Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

Ames assay was performed to investigate the potential of 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) 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 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Chromosome aberration:

test chemical failed to induce chromosome aberrations in the Chinese hamster ovary cells.

Hence the test chemical is not likely to classify as in-vitro gene toxicity as per the criteria mentioned in CLP regulation.

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:
27-02-2018 to 30-03-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
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 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no.1533-45-5) 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
Target gene:
Histidine
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
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
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:
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

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


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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data of read across substances
Justification for type of information:
Weight of evidence approach based on structurally similar chemicals
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: as below
Principles of method if other than guideline:
WoE report is based on two in-vitro gene toxicity studies
1. In vitro mammalian chromosome aberration test was performed to determine the test chemical
2.In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
other: In vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1, 1
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy’s 5a medium supplemented with 10% fetal calf serum, L-glutamine,
and antibiotics.- Properly maintained: No data available- Periodically checked for Mycoplasma cont
amination: No data available- Periodically checked for karyotype stability: No data available- Peri
odically "cleansed" against high spontaneous background: No data available
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
mammalian cell line, other: Chinese hamster bone marrow cells
Remarks:
2.
Details on mammalian cell type (if applicable):
not specified
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
The S9 mix consisted of 15 μl/ml liver homogenate (from male Sprague-Dawley rats, induced with Arocl or 1254), 2.4 mg/ml NADP, and 4.5 mg/ml isocitric acid in serum-free medium.
Test concentrations with justification for top dose:
1.0.04-0.8 μg/mL
2.500, 1000 or 2000 mg/Kg
Vehicle / solvent:
1.- Vehicle(s)/solvent(s) used: Ethanol - Justification for choice of solvent/vehicle: The chemical was soluble in ethanol.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Triethylenemelamine
Remarks:
1.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
2.
Details on test system and experimental conditions:
1. METHOD OF APPLICATION: In mediumDURATION- Preincubation period: No data available- Exposure duration: Cells were exposed to the test chemical for 2 hr in the presence of S9 or for 20 hrs without S9.-
Expression time (cells in growth medium): 28.5 – 37.3 hrs during the delayed harvest time- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): The cell harvest time for the aberration test was 8-12 hr after the beginning of treatme nt. This yielded cells in their first mitosis. Depending on the amount of delay seen in the SCE test, later harvest times, eg, 28.5 – 37.3 hr, were used to allow delayed cells to reach mitosis.SELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): GiemsaNUMBER OF REPLICATIONS: No data availableNUMBER OF CELLS
EVALUATED: 100 cells were scored from each of the three highest dose groups having sufficient meta phases for analysisDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: No data availableOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No dataOTHER: No data available.

2. METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time (cells in growth medium): No data
- 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: No data
NUMBER OF CELLS EVALUATED: No data
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data
OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other:
OTHER: No data
Evaluation criteria:
1. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of “simple” (breaks and terminal deletions), “complex” (exchanges and rearrangements), “other” (includes pulverized chromosomes), and “total. ” Gaps and endoreduplications were recorded but were not included in the totals. We did not score aberrations in polyploidy cells but used metaphases with 19-23 chromosomes (the modal number being 21).

2. The cell line was observed for chromatid breaks, chromatid exchanges, acentric fragments, pulverisations, etc. and anomolous interphase cells
Statistics:
1. Linear regression analysis of the percentage of cells with aberrations vs the log-dose was used as the test for trend. To examine absolute increases over control levels at each dose, a binomial sampling assumption (as opposed to Poisson) was used. The P values were adjusted by Dunnett’s method to take into account the multiple dose comparisons. For data analysis, we used the “total” aberration category, and the criterion for a positive response was that the adjusted P value be < 0.05.
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-W-B1, 1
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
Species / strain:
mammalian cell line, other: Chinese hamster bone marrow cells
Remarks:
2.
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available
- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No available data
RANGE-FINDING/SCREENING
STUDIES: Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hr after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations. In some cases, test chemical precipitate was observed at the higher dose levels. Dose selection for repeat trials involved a range of doses based on observations from the first trial.

COMPARISON WITH HISTORICAL CONTROL DATA: No data available
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data available
Remarks on result:
other: Non mutagenic
Conclusions:
2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5)does not exhibit gene toxicity toxicity.
Executive summary:

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5). When administered to Chinese Hamster Ovary (CHO) cells. 

Study 1:

An in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical using Chinese hamster ovary cells (CHO-W-B1) at dose levels of 0.04- 0.8 µg/mL. Cells were exposed to the test chemical for 2 hr in the presence of S9 or for 20hrs without S9 and were incubated for 28.5 – 37.3 hrs during the delayed harvest time. One hundred cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. Based on the results noted, test chemical failed to induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system, and hence is not classified as a gene mutant in vitro.

Study 2:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster bone marrow cells. The test chemical was tested at dose levels of 500, 1000 or 2000 mg/Kg and cyclophosphamide at 128 mg/kg was used as the positive control chemical. The treated cell line wad scored for aberrant metaphases (chromatid break, chromatid exchanges, acentric fragments, pulverisations, etc.) and anomolous interphase cells. The test chemical did not cause a significant enhancement of the aberrant metaphases or of anomalous interphase cells in the Chinese hamster bone marrow cells and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5)does not exhibit gene toxicity toxicity. Hence the test chemical is not likely to classify as in-vitro gene toxicity as per the criteria mentioned in CLP regulation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Data availabe for the target chemical and from the two read across have been reviewed to determine the mutagenic nature of 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole) (CAS no 1533 -45 -5; IUPAC name: 2,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole)). The studies are as mentioned below:

Ames assay was performed (Sustainability Support Services (Europe) AB, 2018) to investigate the potential of 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) 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 2,2’-(vinylenedi-p-phenylene)bisbenzoxazole (CAS no. 1533 -45 -5) did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Based on the data available for the target chemical, 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole) (CAS no 1533 -45 -5) is not likely to exhibit genetic toxicity in vitro. Thus, the chemical is not classified as a genetic toxicant as per as per the criteria mentioned in CLP regulation.

Chromosome aberration:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5). When administered to Chinese Hamster Ovary (CHO) cells. 

Study 1:

An in vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of test chemical using Chinese hamster ovary cells (CHO-W-B1) at dose levels of 0.04- 0.8 µg/mL. Cells were exposed to the test chemical for 2 hr in the presence of S9 or for 20hrs without S9 and were incubated for 28.5 – 37.3 hrs during the delayed harvest time. One hundred cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. Based on the results noted, test chemical failed to induce chromosome aberrations in the Chinese hamster ovary cells (CHO-W-B1) in the presence and absence of S9 metabolic activation system, and hence is not classified as a gene mutant in vitro.

Study 2:

In vitro mammalian chromosome aberration test was performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster bone marrow cells. The test chemical was tested at dose levels of 500, 1000 or 2000 mg/Kg and cyclophosphamide at 128 mg/kg was used as the positive control chemical. The treated cell line wad scored for aberrant metaphases (chromatid break, chromatid exchanges, acentric fragments, pulverisations, etc.) and anomolous interphase cells. The test chemical did not cause a significant enhancement of the aberrant metaphases or of anomalous interphase cells in the Chinese hamster bone marrow cells and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for 2 ,2'-(ethene-1,2-diyldi-4,1-phenylene)bis(1,3-benzoxazole) (CAS no 1533-45-5)does not exhibit gene toxicity toxicity. Hence the test chemical is not likely to classify as in-vitro gene toxicity as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

Based on the data available for the target chemical, 2,2'-(Vinylenedi-4-phenylene)bis(benzoxazole) (CAS no 1533 -45 -5) is not likely to exhibit genetic toxicity in vitro. Thus, the chemical is not classified as a genetic toxicant as per as per the criteria mentioned in CLP regulation.