Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 December 2017 to 18 July 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by the sponsor, batch 99671
- Expiration date of the lot/batch: 9 October 2019
- Purity test date: 6 November 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: the test item was stored in the test facility in a closed vessel at room temperature (20±5°C)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: the test item was completely soluble in acetone, only
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No reactivity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: a stock solution containing 50 mL/L of the test item in acetone was prepared. Nominal concentrations were prepared
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel: 50 mL/L
- Final preparation of a solid: not applicable

FORM AS APPLIED IN THE TEST (if different from that of starting material)
In solution with acetone

Method

Species / strain
Species / strain / cell type:
other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: See tables in "any other information on material and methods including tables" section
Metabolic activation:
with and without
Metabolic activation system:
S9 was obtained produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
Test concentrations with justification for top dose:
On the day of the start of the experiment 1a, a stock solution containing 50 mL/L of the test item in acetone was prepared. The following nominal concentrations were prepared for the first experiment: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate and 0.05 µL/plate.

The following nominal concentrations were prepared for the experiment 1b for the bacteria strains TA97a, TA98, TA100 and TA102: 5 µL/plate, 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate and 0.015 µL/plate.
The following nominal concentrations were prepared for the experiment 1b for the bacteria strain TA1535: 1.5 µL/plate, 0.5 µL/plate, 0.15 µL/plate, 0.05 µL/plate, 0.015 µL and 0.005 µL/plate.

The following nominal concentrations were prepared for the experiment 1b for the bacteria strains TA97a, TA98, TA100 and TA102: 5 µL/plate, 2.5 µL/plate, 1.25 µL/plate, 0.63 µL/plate, 0.31 µL/plate, 0.16 µL/plate, 0.08 µL/plate and 0.04 µL/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle:In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 mL/L in demineralized water, dimethyl sulfoxide (DMSO) and acetone.
The test item was completely soluble in acetone, only.
Acetone was chosen as vehicle, because the test item was sufficiently soluble, and this solvent does not have any effects on the viability of the bacteria or the number of sponta-neous revertants in the tested concentrations.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
benzo(a)pyrene
other: 4-nitro-1,2-phenylene Diamine ; 2-Amino-anthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 10E9 bacteria/mL

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours after treatment

NUMBER OF REPLICATIONS: three replicates were used per condition

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The colonies were counted visually and the numbers were recorded.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: bacterial background lawn
Evaluation criteria:
A substance is considered to have mutagenic potential, if a reproducible increase of re-vertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. An increase factor of 3 should be taken into account for the bacteria strain TA1535. 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 spread-sheet software (Microsoft Excel®) was used to calculate mean values and standard devia-tions of each treatment, solvent control and positive control.
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.

Results and discussion

Test results
Key result
Species / strain:
other: S. Typhimurium TA97a, TA98, TA100, TA102 and TA1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Experiment 1a:
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants (one exception) of the negative controls were in the normal range of the test laboratory. All positive controls (diagnostic muta-gens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges.
In the first experiment, the test item showed no precipitates on the plates in all tested concentrations. In the highest concentration (5 µL/plate), no bacteria growth and no bacterial background lawn was observed. Towards the bacteria strain TA1535, signs of toxicity (decrease in the number of revertants) were observed in the next lower concentration (1.5 µL/plate), too. No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found.

Experiment 1b
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls (one exception in the negative control acetone) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges. In this experiment, the test item showed no precipitates on the plates in all tested concentrations. In the highest concentration (5 µL/plate), no bacteria growth and no bacterial background lawn was observed. Towards the bacteria strain TA1535, signs of toxicity (decrease in the number of revertants) were observed in the next lower concentration (1.5 µL/plate), too. No increase of the number of revertant colonies in the treatments with and without meta-bolic activation could be observed. No concentration-related increase over the tested range was found.

Experiment 2:
All strains met the criterion of at least 109 bacteria/mL, and no inconsistencies were found in the sterility control. Nearly all determined values for the spontaneous revertants of the negative controls (one exception in the negative control acetone) were in the normal range of the test laboratory. All positive controls (diagnostic mutagens) showed mutagenic effects with and without metabolic activation and were within the historical control data ranges. In this experiment, the test item showed no precipitates on the plates in all tested concentrations. Signs of toxicity were observed in the following concentrations with and without metabolic activation towards the resp. bacteria strains:
TA97a: no toxicity was observed
TA98: 5 µL/plate (decrease in the number of revertants)
TA100: 5 and 2.5 µL/plate (decrease in the number of revertants)
TA102: no toxicity was observed
TA1535: 1.5 µL/plate (decrease in the number of revertants)

The bacterial background lawn was visible in all concentrations.

No significant increase of the number of revertant colonies in the treatments with and without metabolic activation could be observed. No concentration-related increase over the tested range was found.

Historical data were presented in table in any other information on results including tables section.

Any other information on results incl. tables

Table2           Mean Revertants Experiment 1a

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

73

84

36

33

92

108

249

259

21

22

sd

7.6

5.1

6.2

5.5

8.0

15.9

26.6

53.3

1.2

1.5

DMSO

Mean

74

110

38

36

89

91

239

249

26

18

sd

5.2

21.1

4.4

11.9

9.2

1.2

28.9

46.0

1.7

0.6

Acetone

Mean

74

109

36

35

99

99

288

272

25

23

sd

6.4

15.5

2.1

4.5

27.2

2.3

38.2

24.0

5.8

2.0

Positive
Controls*

Mean

479

463

268

176

379

1001

657

1323

220

175

sd

67.2

132.3

28.0

24.3

62.3

0.0

9.2

60.0

10.6

31.1

f(I)

6.47

4.21

7.05

4.89

4.12

11.00

2.75

5.31

10.48

9.72

5 µL/plate

Mean

0

0

0

0

0

0

0

0

0

0

sd

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

f(I)

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

0.00

1.5 µL/plate

Mean

72

83

26

36

80

95

201

217

11

14

sd

8.6

1.2

6.0

6.9

9.8

11.7

55.2

60.7

1.7

3.5

f(I)

0.97

0.76

0.72

1.03

0.81

0.96

0.70

0.80

0.44

0.61

0.5 µL/plate

Mean

85

95

32

33

83

104

251

252

18

16

sd

4.6

13.3

4.7

7.4

15.1

20.0

19.7

93.0

4.0

7.2

f(I)

1.15

0.87

0.89

0.94

0.84

1.05

0.87

0.93

0.72

0.70

0.15 µL/plate

Mean

78

86

36

35

95

95

224

243

13

17

sd

10.4

27.1

6.4

9.6

1.2

7.0

48.0

39.3

2.6

3.2

f(I)

1.05

0.79

1.00

1.00

0.96

0.96

0.78

0.89

0.52

0.74

0.05 µL/plate

Mean

79

74

35

35

92

97

203

188

25

24

sd

15.1

9.2

1.2

1.5

9.2

24.3

12.9

25.0

5.3

5.6

f(I)

1.07

0.68

0.97

1.00

0.93

0.98

0.70

0.69

1.00

1.04

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

 

Table3Mean Revertants Experiment 1b

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

77

86

35

34

86

80

335

329

29

25

sd

14.8

18.9

3.2

6.1

12.0

15.4

30.0

20.1

5.0

4.0

DMSO

Mean

79

71

34

38

75

87

328

283

21

19

sd

15.6

9.5

2.6

6.5

14.2

20.7

36.0

35.9

2.6

5.9

Acetone

Mean

73

77

36

41

81

112

325

300

24

24

sd

5.8

2.6

5.6

5.9

7.6

14.2

15.1

58.9

6.9

4.0

Positive
Controls*

Mean

584

669

443

451

547

1001

1371

1533

343

243

sd

65.5

6.1

72.6

68.0

196.3

0.0

59.0

53.3

44.1

11.5

f(I)

7.39

9.42

13.03

11.87

6.36

11.51

4.18

5.42

11.83

12.79

5 µL/plate

Mean

72

89

8

11

20

36

41

43

n.d.

n.d.

sd

12.0

11.8

1.0

1.0

6.1

20.8

0.6

8.7

n.d.

n.d.

f(I)

0.99

1.16

0.22

0.27

0.25

0.32

0.13

0.14

n.d.

n.d.

1.5 µL/plate

Mean

84

101

34

34

79

106

320

293

7

4

sd

4.0

14.5

7.6

6.4

3.6

18.6

52.5

134.5

0.6

2.3

f(I)

1.15

1.31

0.94

0.83

0.98

0.95

0.98

0.98

0.29

0.17

0.5 µL/plate

Mean

88

119

34

35

82

88

231

340

30

30

sd

8.0

4.2

4.4

4.5

10.1

16.5

56.6

66.1

9.2

6.7

f(I)

1.21

1.55

0.94

0.85

1.01

0.79

0.71

1.13

1.25

1.25

0.15 µL/plate

Mean

94

97

33

39

82

105

311

328

29

31

sd

1.7

6.4

7.0

10.4

12.8

6.4

112.0

0.0

9.1

7.0

f(I)

1.29

1.26

0.92

0.95

1.01

0.94

0.96

1.09

1.21

1.29

0.05 µL/plate

Mean

70

89

43

38

111

112

293

395

35

28

sd

12.1

18.6

1.5

6.4

13.0

14.2

48.9

51.4

4.6

7.5

f(I)

0.96

1.16

1.19

0.93

1.37

1.00

0.90

1.32

1.46

1.17

0.015 µL/plate

Mean

78

76

36

36

97

100

384

403

26

31

sd

8.7

8.7

1.7

5.0

15.1

18.3

52.5

41.1

3.0

6.1

f(I)

1.07

0.99

1.00

0.88

1.20

0.89

1.18

1.34

1.08

1.29

0.005 µL/plate

Mean

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

32

34

sd

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

6.7

6.4

f(I)

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

n.d.

1.33

1.42

1001 colonies per plate means the bacteria growth was too strong for counting.

n.d. = not determined, due to the toxicity effect in experiment 1a

 f(I) = increase factor

 


Table4          Mean Revertants Experiment 2

Strain

TA97a

TA98

TA100

TA102

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

Demin. water

Mean

80

75

34

45

101

117

317

325

sd

14.0

8.3

4.0

2.1

14.2

2.3

22.7

18.0

DMSO

Mean

71

81

41

42

87

92

304

300

sd

9.0

1.2

7.5

3.8

5.9

10.4

38.2

41.6

Acetone

Mean

86

79

39

42

84

120

331

291

sd

13.5

12.5

5.3

3.6

3.5

5.3

10.1

36.3

Positive
Controls*

Mean

268

368

307

328

372

1001

663

1267

sd

50.1

26.2

59.9

52.9

32.7

0.0

52.8

125.6

f(I)

3.77

4.54

7.49

7.81

3.68

10.88

2.18

4.22

5 µL/plate

Mean

72

93

15

12

12

26

296

343

sd

19.1

25.4

1.2

5.2

1.0

9.9

55.0

20.1

f(I)

0.84

1.18

0.38

0.29

0.14

0.22

0.89

1.18

2.5 µL/plate

Mean

78

89

37

24

38

45

267

256

sd

12.5

5.3

7.5

5.6

13.5

13.1

82.6

24.3

f(I)

0.91

1.13

0.95

0.57

0.45

0.38

0.81

0.88

1.25 µL/plate

Mean

77

74

37

29

74

76

333

369

sd

4.7

7.5

5.2

10.0

10.5

6.4

24.1

100.4

f(I)

0.90

0.94

0.95

0.69

0.88

0.63

1.01

1.27

0.63 µL/plate

Mean

69

77

36

34

75

104

316

329

sd

14.2

3.1

4.2

3.0

4.5

8.5

50.0

56.6

f(I)

0.80

0.97

0.92

0.81

0.89

0.87

0.95

1.13

0.31 µL/plate

Mean

100

77

34

38

86

96

320

376

sd

10.1

9.6

8.4

3.5

8.7

15.0

48.5

17.4

f(I)

1.16

0.97

0.87

0.90

1.02

0.80

0.97

1.29

0.16 µL/plate

Mean

82

72

35

33

111

110

352

388

sd

1.5

3.2

14.0

3.8

12.7

11.7

24.3

82.7

f(I)

0.95

0.91

0.90

0.79

1.32

0.92

1.06

1.33

0.08 µL/plate

Mean

86

91

37

34

108

105

397

421

sd

18.0

4.0

2.5

1.7

8.9

18.0

73.4

26.6

f(I)

1.00

1.15

0.95

0.81

1.29

0.88

1.20

1.45

0.04 µL/plate

Mean

80

81

42

36

93

89

297

389

sd

2.0

1.0

12.5

2.6

16.8

8.7

75.6

68.6

f(I)

0.93

1.03

1.08

0.86

1.11

0.74

0.90

1.34

1001 colonies per plate means the bacteria growth was too strong for counting.

f(I) = increase factor

 

Strain

TA1535

Induction

-S9

+S9

Demin. water

Mean

14

12

sd

5.2

1.7

DMSO

Mean

11

16

sd

5.1

4.2

Acetone

Mean

16

15

sd

2.6

5.1

Positive
Controls*

Mean

333

197

sd

56.8

76.4

f(I)

23.79

12.31

1.5 µL/plate

Mean

5

3

sd

0.6

0.6

f(I)

0.31

0.20

0.75 µL/plate

Mean

13

14

sd

3.6

2.5

f(I)

0.81

0.93

0.38 µL/plate

Mean

14

12

sd

1.0

2.5

f(I)

0.88

0.80

0.19 µL/plate

Mean

12

15

sd

1.5

3.1

f(I)

0.75

1.00

0.09 µL/plate

Mean

13

8

sd

2.9

1.2

f(I)

0.81

0.53

0.05 µL/plate

Mean

13

11

sd

4.2

2.6

f(I)

0.81

0.73

0.02 µL/plate

Mean

13

14

sd

2.9

3.2

f(I)

0.81

0.93

0.01 µL/plate

Mean

17

13

sd

4.6

1.5

f(I)

1.06

0.87

f(I) = increase factor

Table5           Historical Data of Spontaneous Revertants

Strain

 

TA97a

TA98

TA100

TA102

TA1535

Induction

 

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

- S9

+ S9

Demin. water

Mean

89

94

20

22

92

96

279

297

18

18

Min

60

63

6

8

51

64

85

67

6

7

Max

144

138

52

51

141

141

425

511

31

33

SD

18

16

11

10

15

14

58

69

6

6

Exp 1a

73

84

36

33

92

108

249

259

21

22

Exp 1b

77

86

35

34

86

80

335

329

29

25

Exp 2

80

75

34

45

101

117

317

325

14

12

DMSO

Mean

88

97

20

21

89

92

278

294

18

17

Min

58

67

7

8

44

62

79

80

8

6

Max

135

144

47

50

136

199

393

459

33

32

SD

17

17

11

10

16

17

56

61

6

6

Exp 1a

74

110

38

36

89

91

239

249

26

18

Exp 1b

79

71

34

38

75

87

328

283

21

19

Exp 2

71

81

41

42

87

92

304

300

11

16

Acetone

Mean

66

107

16

19

68

76

335

338

16

16

Min

34

61

6

11

52

54

227

223

10

10

Max

88

139

52

51

109

106

525

485

24

25

SD

17

25

11

10

14

14

83

83

4

4

Exp 1a

74

109

36

35

99

99

288

272

25

23

Exp 1b

73

77

36

41

81

112

325

300

24

24

Exp 2

86

79

39

42

84

120

331

291

16

15

Positive Controls*

Mean

537

520

407

113

490

764

1110

1213

265

132

Min

264

237

100

39

220

273

491

408

55

45

Max

1165

1181

1001

487

984

1912

2331

6083

515

712

SD

173

160

163

90

153

288

423

585

88

82

Exp 1a

479

463

268

176

379

1001

657

1323

220

175

Exp 1b

584

669

443

451

547

1001

1371

1533

343

243

Exp 2

268

368

307

328

372

1001

663

1267

333

197

* Different positive controls were used

1001 colonies per plate means the bacteria growth was too strong for counting.

 

Applicant's summary and conclusion

Conclusions:
Under the experimental condition of the study, the results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Hence, the test item Triisononylamine was not considered as mutagenic for bacteria strain according to CLP criteria.
Executive summary:

In this GLP compliant in vitro test, the test item Triisononylamine was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535) according to OECD TG 471 method.

The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation.

Experiment 1a:

In this experiment, the test item (dissolved in acetone) was tested up to concentrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. In the highest concentration (5 µL/plate), no bacteria growth and no bacterial background lawn was observed. Towards the bacteria strain TA1535, signs of toxicity (decrease in the number of revertants) were observed in the next lower concentration (1.5 µL/plate), too.

The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 1b:

In this experiment (repetititon of experiment 1a with lower concentrations), the test item was tested up to concentrations of 5  µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100 and TA102 and up to concentrations of 1.5  µL/plate in the absence and presence of S9-mix in the strain TA1535 using the plate incorporation method, too. The test item showed no precipitates on the plates at any of the concentrations. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation.

Experiment 2:

Based on the toxicity results of experiment 1a and 1b, the test item was tested up to con-centrations of 5 µL/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100 and TA102 and up to concentrations of 1.5  µL/plate in the absence and presence of S9-mix in the strain TA1535 using the pre-incubation method.

Signs of toxicity were observed in the following concentrations with and without metabolic activation towards the resp. bacteria strains:

TA97a: no toxicity was observed

TA98: 5 µL/plate (decrease in the number of revertants)

TA100: 5 and 2.5 µL/plate (decrease in the number of revertants)

TA102: no toxicity was observed

TA1535: 1.5 µL/plate (decrease in the number of revertants)

The results of this experiments showed that the test item caused no increase in the num-ber of revertants in all bacteria strains compared to the solvent control, in both the ab-sence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertants colonies in all strains, in the presence and absence of metabolic activation.

Based on the results of this study it is concluded that Triisononylamine is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study. Hence, the test item Triisononylamine was not considered as mutagenic for bacteria strain according to CLP criteria.