. Precipitation was noted at the highest test substance concentration (665.0 μg/mL). Turbidity occurred down to a concentration of 166.3 μg/mL. Relevant cytotoxic effects were determined in the first experiment at 665.0 μg/mL following 4 h treatment with metabolic activation and at 665.0 μg/mL and 332.5 μg/mL following 4 h treatment without metabolic activation. In the second experiment without metabolic activation a cytotoxic effect was observed at concentrations of 166.3 μg/mL, 83.1 μg/mL and 41.6 μg/mL. These concentrations were not evaluated for mutagenicity. There was no significant increase in the number of forward mutations at the thymidine kinase locus of L5178Y mouse lymphoma cells treated with the test subtsance with and without metabolic activation. Under the conditions of the study, 4-(octadecylamino)-4-oxoisocrotonic acid did not show gene mutation activity in this test performed in L5178Y mouse lymphoma cells in vitro.

Conclusion

Taken together, the available data on genetic toxicity of 4-(octadecylamino)-4-oxoisocrotonic acid do not indicate any mutagenic and clastogenic potential in vitro.

Justification for classification or non-classification

The available data on genetic toxicity of the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008 and are, therefore, conclusive but not sufficient for classification.