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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 test item 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.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of the test material: 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole)
- IUPAC name: 2,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole)
- Molecular formula: C28H18N2O2
- Molecular Weight: 414.462 g/mol
- Substance type: Organic
- Smiles: n1c2ccccc2oc1c1ccc(\C=C\c2ccc(c3oc4ccccc4n3)cc2)cc1
- Physical state: Solid

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
Additional strain / cell type characteristics:
other:
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 metabolic activation system
Test concentrations with justification for top dose:
0 (NC), 0 (VC), 0.003, 0.008, 0.025, 0.079 and 0.250 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
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:
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:
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, 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). 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 0.001 – 2.5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 2.5 mg/plate (T8), 0.791 mg/plate (T7) both in absence and in the presence of metabolic activation and no reduction in colony count as well as in background lawn in treated concentrations (0.250 (T6) mg/plate – 0.001 (T1) mg/plate) 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, 0.003, 0.008, 0.025, 0.079 and 0.250 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

TABLE1- 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

108

20

109

17

R2

110

18

113

21

R3

114

18

117

21

VC

(0.00)

R1

128

26

140

29

R2

136

26

135

28

R3

132

24

131

27

T1

(0.001)

R1

110

19

112

21

R2

114

20

109

19

R3

112

20

111

21

T2

(0.003)

R1

112

22

115

21

R2

112

20

119

22

R3

114

20

112

19

T3

(0.008)

R1

116

22

115

23

R2

114

24

119

19

R3

112

20

119

22

T4

(0.025)

R1

118

22

121

21

R2

114

24

123

23

R3

116

22

127

22

T5

(0.079)

R1

116

24

129

24

R2

120

24

131

22

R3

118

22

127

23

T6

(0.250)

R1

122

26

125

23

R2

118

22

129

22

R3

120

24

132

25

T7

(0.791)

R1

124

24

133

26

R2

120

26

135

24

R3

122

24

137

23

T8

(2.5)

R1

128

26

138

25

R2

126

26

137

26

R3

128

22

136

24

PC

R1

1176

1038

1352

1256

R2

1160

1014

1376

1304

R3

1112

992

1304

1288

NC           =     Negative control

VC           =   Vehicle 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

5

10

17

109

232

R2

4

9

21

113

228

R3

5

11

21

117

234

VC

(0.00)

R1

7

14

29

140

260

R2

7

16

28

135

248

R3

6

12

27

131

254

T1

(0.003)

R1

5

10

21

115

230

R2

6

12

22

119

236

R3

5

10

19

112

238

T2

(0.008)

R1

6

12

23

115

234

R2

4

10

19

119

240

R3

5

12

22

119

238

T3

(0.025)

R1

6

14

21

121

236

R2

6

10

23

123

242

R3

5

12

22

127

240

T4

(0.079)

R1

7

14

24

129

246

R2

6

12

22

131

238

R3

5

12

23

127

244

T5

(0.250)

R1

7

12

23

125

252

R2

6

14

22

129

248

R3

6

14

25

132

242

PC

R1

188

520

1256

1352

1472

R2

177

508

1304

1376

1560

R3

165

492

1288

1304

1640

 

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

5

11

20

108

230

R2

5

9

18

110

214

R3

4

11

18

114

223

VC

(0.00)

R1

7

14

26

128

248

R2

6

12

26

136

256

R3

8

14

24

132

252

T1

(0.003)

R1

5

10

22

112

224

R2

5

12

20

112

224

R3

5

12

20

114

226

T2

(0.008)

R1

5

10

22

116

228

R2

6

14

24

114

222

R3

6

12

20

112

226

T3

(0.025)

R1

6

13

22

118

224

R2

5

12

24

114

228

R3

5

10

22

116

232

T4

(0.079)

R1

6

10

24

116

226

R2

6

12

24

120

232

R3

6

14

22

118

236

T5

(0.250)

R1

7

12

26

122

228

R2

6

14

22

118

234

R3

6

12

24

120

238

PC

R1

167

1024

1038

1176

1608

R2

179

1032

1014

1160

1656

R3

188

1040

992

1112

1688

NC= Negative Control,VC= Vehicle 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

5

10

21

98

240

R2

5

11

19

100

238

R3

5

10

18

88

232

VC

(0.00)

R1

8

15

28

117

256

R2

7

16

26

120

248

R3

8

17

25

123

240

T1

(0.003)

R1

5

10

19

98

236

R2

5

11

20

100

244

R3

6

12

20

102

240

T2

(0.008)

R1

5

10

22

106

242

R2

6

13

21

102

238

R3

6

12

21

100

250

T3

(0.025)

R1

6

12

23

104

248

R2

5

12

22

106

244

R3

7

13

20

104

246

T4

(0.079)

R1

7

13

23

105

242

R2

6

14

24

107

248

R3

7

13

24

104

252

T5

(0.250)

R1

7

14

26

110

248

R2

8

15

25

106

242

R3

7

16

24

108

250

PC

R1

178

408

1104

1408

1280

R2

183

366

1128

1384

1352

R3

187

378

1176

1324

1312

 

 

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

5

11

21

96

255

R2

4

12

19

104

247

R3

4

11

18

89

259

VC

(0.00)

R1

7

15

27

121

272

R2

8

16

26

117

268

R3

8

16

22

108

260

T1

(0.003)

R1

5

11

21

100

250

R2

4

11

19

104

254

R3

5

12

22

106

260

T2

(0.008)

R1

5

12

21

100

260

R2

6

11

20

104

252

R3

5

12

22

108

258

T3

(0.025)

R1

6

10

21

106

264

R2

6

12

22

104

254

R3

5

14

21

104

258

T4

(0.079)

R1

6

12

23

105

260

R2

7

14

22

104

254

R3

6

12

23

108

266

T5

(0.250)

R1

6

14

24

106

262

R2

8

12

22

110

268

R3

6

14

25

112

254

PC

R1

189

1384

984

1320

1552

R2

197

1392

888

1344

1624

R3

192

1376

876

1240

1584

NC= Negative Control,VC= Vehicle 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)

4.67

0.58

10.00

1.00

19.67

2.31

113.00

4.00

231.33

3.06

VC

(0.00)

6.67

0.58

14.00

2.00

28.00

1.00

135.33

4.51

254.00

6.00

T1

(0.003)

5.33

0.58

10.67

1.15

20.67

1.53

115.33

3.51

234.67

4.16

T2

(0.008)

5.00

1.00

11.33

1.15

21.33

2.08

117.67

2.31

237.33

3.06

T3

(0.025)

5.67

0.58

12.00

2.00

22.00

1.00

123.67

3.06

239.33

3.06

T4

(0.079)

6.00

1.00

12.67

1.15

23.00

1.00

129.00

2.00

242.67

4.16

T5

(0.250)

6.33

0.58

13.33

1.15

23.33

1.53

128.67

3.51

247.33

5.03

PC

176.67

11.50

506.67

14.05

1282.67

24.44

1344.00

36.66

1557.33

84.03

 

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)

4.67

0.58

10.33

1.15

18.67

1.15

110.67

3.06

222.33

8.02

VC

(0.00)

7.00

1.00

13.33

1.15

25.33

1.15

132.00

4.00

252.00

4.00

T1

(0.003)

5.00

0.00

11.33

1.15

20.67

1.15

112.67

1.15

224.67

1.15

T2

(0.008)

5.67

0.58

12.00

2.00

22.00

2.00

114.00

2.00

225.33

3.06

T3

(0.025)

5.33

0.58

11.67

1.53

22.67

1.15

116.00

2.00

228.00

4.00

T4

(0.079)

6.00

0.00

12.00

2.00

23.33

1.15

118.00

2.00

231.33

5.03

T5

(0.250)

6.33

0.58

12.67

1.15

24.00

2.00

120.00

2.00

233.33

5.03

PC

178.00

10.54

1032.00

8.00

1014.67

23.01

1149.33

33.31

1650.67

40.27

NC= Negative Control,VC= Vehicle 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-INCUBATION METHOD
(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)

5.00

0.00

10.33

0.58

19.33

1.53

95.33

6.43

236.67

4.16

VC

(0.00)

7.67

0.58

16.00

1.00

26.33

1.53

120.00

3.00

248.00

8.00

T1

(0.003)

5.33

0.58

11.00

1.00

19.67

0.58

100.00

2.00

240.00

4.00

T2

(0.008)

5.67

0.58

11.67

1.53

21.33

0.58

102.67

3.06

243.33

6.11

T3

(0.025)

6.00

1.00

12.33

0.58

21.67

1.53

104.67

1.15

246.00

2.00

T4

(0.079)

6.67

0.58

13.33

0.58

23.67

0.58

105.33

1.53

247.33

5.03

T5

(0.250)

7.33

0.58

15.00

1.00

25.00

1.00

108.00

2.00

246.67

4.16

PC

182.67

4.51

384.00

21.63

1136.00

36.66

1372.00

43.27

1314.67

36.07

 

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)

4.33

0.58

11.33

0.58

19.33

1.53

96.33

7.51

253.67

6.11

VC

(0.00)

7.67

0.58

15.67

0.58

25.00

2.65

115.33

6.66

266.67

6.11

T1

(0.003)

4.67

0.58

11.33

0.58

20.67

1.53

103.33

3.06

254.67

5.03

T2

(0.008)

5.33

0.58

11.67

0.58

21.00

1.00

104.00

4.00

256.67

4.16

T3

(0.025)

5.67

0.58

12.00

2.00

21.33

0.58

104.67

1.15

258.67

5.03

T4

(0.079)

6.33

0.58

12.67

1.15

22.67

0.58

105.67

2.08

260.00

6.00

T5

(0.250)

6.67

1.15

13.33

1.15

23.67

1.53

109.33

3.06

261.33

7.02

PC

192.67

4.04

1384.00

8.00

916.00

59.19

1301.33

54.45

1586.67

36.07

NC= Negative Control,VC= Vehicle 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

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


Applicant's summary and conclusion

Conclusions:
Test substance 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 test substance  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, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz.,0 (NC), 0, (VC) 0.001, 0.003, 0.008, 0.025, 0.079, 0.250, 0.791 and 2.5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0 (NC), 0 (VC), 0.003, 0.008, 0.025, 0.079 and 0.250 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 Methyl-2-napthyl ether (CAS no. 93-04-9) 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, vehicle and positive controls are within the range of our historical data. The spontaneous reversion rates in the negative, vehicle and positive controls were within the range of inhouse historical data. Whereas reference mutagens showed a distinct increase in induced revertant colonies in all the tester strains both in the presence as well as in the absence of metabolic activation without showing cytotoxicity.

In conclusion, it is stated that during the described mutage­nicity test and under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.