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Toxicological information

Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on data from various test chemicals
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27-02-2018 - 26-03-2018
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
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of Disodium 6-hydroxy-5-[(4-sulphonatophenyl)azo]naphthalene-2-sulphonate (CAS no. 2783-94-0) 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
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:
0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
RO 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
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 precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- 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 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 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. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (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: 0.050, 0.158, 0.501, 1.582 and 5 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

110

26

124

28

R2

106

24

122

26

R3

104

25

126

24

T1

(0.002)

R1

91

14

86

16

R2

84

13

82

18

R3

87

13

88

16

T2

(0.005)

R1

86

14

90

19

R2

92

17

88

21

R3

85

18

86

17

T3

(0.016)

R1

90

16

102

19

R2

96

14

98

20

R3

84

16

106

21

T4

(0.050)

R1

90

18

106

21

R2

88

16

98

23

R3

98

16

90

19

T5

(0.158)

R1

95

18

104

20

R2

88

19

100

18

R3

96

18

108

22

T6

(0.501)

R1

94

20

108

18

R2

101

21

110

20

R3

98

19

114

24

T7

(1.582)

R1

96

20

112

18

R2

104

23

116

22

R3

98

20

118

24

T8

(5)

R1

102

23

118

23

R2

98

25

114

25

R3

100

24

120

24

PC

R1

1232

896

1458

1242

R2

1280

944

1432

1180

R3

1296

936

1488

1208

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

15

28

124

284

R2

8

14

26

122

278

R3

6

16

24

126

284

T1

(0.005)

R1

6

10

19

90

228

R2

5

11

21

88

238

R3

5

12

17

86

232

T2

(0.016)

R1

7

12

19

102

227

R2

5

11

20

98

244

R3

6

12

21

106

236

T3

(0.050)

R1

6

13

21

106

242

R2

7

12

23

98

254

R3

6

12

19

90

236

T4

(0.158)

R1

6

14

20

104

254

R2

5

13

18

100

260

R3

5

12

22

108

262

T5

(0.501)

R1

7

14

18

108

268

R2

5

15

20

110

274

R3

6

15

24

114

270

PC

R1

172

398

1242

1458

1336

R2

186

408

1180

1432

1384

R3

190

440

1208

1488

1344

 

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

16

26

110

272

R2

6

15

24

106

296

R3

8

14

25

104

268

T1

(0.005)

R1

4

11

14

86

226

R2

5

10

17

92

231

R3

5

11

18

85

218

T2

(0.016)

R1

5

13

16

90

226

R2

6

12

14

96

234

R3

5

10

16

84

232

T3

(0.050)

R1

6

14

18

90

242

R2

6

13

16

88

238

R3

6

11

16

98

250

T4

(0.158)

R1

6

14

18

95

244

R2

6

15

19

88

252

R3

5

13

18

96

260

T5

(0.501)

R1

7

14

20

94

268

R2

7

16

21

101

274

R3

6

14

19

98

284

PC

R1

168

1218

896

1232

1624

R2

183

1230

944

1280

1688

R3

175

1170

936

1296

1680

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

8

17

28

121

278

R2

6

16

27

119

262

R3

7

16

27

120

272

T1

(0.005)

R1

5

12

19

104

234

R2

5

12

21

98

240

R3

4

11

22

102

230

T2

(0.016)

R1

5

13

23

106

240

R2

6

13

22

110

236

R3

5

12

24

104

242

T3

(0.050)

R1

5

12

20

112

250

R2

6

15

21

108

240

R3

4

13

23

112

258

T4

(0.158)

R1

5

14

23

114

266

R2

6

13

23

108

260

R3

6

14

24

112

258

T5

(0.501)

R1

7

15

26

117

260

R2

7

14

25

115

272

R3

6

14

23

118

258

PC

R1

179

398

1360

1518

1736

R2

158

418

1384

1448

1704

R3

184

376

1328

1480

1712

 

 

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

16

28

114

264

R2

7

15

26

112

256

R3

6

13

26

118

274

T1

(0.005)

R1

5

10

20

102

229

R2

4

10

22

108

237

R3

4

11

18

110

240

T2

(0.016)

R1

6

12

21

104

242

R2

4

11

23

110

246

R3

5

14

22

108

256

T3

(0.050)

R1

5

13

19

106

260

R2

4

13

23

110

252

R3

5

12

22

111

254

T4

(0.158)

R1

5

11

25

116

262

R2

5

12

20

112

250

R3

6

13

23

108

260

T5

(0.501)

R1

6

13

26

116

266

R2

6

14

23

112

252

R3

7

12

21

114

258

PC

R1

187

1208

928

1086

1608

R2

178

1160

894

1164

1656

R3

182

1224

952

1128

1568

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

15.00

1.00

26.00

2.00

124.00

2.00

282.00

3.46

T1

(0.005)

5.33

0.58

11.00

1.00

19.00

2.00

88.00

2.00

232.67

5.03

T2

(0.016)

6.00

1.00

11.67

0.58

20.00

1.00

102.00

4.00

235.67

8.50

T3

(0.050)

6.33

0.58

12.33

0.58

21.00

2.00

98.00

8.00

244.00

9.17

T4

(0.158)

5.33

0.58

13.00

1.00

20.00

2.00

104.00

4.00

258.67

4.16

T5

(0501)

6.00

1.00

14.67

0.58

20.67

3.06

110.67

3.06

270.67

3.06

PC

182.67

9.45

415.33

21.94

1210.00

31.05

1459.33

28.02

1354.67

25.72

 

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)

7.00

1.00

15.00

1.00

25.00

1.00

106.67

3.06

278.67

15.14

T1

(0.005)

4.67

0.58

10.67

0.58

16.33

2.08

87.67

3.79

225.00

6.56

T2

(0.016)

5.33

0.58

11.67

1.53

15.33

1.15

90.00

6.00

230.67

4.16

T3

(0.050)

6.00

0.00

12.67

1.53

16.67

1.15

92.00

5.29

243.33

6.11

T4

(0.158)

5.67

0.58

14.00

1.00

18.33

0.58

93.00

4.36

252.00

8.00

T5

(0501)

6.67

0.58

14.67

1.15

20.00

1.00

97.67

3.51

275.33

8.08

PC

175.33

7.51

1206.00

31.75

925.33

25.72

1269.33

33.31

1664.00

34.87

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)

7.00

1.00

16.33

0.58

27.33

0.58

120.00

1.00

270.67

8.08

T1

(0.005)

4.67

0.58

11.67

0.58

20.67

1.53

101.33

3.06

234.67

5.03

T2

(0.016)

5.33

0.58

12.67

0.58

23.00

1.00

106.67

3.06

239.33

3.06

T3

(0.050)

5.00

1.00

13.33

1.53

21.33

1.53

110.67

2.31

249.33

9.02

T4

(0.158)

5.67

0.58

13.67

0.58

23.33

0.58

111.33

3.06

261.33

4.16

T5

(0501)

6.67

0.58

14.33

0.58

24.67

1.53

116.67

1.53

263.33

7.57

PC

173.67

13.80

397.33

21.01

1357.33

28.10

1482.00

35.04

1717.33

16.65

 

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

14.67

1.53

26.67

1.15

114.67

3.06

264.67

9.02

T1

(0.005)

4.33

0.58

10.33

0.58

20.00

2.00

106.67

4.16

235.33

5.69

T2

(0.016)

5.00

1.00

12.33

1.53

22.00

1.00

107.33

3.06

248.00

7.21

T3

(0.050)

4.67

0.58

12.67

0.58

21.33

2.08

109.00

2.65

255.33

4.16

T4

(0.158)

5.33

0.58

12.00

1.00

22.67

2.52

112.00

4.00

257.33

6.43

T5

(0501)

6.33

0.58

13.00

1.00

23.33

2.52

114.00

2.00

258.67

7.02

PC

182.33

4.51

1197.33

33.31

924.67

29.14

1126.00

39.04

1610.67

44.06

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

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:
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 negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using th e 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, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 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), 0.005, 0.016, 0.050, 0.158, 0.501 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, positive controls are 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 is 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.  

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Including Prival modification
Principles of method if other than guideline:
Ames Salmonella typhimurium mutagenicity test including the Prival modification was conducted to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA98, TA100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Liver S9 homogenate was prepared from Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 mL/mL of mix
- source of S9 : Liver S9 homogenate was prepared from Syrian golden hamsters
- method of preparation of S9 mix : The postmitochondrial (microsomal) enzyme fractions were prepared as described by Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium : Uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate),
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
0, 10, 33, 100, 333, 1000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble 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:
congo red
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 30 mins without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced.
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available

STAIN (for cytogenetic assays): No data available

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was checked in preliminary dose range finding study

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available
Rationale for test conditions:
No data
Evaluation criteria:
The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100) in the mean number of revertants per plate of at least one tester strain. This increase in the mean revertants per plate had to be accompanied by a dose response to increasing concentrations of the test chemical.
Statistics:
No data available
Species / strain:
S. typhimurium, other: TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES:
The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxicity observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data
Remarks on result:
other: No mutagenic potential

Mutagenic response of the test chemical:

Prival Modification

Doses ug/plate

TA98

TA100

Negative

Negative

DMSO

24 ± 6

139 ± 24

10ug

33 ± 7

142 ± 19

33ug

24 ± 4

166 ± 52

100ug

39 ± 13

124 ± 19

333ug

31 ± 4

161 ± 13

1000ug

27 ± 4

193± 9

Positive 1

190 ± 24

368 ± 50

Positive 2

522 ± 67

1150 ± 55

Conclusions:
The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.
Executive summary:

Ames mutagenicity test with Prival modification was conducted as per OECD 471 for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98 and TA100 with dose concentration of 0, 10, 33, 100, 333, 1000 µg/plate in pre-incubation assay. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter or Minicount colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
Data is from secondary source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonells strains and Tryptophan for E. coli strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats
Test concentrations with justification for top dose:
0, 33, 100, 333, 1000, 2500 or 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (TA1537, TA98, -S9); 2-aminoanthracene (TA1535, TA1537, TA98, TA100, WP2 uvrA + S9)
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added : in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data
Rationale for test conditions:
No data
Evaluation criteria:
The plates were observed for the an increase in the number of revertants
Statistics:
No data
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with strains TA1535, TA1537, TA98, TA100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn. Based upon the results of the pre-experiment the concentrations applied in the main experiments were chosen. According to the dose selection criteria the test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The results of the pre-experiment ate included are reported as a part of the main experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Ames test:
- Signs of toxicity : The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
- Individual plate counts : No data
- Mean number of revertant colonies per plate and standard deviation : No data
Remarks on result:
other: No mutagenic potential
Conclusions:
The test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and the Escherichia coli strain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and Escherichia coli strain WP2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical dissolved in water was used at dose level of 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate. The doses for the main study was based on pre-experiment conducted. Main study was performed as per plate incorporation and pre-incubation assay in two independent experiments with triplicate concentrations used. Concurrent untreated, solvent and positive control plates were also included in the study. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test chemical at any dose level, 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. Appropriate reference mutagens used as positive controls showed a distinct increase of induced revertant colonies. Based on the details of the study, the test chemical did not induce gene mutation in Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and Escherichia coli strain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Data source

Reference
Reference Type:
publication
Title:
WoE of gene mutation in vitro toxicity study for CAS no 73507-17-2
Author:
Sustainability Support Services (Europe) AB
Year:
2019
Bibliographic source:
WoE report, Sustainability Support Services (Europe) AB, 2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Including Prival modification / 3
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)
EC Number:
277-492-0
EC Name:
Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)
Cas Number:
73507-17-2
Molecular formula:
C52H28Cl2CrN14O18S44Na
IUPAC Name:
Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)
Details on test material:
- Name of the test chemical: Hydrogen tetrasodium bis[2-[[6-[[4-chloro-6-[3-sulphoanilino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3-sulpho-2-naphthyl]azo]benzoato(4-)]chromate(5-)
- Molecular formula: C52H28Cl2CrN14O18S4.H.4Na
- Molecular weight: 2693.61 g/mol
- Substance type: Organic

Method

Target gene:
Histidine for Salmonella strains and Tryptophan for E. coli strains
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium, other: TA98, TA100
Remarks:
3 / 5
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Remarks:
4
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Remarks:
4
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
2. Aroclor 1254 induced S9 metabolic activation system

3/5. Type and composition of metabolic activation system: Liver S9 homogenate was prepared from Syrian golden hamsters that had been injected with Aroclor 1254 at 500 mg/kg body weight. The components of the S9 mix were 8 mM MgCl2, 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADP, 100 mM sodium phosphate (pH 7.4), and the appropriate S9 homogenate at a concentration of 0.1 m L/mL of mix
- source of S9 : Liver S9 homogenate was prepared from Syrian golden hamsters
- method of preparation of S9 mix : The postmitochondrial (microsomal) enzyme fractions were prepar ed as described by Ames et al.
- concentration or volume of S9 mix and S9 in the final culture medium : Uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate)
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data

4. The S9 liver microsomal fraction was obtained from the livers of 8 - 12 weeks old male Wistar rats
Test concentrations with justification for top dose:
2. 0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
3. 0, 10, 33, 100, 333, 1000 µg/plate
4. 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate
5. 0, 10, 33, 100, 333, 1000, 3333 or 10000 µg/plate
Vehicle / solvent:
2. - Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was solulble in RO water

3. - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO

4. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water

5. - Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle: The test chemical was soluble in water
Controlsopen allclose all
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
RO 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)
Remarks:
2
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
congo red
Remarks:
3
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine (TA1537, TA98, -S9); 2-aminoanthracene (TA1535, TA1537, TA98, TA100, WP2 uvrA + S9)
Remarks:
4
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
congo red
Remarks:
5
Details on test system and experimental conditions:
2. 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

3/5. METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 30 mins without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): No data available
- Fixation time (start of exposure up to fixation or harvest of cells): No data available

SELECTION AGENT (mutation assays): No data available
SPINDLE INHIBITOR (cytogenetic assays): No data available
STAIN (for cytogenetic assays): No data available
NUMBER OF REPLICATIONS: Triplicate
NUMBER OF CELLS EVALUATED: No data available

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Cytotoxicity was checked in preliminary dose range finding study

OTHER EXAMINATIONS:
- Determination of polyploidy: No data available
- Determination of endoreplication: No data available
- Other: No data available

OTHER: No data available

4. NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): No data
- Test substance added : in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition
- Any supplementary information relevant to cytotoxicity: No data
Rationale for test conditions:
No data
Evaluation criteria:
2. 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.

3. The criteria used to evaluate a test were as follows: for a test article to be considered positive, it had to induce at least a doubling (TA98, TA100) in the mean number of revertants per plate of at least on e tester strain. This increase in the mean revertants per plate had to be accompanied by a dose res ponse to increasing concentrations of the test chemical.

4. The plates were observed for the an increase in the number of revertants
Statistics:
No data

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Remarks:
2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA98, TA100
Remarks:
3
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
S. typhimurium, other: TA1535, TA1537, TA98, TA100
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Remarks:
4
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
2. 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 precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- 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 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 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. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) mg/plate – 0.002 (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: 0.050, 0.158, 0.501, 1.582 and 5 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

3. 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
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: The doses that were tested in the mutagenicity assay were selected based on the levels of cytotoxici ty observed in a preliminary dose range-finding study using strain TA100. Ten dose levels of the ch emical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used. Ten dose levels of the chemical, one plate per dose, were tested in both the presence and the absence of induced hamster S9. If no toxicity was observed, a total maximum dose of 10 mg of test chemical per plate was used.

COMPARISON WITH HISTORICAL CONTROL DATA: No data
ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result
other: No mutagenic potential

4. TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): The pre-experiment was performed with strains TA1535, TA1537, TA98, TA100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as described below for the experiment I (plate incorporation test). Toxicity of the test article results in a reduction in the number of spontaneous revertants or a clearing of the bacterial ba ckground lawn. Based upon the results of the pre-experiment the concentrations applied in the main experiments were chosen. According to the dose selection criteria the test article was tested at the following concentrations: 33; 100; 333; 1000; 2500; and 5000 µg/plate The results of the pre-expe riment ate included are reported as a part of the main experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies

Ames test:
- Signs of toxicity : The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments.
- Individual plate counts : No data
- Mean number of revertant colonies per plate and standard deviation : No data

Remarks on result other: No mutagenic potential
Remarks on result:
other: No mutagenic potential

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 and E. coli strains 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:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The summaries are as mentioned below:

Ames assay 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 th e 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, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 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), 0.005, 0.016, 0.050, 0.158, 0.501 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, positive controls are 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 is 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.  

Ames mutagenicity test with Prival modifications was also conducted as per the OECD 471 guideline for the test chemical to evaluate its genetoxic effects. In the FMN-modified assay, the test chemical was exposed toSalmonella typhimuriumstrains TA98 and TA100 at dose concentrations of 0, 10, 33, 100, 333, 1000  µg/plate. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control used for the experiment was Congo red. All plates were counted with an Artek automated colony counter or Mini-count colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

In another study, Bacterial reverse mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed usingSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system. The test chemical dissolved in water was used at dose level of 0, 33, 100, 333, 1000, 2500 or 5000 µg/plate. The doses for the main study was based on pre-experiment conducted. Main study was performed as per plate incorporation and pre-incubation assay in two independent experiments with triplicate concentrations used. Concurrent untreated, solvent and positive control plates were also included in the study. The plates incubated with the test article showed normal background growth up to 5000 µg/plate with and without S9 mix in both experiments. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test chemical at any dose level, 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. Appropriate reference mutagens used as positive controls showed a distinct increase of induced revertant colonies. Based on the details of the study, the test chemical did not induce gene mutation inSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100, andEscherichia colistrain WP2 uvrA in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In the same study as mentioned above, Ames mutagenicity test with Prival modification was conducted as per OECD 471 guideline for the test chemical to evaluate its genetoxic effects when exposed to Salmonella typhimurium strains TA98 and TA100 with dose concentration of 0, 10, 33, 100, 333, 1000, 3333 or 10000  µg/plate in pre-incubation assay. The doses for the main study were based on preliminary dose range study conducted. The bacteria, uninduced hamster liver S9 (30% v/v), cofactors (FMN, NADH, glucose-6-phosphate dehydrogenase, and glucose-6-phosphate), and test chemical were added, mixed, and incubated at 30 °C for 30 min without shaking. Nitrogen was blown over the preincubation tube to keep the atmosphere reduced. At the end of the incubation period, 2 mL of molten top agar was added to each sample tube and the mixture was poured on a minimal agar plate containing 0.5% glucose rather than the 2% glucose specified by Ames et al.. The plates were then incubated at 37 °C for 48 h. The positive control in all FMN experiments was Congo red. All plates were counted with an Artek automated colony counter or Minicount colony counter, which was calibrated prior to use. The test chemical did not induce gene mutation in the Salmonella typhimurium strains TA98 and TA100 both in the presence and absence of hamster S9 activation system and hence the chemical is not likely to be a genotoxic in vitro.

Based on the data available and applying the weight of evidence approach, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the Salmonella typhimurium and E. coli strains 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.