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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

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)
Principles of method if other than guideline:
This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Tetrabutylammonium bromide
- IUPAC name: 1-Butanaminium, N,N,N-tributyl-, bromide
- Molecular formula: C16H36BNr
- Molecular weight: 322.37 g/mol
- Substance type: Organic
- Physical state: White crystalline powder
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Aroclor 1254 induced S9 metabolic activation system
Test concentrations with justification for top dose:
0.0, 0.050, 0.158, 0.501, 1.582 or 5 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)] : Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in distilles water
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

DURATION
- Preincubation period: Trial I: Not applicable Trial II: 60 min
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: Each concentration, including the negative, vehicle and positive controls was tested in triplicate in two independent experiments performed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): No data

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
- Any supplementary information relevant to cytotoxicity: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): No data

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.

A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.

An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.0 (N.C), 0.002(T1), 0.005(T2), 0.016(T3), 0.050(T4), 0.158(T5), 0.501(T6), 1.582(T7) and 5(T8) mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 - 5 mg/plate based on the solubility and precipitation test. In treated concentrations 5 - 0.002 mg/plate (T8-T1) no colony reduction or back ground lawn reduction was observed both, in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.050(T1), 0.158(T2), 0.501(T3), 1.582(T4) and 5(T5) mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Any other information on results incl. tables

Table 1: REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Without metabolic activation (-S9)

With metabolic activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

104

22

116

28

R2

106

20

120

25

R3

108

24

118

21

T1

(0.002)

R1

82

12

90

20

R2

84

14

86

18

R3

90

12

88

16

T2

(0.005)

R1

85

14

84

19

R2

80

12

82

21

R3

86

14

80

15

T3

(0.016)

R1

88

18

96

18

R2

94

15

92

21

R3

82

14

88

18

T4

(0.050)

R1

92

18

86

20

R2

88

16

90

18

R3

90

16

82

20

T5

(0.158)

R1

86

14

84

18

R2

92

18

90

22

R3

86

16

82

20

T6

(0.501)

R1

90

18

92

20

R2

92

19

88

18

R3

96

15

84

18

T7

(1.582)

R1

94

20

88

20

R2

90

16

86

22

R3

92

18

86

22

T8

(5)

R1

98

16

94

24

R2

96

20

104

22

R3

92

20

102

24

PC

R1

1240

976

1456

1008

R2

1288

1008

1480

1128

R3

1256

1032

1448

1104

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

T              =     Test concentration (T8: Highest, T1: Lowest)

4-Nitro-o-phenylenediamine [10μg/plate]: TA 98

Sodium azide [10μg/plate]: TA 100,

2-Aminoanthracene [2.5μg/plate]: TA98, TA100

 

TABLE 2 - REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIAL I)

Dose (mg/plate)

R

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

8

14

28

116

282

R2

6

12

25

120

288

R3

8

14

21

118

282

T1

(0.050)

R1

5

10

20

86

226

R2

5

10

18

90

240

R3

5

10

20

82

234

T2

(0.158)

R1

7

10

18

84

236

R2

5

11

22

90

252

R3

6

11

20

82

246

T3

(0.501)

R1

5

10

20

92

240

R2

7

10

18

88

248

R3

5

11

18

84

232

T4

(1.582)

R1

6

12

20

88

260

R2

7

10

22

86

248

R3

6

12

22

86

232

T5

(5)

R1

7

12

24

94

282

R2

7

10

22

104

268

R3

6

14

24

102

272

PC

R1

142

448

1008

1456

1266

R2

164

472

1128

1480

1544

R3

176

456

1104

1448

1368

 

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

12

22

104

274

R2

6

15

20

106

268

R3

7

15

24

108

288

T1

(0.050)

R1

5

10

18

92

232

R2

4

10

16

88

244

R3

4

8

16

90

238

T2

(0.158)

R1

5

12

14

86

228

R2

5

10

18

92

240

R3

4

10

16

86

234

T3

(0.501)

R1

5

12

18

90

242

R2

6

15

19

92

238

R3

5

10

15

96

244

T4

(1.582)

R1

5

12

20

94

240

R2

5

10

16

90

236

R3

5

14

18

92

260

T5

(5)

R1

6

12

16

98

264

R2

6

14

20

96

252

R3

7

12

20

92

240

PC

R1

168

1208

976

1240

1584

R2

156

1232

1008

1288

1648

R3

184

1264

1032

1256

1632

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA 98, TA 100        
2- Aminoanthracene [10μg/plate]:TA 102                                             Sodium azide [10μg/plate]: TA 1535, TA 100                                             

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98[10μg/plate]      Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 3 - REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose (mg/plate)

R

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

6

12

27

114

256

R2

5

15

24

122

264

R3

7

14

24

120

257

T1

(0.050)

R1

4

10

20

102

244

R2

3

12

22

110

232

R3

4

12

24

104

238

T2

(0.158)

R1

5

12

22

102

242

R2

4

10

20

106

238

R3

5

14

22

110

244

T3

(0.501)

R1

5

13

24

100

240

R2

4

10

20

108

236

R3

4

12

24

105

242

T4

(1.582)

R1

6

13

22

110

246

R2

4

12

24

106

238

R3

5

12

24

108

242

T5

(5)

R1

4

12

22

112

246

R2

6

12

24

110

254

R3

6

14

26

112

250

PC

R1

178

448

1482

1444

1712

R2

189

496

1504

1460

1796

R3

202

502

1548

1528

1808

 

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

4

14

25

122

246

R2

6

12

24

118

252

R3

7

15

26

112

264

T1

(0.050)

R1

3

10

18

100

236

R2

4

8

20

110

242

R3

4

12

18

106

240

T2

(0.158)

R1

5

10

22

112

240

R2

3

8

18

106

244

R3

4

14

20

108

246

T3

(0.501)

R1

5

12

20

106

246

R2

4

13

18

106

240

R3

5

10

20

106

236

T4

(1.582)

R1

4

12

22

108

248

R2

4

9

20

114

232

R3

5

12

22

110

256

T5

(5)

R1

6

10

24

112

252

R2

4

12

22

110

248

R3

6

14

22

118

244

PC

R1

184

1076

808

1208

1504

R2

196

1136

916

1364

1620

R3

210

1208

1024

1344

1748

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),R= Replicate

PC= Positive control                                                                       2-Aminoanthracene [2.5μg/plate]: TA 1537, TA1535, TA98, TA100        
2-Aminoanthracene [10μg/plate]:TA 102                                              Sodium azide [10μg/plate]: TA 1535, TA 100,                                            

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate] TA 98[10μg/plate]        Methyl methanesulfonate [4μl/plate]: TA 102

 

TABLE 4 - MEAN REVERTANT COUNT IN PLATE INCORPORATION METHOD (TRIALI)

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

7.33

1.15

13.33

1.15

24.67

3.51

118.00

2.00

284.00

3.46

T1

(0.050)

5.00

0.00

10.00

0.00

19.33

1.15

86.00

4.00

233.33

7.02

T2

(0.158)

6.00

1.00

10.67

0.58

20.00

2.00

85.33

4.16

244.67

8.08

T3

(0.501)

5.67

1.15

10.33

0.58

18.67

1.15

88.00

4.00

240.00

8.00

T4

(1.582)

6.33

0.58

11.33

1.15

21.33

1.15

86.67

1.15

246.67

14.05

T5

(5)

6.67

0.58

12.00

2.00

23.33

1.15

100.00

5.29

274.00

7.21

PC

160.67

17.24

458.67

12.22

1080.00

63.50

1461.33

16.65

1392.67

140.63

 

 

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

6.67

0.58

14.00

1.73

22.00

2.00

106.00

2.00

276.67

10.26

T1

(0.050)

4.33

0.58

9.33

1.15

16.67

1.15

90.00

2.00

238.00

6.00

T2

(0.158)

4.67

0.58

10.67

1.15

16.00

2.00

88.00

3.46

234.00

6.00

T3

(0.501)

5.33

0.58

12.33

2.52

17.33

2.08

92.67

3.06

241.33

3.06

T4

(1.582)

5.00

0.00

12.00

2.00

18.00

2.00

92.00

2.00

245.33

12.86

T5

(5)

6.33

0.58

12.67

1.15

18.67

2.31

95.33

3.06

252.00

12.00

PC

169.33

14.05

1234.67

28.10

1005.33

28.10

1261.33

24.44

1621.33

33.31

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATIONMETHOD (TRIAL II)

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

6.00

1.00

13.67

1.53

25.00

1.73

118.67

4.16

259.00

4.36

T1

(0.050)

3.67

0.58

11.33

1.15

22.00

2.00

10.33

4.16

238.00

6.00

T2

(0.158)

4.67

0.58

12.00

2.00

21.33

1.15

106.00

4.00

241.33

3.06

T3

(0.501)

4.33

0.58

11.67

1.53

22.67

2.31

104.33

4.04

239.33

3.06

T4

(1.582)

5.00

1.00

12.33

0.58

23.33

1.15

108.00

2.00

242.00

4.00

T5

(5)

5.33

1.15

12.67

1.15

24.00

2.00

111.33

1.15

250.00

4.00

PC

189.67

12.01

482.00

29.60

1511.33

33.61

1477.33

44.60

1772.00

52.31

 

 

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.67

1.53

13.67

1.53

25.00

1.00

117.33

5.03

254.00

9.17

T1

(0.050)

3.67

0.58

10.00

2.00

18.67

1.15

105.33

5.03

239.33

3.06

T2

(0.158)

4.00

1.00

10.67

3.06

20.00

2.00

108.67

3.06

243.33

3.06

T3

(0.501)

4.67

0.58

11.67

1.53

19.33

1.15

106.00

0.00

240.67

5.03

T4

(1.582)

4.33

0.58

11.00

1.73

21.33

1.15

110.67

3.06

245.33

12.22

T5

(5)

5.33

1.15

12.00

2.00

22.67

1.15

113.33

4.16

248.00

4.00

PC

196.67

13.01

1140.00

66.09

916.00

108.00

1305.33

84.88

1624.00

122.05

NC= Negative Control,T =Test concentration (T5: Highest, T1: Lowest),SD= Standard Deviation

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

2-Aminoanthracene [10μg/plate]:TA 102                                           

Sodium azide [10μg/plate]: TA 1535, TA 100

4-Nitro-o-phenylenediamine: TA 1537[50μg/plate], TA 98 [10μg/plate]

Historical Control Data        

These data represent the laboratory´s historical control data.

Trial I (Plate Incorporation Method)

Strains

Metabolic Activation

Treatment

Mean

SD

Max

Min

TA 1537

S9 +

Negative control

6

2

10

2

S9 -

6

2

10

2

S9 +

Positive control

168

38

245

92

S9 -

175

43

261

89

TA 1535

S9 +

Negative control

12

3

18

7

S9 -

12

3

18

7

S9 +

Positive control

336

211

757

86

S9 -

1200

263

1726

674

TA 98

S9 +

Negative control

24

6

36

11

S9 -

23

6

35

11

S9 +

Positive control

1099

312

1722

476

S9 -

815

284

1383

248

TA 100

S9 +

Negative control

117

28

173

61

S9 -

114

26

166

62

S9 +

Positive control

1488

390

2268

709

S9 -

1311

298

1906

715

TA 102

S9 +

Negative control

274

42

358

190

S9 -

271

55

382

161

S9 +

Positive control

1648

305

2258

1037

S9 -

1896

364

2624

1168

Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

 

 HISTORICAL CONTROL DATA (Contd.)

Trial II (Pre-Incubation Method)

Strains

Metabolic Activation

Treatment

Mean

SD

Max

Min

TA 1537

S9 +

Negative control

6

2

10

2

S9 -

6

2

10

2

S9 +

Positive control

170

39

249

91

S9 -

182

43

268

96

TA 1535

S9 +

Negative control

13

3

18

7

S9 -

12

3

18

7

S9 +

Positive control

299

197

694

145

S9 -

1244

260

1765

724

TA 98

S9 +

Negative control

24

6

35

13

S9 -

23

5

33

13

S9 +

Positive control

1269

275

1819

719

S9 -

740

210

1160

320

TA 100

S9 +

Negative control

117

25

166

67

S9 -

113

23

159

66

S9 +

Positive control

1469

347

2163

775

S9 -

1352

263

1878

827

TA 102

S9 +

Negative control

281

32

345

218

S9 -

276

28

331

220

S9 +

Positive control

1595

287

2168

1022

S9 -

1753

248

2248

1258

Mean = mean value of revertants/plate, SD = standard deviation, Min = -2SD, Max = +2SD

Applicant's summary and conclusion

Conclusions:
The test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

Ames assay was performed to investigate the potential of the test chemical to induce gene muta­tions in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (N.C), 0.002(T1), 0.005(T2), 0.016(T3), 0.050(T4), 0.158(T5), 0.501(T6), 1.582(T7) and 5(T8) mg/plate were selected for pre-experiment.Based on the pre-experiment results, the test item was tested with the following concentrations: 0.0 (NC), 0.050(T1), 0.158(T2), 0.501(T3), 1.582(T4) and 5(T5) mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative and positive controls were within the range of our historical data. The positive controls used for various strains showed a distinct increase in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.