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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

Ames assay as per the OECD 471 guideline was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC),  0.002, 0.005, 0.016, 0.050 and 0.158 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, vehicle and positive controls were within the range of 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 frameshifts in the genome of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 used.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to evaluate the mutagenic potential of the test chemical in Chinese hamster lung (CHL) cells by chromosomal aberration test. The test material was exposed to Chinese hamster lung-derived fiibroblast cell line (CHL) in the presence and absence of metabolic activation S9. The test chemical was dissolved in physiological saline and used at dose level of 0, 625, 1250, 2500, 5000 µg/plate in the short term treatment (6 hrs) and continuous treatment method (24 and 48 hrs). The doses for the main study were based on preliminary dose range finding study conducted. Two hours before the end of culturing, colcemid was added to a final concentration of 0.2 μg / mL. Cells were detached by trypsin treatment and cells were collected by centrifugation. After hypotonic treatment with a 75 mM potassium chloride aqueous solution, the cells were fixed with a cooled methanol-acetic acid (3: 1) mixed solution prepared at the time of use. Chromosome specimens were prepared by air drying method and then stained with 1.4% Giemsa solution for about 15 minutes. Three slide specimens were prepared for each dish. Mid-metaphase images of 100 cells per dish, that is, 200 cells of two dishes per concentration, were observed under a microscope with a total magnification of 600 times. The number of cells observed, the type and number of structural abnormalities and the number of ploidy cells were counted. For cells with structural abnormality, the case where cells having a gap only were included (+ g) and not included (- g) was recorded separately. Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster lung-derived fibroblast cell line (CHL) 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 vitro mammalian cell gene mutation assay:

The registered substance, Sodium 3-nitrobenzene-1-sulfonate (CAS 127-68-4) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
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 the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
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), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in Distilled water
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

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

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

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

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

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

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

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

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

In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentration 5, 1.582, 0.501 mg/plate but slight reduction in background lawn was observed in treated concentration 0.158 mg/plate and no reduction in colony count as well as in background lawn in treated concentrations 0.050 mg/plate-0.002 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 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 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

111

23

114

21

R2

115

21

113

24

R3

118

20

116

22

T1

(0.002)

R1

85

21

111

22

R2

92

19

113

20

R3

89

17

112

20

T2

(0.005)

R1

110

18

97

17

R2

106

21

107

16

R3

108

20

111

18

T3

(0.016)

R1

111

17

90

19

R2

103

19

92

22

R3

99

22

97

20

T4

(0.050)

R1

96

16

106

21

R2

95

20

110

19

R3

104

20

111

15

T5

(0.158)

R1

113

16

106

17

R2

108

19

100

22

R3

104

18

99

25

T6

(0.501)

R1

99

23

109

23

R2

105

18

104

20

R3

116

21

98

19

T7

(1.582)

R1

105

18

100

18

R2

107

23

107

24

R3

99

20

114

24

T8

(5)

R1

120

17

109

19

R2

110

21

110

17

R3

109

22

112

18

PC

R1

1256

946

1488

1128

R2

1048

1008

1396

1096

R3

1312

1048

1420

1024

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

7

15

21

114

262

R2

7

14

24

113

284

R3

8

16

22

116

276

T1

(0.002)

R1

5

10

22

111

226

R2

5

12

20

113

214

R3

5

10

20

112

220

T2

(0.005)

R1

4

11

17

97

228

R2

5

11

16

107

246

R3

5

12

18

111

238

T3

(0.016)

R1

6

13

19

90

254

R2

7

14

22

92

256

R3

5

12

20

97

264

T4

(0.050)

R1

7

14

21

106

248

R2

6

15

19

110

250

R3

7

14

15

111

268

T5

(0.158)

R1

6

15

17

106

274

R2

7

15

22

100

266

R3

6

14

25

99

269

PC

R1

173

434

1128

1488

1400

R2

184

410

1096

1396

1320

R3

160

388

1024

1420

1368

 

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

6

16

23

111

274

R2

8

15

21

115

268

R3

7

16

20

118

294

T1

(0.002)

R1

5

9

21

85

224

R2

4

11

19

92

218

R3

4

11

17

89

234

T2

(0.005)

R1

5

10

18

110

212

R2

5

12

21

106

236

R3

4

11

20

108

224

T3

(0.016)

R1

6

12

17

111

218

R2

5

11

19

103

234

R3

5

12

22

99

240

T4

(0.050)

R1

6

11

16

96

236

R2

7

14

20

95

244

R3

6

13

20

104

252

T5

(0.158)

R1

5

15

16

113

242

R2

6

14

19

108

276

R3

7

13

18

104

254

PC

R1

176

1176

946

1256

1872

R2

187

1248

1008

1048

1640

R3

167

1224

1048

1312

1704

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

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

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

 

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

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

6

16

27

120

248

R2

7

15

28

122

266

R3

8

16

27

118

256

T1

(0.002)

R1

5

11

19

101

229

R2

4

10

21

104

234

R3

5

10

21

108

239

T2

(0.005)

R1

4

12

23

112

247

R2

5

11

22

110

234

R3

4

10

21

117

249

T3

(0.016)

R1

5

11

24

107

254

R2

5

12

25

113

244

R3

6

13

26

114

253

T4

(0.050)

R1

4

12

25

112

259

R2

6

11

23

116

276

R3

5

14

24

115

252

T5

(0.158)

R1

5

13

27

120

260

R2

7

14

26

119

264

R3

7

15

25

117

258

PC

R1

166

344

1336

1464

1744

R2

174

432

1368

1496

1728

R3

182

378

1400

1544

1736

 

 

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

6

15

27

121

254

R2

6

17

24

118

260

R3

7

15

26

117

272

T1

(0.002)

R1

4

10

18

105

228

R2

5

11

19

102

232

R3

4

12

18

108

234

T2

(0.005)

R1

4

11

21

103

246

R2

4

10

19

105

238

R3

4

11

20

106

252

T3

(0.016)

R1

5

14

21

109

258

R2

6

12

22

112

246

R3

4

13

19

109

254

T4

(0.050)

R1

5

14

23

113

268

R2

5

13

22

114

262

R3

6

15

21

116

268

T5

(0.158)

R1

6

15

25

119

264

R2

6

16

24

120

258

R3

5

14

23

114

270

PC

R1

177

1160

900

1144

1576

R2

185

1248

882

1088

1632

R3

168

1272

942

1176

1592

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

0.58

15.00

1.00

22.33

1.53

114.33

1.53

274.00

11.14

T1

(0.002)

5.00

0.00

10.67

1.15

20.67

1.15

112.00

1.00

220.00

6.00

T2

(0.005)

4.67

0.58

11.33

0.58

17.00

1.00

105.00

7.21

237.33

9.02

T3

(0.016)

6.00

1.00

13.00

1.00

20.33

1.53

93.00

3.61

258.00

5.29

T4

(0.050)

6.67

0.58

14.33

0.58

18.33

3.06

109.00

2.65

255.33

11.02

T5

(0.158)

6.33

0.58

14.67

0.58

21.33

4.04

101.67

3.79

269.67

4.04

PC

172.33

12.01

410.67

23.01

1082.67

53.27

1434.67

47.72

1362.67

40.27

 

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

15.67

0.58

21.33

1.53

114.67

3.51

278.67

13.61

T1

(0.002)

4.33

0.58

10.33

1.15

19.00

2.00

88.67

3.51

225.33

8.08

T2

(0.005)

4.67

0.58

11.00

1.00

19.67

1.53

108.00

2.00

224.00

12.00

T3

(0.016)

5.33

0.58

11.67

0.58

19.33

2.52

104.33

6.11

230.67

11.37

T4

(0.050)

6.33

0.58

12.67

1.53

18.67

2.31

98.33

4.93

244.00

8.00

T5

(0.158)

6.00

1.00

14.00

1.00

17.67

1.53

108.33

4.51

257.33

17.24

PC

176.67

10.02

1216.00

36.66

1000.67

51.39

1205.33

139.10

1738.67

119.82

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

 

 

 

 

 

 

 

 

 

 

 

 

 

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

15.67

0.58

27.33

0.58

120.00

2.00

256.67

9.02

T1

(0.002)

4.67

0.58

10.33

0.58

20.33

1.15

104.33

3.51

234.00

5.00

T2

(0.005)

4.33

0.58

11.00

1.00

22.00

1.00

113.00

3.61

243.33

8.14

T3

(0.016)

5.33

0.58

12.00

1.00

25.00

1.00

111.33

3.79

250.33

5.51

T4

(0.050)

5.00

1.00

12.33

1.53

24.00

1.00

114.33

2.08

262.33

12.34

T5

(0.158)

6.33

1.15

14.00

1.00

26.00

1.00

118.67

1.53

260.67

3.06

PC

174.00

8.00

384.67

44.38

1368.00

32.00

1501.33

40.27

1736.00

8.00

 

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)

6.33

0.58

15.67

1.15

25.67

1.53

118.67

2.08

262.00

9.17

T1

(0.002)

4.33

0.58

11.00

1.00

18.33

0.58

105.00

3.00

231.33

3.06

T2

(0.005)

4.00

0.00

10.67

0.58

20.00

1.00

104.67

1.53

245.33

7.02

T3

(0.016)

5.00

1.00

13.00

1.00

20.67

1.53

110.00

1.73

252.67

6.11

T4

(0.050)

5.33

0.58

14.00

1.00

22.00

1.00

114.33

1.53

266.00

3.46

T5

(0.158)

5.67

0.58

15.00

1.00

24.00

1.00

117.67

3.21

264.00

6.00

PC

176.67

8.50

1226.67

58.97

908.00

30.79

1136.00

44.54

1600.00

28.84

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 frameshifts 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 mutagen as per the criteria mentioned in CLP regulation.
Executive summary:

Ames assay as per the OECD 471 guideline was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC),  0.002, 0.005, 0.016, 0.050 and 0.158 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, vehicle and positive controls were within the range of 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 frameshifts in the genome of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 used.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
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
Justification for type of information:
Data is from authoritative database
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Principles of method if other than guideline:
In vitro mammalian chromosome aberration study was performed to evaluate the mutagenic potential of the test chemical in Chinese hamster lung (CHL) cells by chromosomal aberration test.
GLP compliance:
not specified
Type of assay:
other: chromosomal aberration test
Target gene:
No data
Species / strain / cell type:
other: Fibroblast cell line (CHL) derived from Chinese hamster lung
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: National Institutes of Health and Testing Laboratories, Mutagenicity Division
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index: No data
- Sex, age and number of blood donors if applicable: No data
- Whether whole blood or separated lymphocytes were used if applicable: No data
- Number of passages if applicable: No data
- Methods for maintenance in cell culture if applicable: No data
- Modal number of chromosomes: No data
- Normal (negative control) cell cycle time: No data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Eagle-MEM powder medium prepared according to a conventional method, it was used for
the inactivated calf serum which was added at a rate of 10%.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically 'cleansed' against high spontaneous background: No data]
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the liver of Sprague-Dawley male rats administered phenobarbital and 5,6- benzoflavone as inducing agents.
Test concentrations with justification for top dose:
-S9 mix(24 and 48-hr continuous treatment): 0, 625, 1250, 2500, 5000 µg/plate
-S9 mix(6-hr short-term treatment): 0, 625, 1250, 2500, 5000, µg/plate
+S9 mix(6-hr short-term treatment): 0, 625, 1250, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: physiological saline
- Justification for choice of solvent/vehicle: The test chemical was soluble in physiolgical saline
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
physiological saline
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: S9 mix, N-methyl-N'-nitro-N-nitrosoguanidine +S9 mix, Benzo[a]pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 4000 cells//mL

DURATION
- Preincubation period: No data
- Exposure duration: Continuous treatment: 24 and 48 hrs
Short term treatment: 6 hrs
- Expression time (cells in growth medium): Short term treatment: 18 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): Colcemid

STAIN (for cytogenetic assays): Giemsa solution

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: No data

NUMBER OF CELLS EVALUATED: Mid-metaphase images of 100 cells per dish, that is, 200 cells of two dishes per concentration, were observed under a microscope with a total magnification of 600 X

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

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:
Not specified
Evaluation criteria:
The number of cells observed, the type and number of structural abnormalities and the number of ploidy cells were counted. For cells with structural abnormality, the case where cells having a gap only were included (+ g) and not included (- g) was recorded separately

When the significant difference (significance level 5% or less) was found by multisample χ ^ 2 test on the occurrence frequency of chromosomal structural abnormal cells and ploidy cells including gaps, Fisher's direct stochastic method was used to calculate the solvent A significant difference test between the control group and each concentration group (significance level divided by 5% or 1% divided by the number of treated groups was used in consideration of multiplicity) was performed.

As a result, when the frequency of appearance of chromosomal abnormal cells by the test substance significantly increased at two concentrations or more and concentration dependence or reproducibility was recognized as compared with the solvent control group, it was judged as positive.
Statistics:
Multi-sample χ ^ 2 test, Fisher's direct probability method were used
Species / strain:
mammalian cell line, other: Fibroblast cell line (CHL) derived from Chinese hamster lung
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
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To determine the treatment concentration of the test substance used for the chromosomal aberration test, the influence of the test substance on cell proliferation was investigated. The density of cells stained with a 0.1% crystal violet aqueous solution was measured using a monolayer culture cell densitometer, and the cell growth rate of the solvent control group was taken as 100% Cell proliferation rate was determined.

As a result, cell proliferation inhibition exceeding 50% was not observed at all concentrations treated with both the continuous treatment method and the short time treatment method.

Based on the results of the cell proliferation inhibition test, in the chromosome aberration test, four concentrations of 0, 625, 1250, 2500 and 5000 μg / mL were set for both the continuous treatment method and the short time treatment method

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: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; No data
- Other observations when applicable: No data
Remarks on result:
other: No mutagenic potential

Table: Chromosome analysis on CHL cells treated with the test chemical without S9 mix

Group

Dose (µg/mL)

Time of exposure (h)

No. of cells analysed

No. of structural aberration

No. of cells with aberrations

Polyploid (%)

Judgement

Gap

Ctb

Cte

Csb

Cse

Oth

total

-g (%)

+g (%)

SA

NA

Solvent

0

24

200

1

0

0

0

1

0

2

1(0.5)

2(1.0)

0

-

-

Test chemical

625

24

200

1

0

0

0

1

0

2

1(0.5)

2(1.0)

0

-

-

1250

24

200

1

0

1

0

0

0

2

1(0.5)

2(1.0)

0

-

-

2500

24

200

1

0

1

0

1

0

3

2(1.0)

3(1.5)

0

-

-

5000

24

200

1

1

2

0

0

0

4

2(1.0)

2(1.0)

0

-

-

MNNG

2.5

48

200

4

27

186

2

3

0

222

188(94.0)

189(94.5)**

0.5

+

-

Solvent

0

48

200

0

0

0

0

0

0

0

0(0.0)

0(0.0))

0

-

-

Test chemical

625

48

200

1

0

0

0

0

0

1

0(0.0)

1(0.5)

0

-

-

1250

48

200

3

0

0

0

0

0

3

0(0.0)

3(1.5)

0

-

-

2500

48

200

1

0

1

0

0

0

2

1(0.5)

2(1.0)

0

-

-

5000

48

200

0

0

2

0

0

0

2

2(1.0)

2(1.0)

0

-

-

MNNG

2.5

48

200

13

55

172

27

21

0

288

188(94.0)

189(94.5)**

0.5

+

-

MNNG: N-methyl-N’-nitro-N-nitrosoguanidine

 

Table: Chromosome analysis oh CHL cells treated with the test chemical with and without S9 mix

Group

Dose (µg/mL)

S9 mix

Time of exposure (h)

No. of cells analysed

No. of structural aberration

No. of cells with aberrations

Polyploid (%)

Judgement

Gap

Ctb

Cte

Csb

Cse

Oth

total

-g (%)

+g (%)

SA

NA

Solvent

0

-

6-(18)

200

1

0

0

0

1

0

2

1(0.5)

2(1.0)

0

-

-

Test chemical

625

-

6-(18)

200

1

0

0

0

0

0

1

0(0.0)

1(0.5)

0

-

-

1250

-

6-(18)

200

2

0

1

0

1

0

4

2(1.0)

4(2.0)

0

-

-

2500

-

6-(18)

200

0

0

0

0

0

0

0

0(0.0)

0(0.0)

0

-

-

5000

-

6-(18)

200

0

0

3

0

0

0

3

3(1.5)

3(1.5)

0

-

-

BP

10

-

6-(18)

200

1

1

0

0

0

0

1

1(0.5)

2(1.0)

0

-

-

Solvent

0

+

6-(18)

200

0

1

0

0

1

0

2

2(1.0)

2(1.0)

0.5

-

-

Test chemical

625

+

6-(18)

200

0

0

0

0

0

0

0

0(0.0)

0(0.0)

0

-

-

1250

+

6-(18)

200

0

0

1

0

2

0

3

2(1.0)

2(1.0)

0.5

-

-

2500

+

6-(18)

200

1

0

0

0

1

0

2

1(0.5)

2(1.0)

0

-

-

5000

+

6-(18)

200

0

0

2

0

1

0

3

3(1.5)

3(1.5)

0

-

-

BP

10

+

6-(18)

200

8

31

148

7

3

0

197

151(75.5)

153(76.5**)

0

+

-

BP: Benzo(a)pyrene

Conclusions:
The test chemical did not induce chromosomal aberration in Chinese hamster lung-derived fibroblast cell line (CHL) 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:

In vitro mammalian chromosome aberration study was performed to evaluate the mutagenic potential of the test chemical in Chinese hamster lung (CHL) cells by chromosomal aberration test. The test material was exposed to Chinese hamster lung-derived fiibroblast cell line (CHL) in the presence and absence of metabolic activation S9. The test chemical was dissolved in physiological saline and used at dose level of 0, 625, 1250, 2500, 5000 µg/plate in the short term treatment (6 hrs) and continuous treatment method (24 and 48 hrs). The doses for the main study were based on preliminary dose range finding study conducted. Two hours before the end of culturing, colcemid was added to a final concentration of 0.2 μg / mL. Cells were detached by trypsin treatment and cells were collected by centrifugation. After hypotonic treatment with a 75 mM potassium chloride aqueous solution, the cells were fixed with a cooled methanol-acetic acid (3: 1) mixed solution prepared at the time of use. Chromosome specimens were prepared by air drying method and then stained with 1.4% Giemsa solution for about 15 minutes. Three slide specimens were prepared for each dish. Mid-metaphase images of 100 cells per dish, that is, 200 cells of two dishes per concentration, were observed under a microscope with a total magnification of 600 times. The number of cells observed, the type and number of structural abnormalities and the number of ploidy cells were counted. For cells with structural abnormality, the case where cells having a gap only were included (+ g) and not included (- g) was recorded separately. Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster lung-derived fibroblast cell line (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The study provides experimental data on the registered substance.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Version / remarks:
Adopted July 29, 2016
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Specific details on test material used for the study:
Purity: 99.15%
Target gene:
Hprt gene
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: NCCS, Pune, India
- Suitability of cells: As per recommendations specified in OECD 476
- Normal cell cycle time (negative control): No data

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: CHO cells were cultured in complete RPMI-1640 medium (10 % Fetal bovine serum), 100 units Penicillin/ml, 10 µg Streptomycin/ml, and incubated at 37±2 °C, 5% CO2 in a CO2 incubator.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-naphthoflavone-induced rat liver microsomal fraction (S9 homogenate) was used.
Test concentrations with justification for top dose:
0 mg/ml (solvent control)
0.25 mg/ml
0.5 mg/ml
1 mg/ml
2 mg/ml
Justification: No limiting cytotoxicity was observed in the preliminary cytotoxicity assay as the relative survival values were ≥ 60% at 2 mg/ml (-S9, +S9).
Vehicle / solvent:
Vehicle: Distilled water
- Justification for choice of solvent/vehicle: The Test Item was found to be soluble in distilled water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Distilled water
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
Cultures of CHO cells were grown in a complete RPMI-1640 medium; cells at a density of 10 x 106/25 cm2 were used for cytotoxicity measurement and mutation frequency calculation. Before the experiment, spontaneous mutant CHO cells were cleansed by the treatment with 100 μM hypoxanthine, 400 µM aminopterin and 16 μM thymidine (HAT medium) for 3 days at 37 ±2°C in a CO2 incubator.

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Triplicates were used.
- Number of independent experiments: One experiment was performed.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 10 x 106 /cm2
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk: Test substance was added in medium.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: NA
- Exposure duration/duration of treatment: 4 hours
- Harvest time after the end of treatment (sampling/recovery times): NA

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):7 days
- Selection time (if incubation with a selective agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells):7 days of expression period +9 day mutant selection period =16 days
- Method used: agar or microwell plates for the mouse lymphoma assay: agar plates were used
- Selective agent used: 2x 105cells / 10 ml of cloning medium were seeded with10 µl/ml 6-thioguanine (6TG)in triplicates.
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
Plating for Cloning efficiency 1 (CE1): 10 ml of cloning media containing 100 cells were dispensed in 60 mm culture plates in triplicates. Plates were incubated at 37±2°C, 5 % CO2, in a CO2 incubator for 7 days.
Plating for expression: 3x105 cells / 5 ml cells were seeded and incubated at 37±2 °C, 5 % CO2 in a CO2 incubator for 7 days to allow phenotypic expression of the induced mutation.
Plating for Cloning efficiency 2 (CE2): At the end of the expression period, cells were trypsinized and counted. Cells at the density of 100 cells / 10 ml of cloning media were plated in 60 mm culture plates in triplicate. The plates were incubated at 37±2 °C, 5 % CO2, in a CO2 incubator for 9 days.
Plating for Mutation Frequency: 2x105 cells / 10 ml of cloning media were seeded in the presence of 10 µg/ml of 6-thioguanine (6TG) in triplicate and incubated at 37±2 °C in a CO2 incubator for 9 days for mutation frequency.
- Fixation and staining: At the end of the incubation, cells in the culture plates were fixed with 2.5 % and 10 % of formaldehyde in water for 10 minutes each. After fixation, colonies were stained with 5 % Giemsa stain for 10 minutes, followed by washing with distilled water.
- Colony counting: Colonies in all the plates for CE1, CE2 and Mutation Frequency (MF) was counted manually and recorded in the raw data.
Rationale for test conditions:
- Solubility:The test Item was found to be soluble in distilled water (200 mg/ml)

- Precipitation check:Precipitation check was performed by adding 50 µl of the test Item (200 mg/ml) to 4.950 ml of culture media to attain a concentration of 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.

- pH check:50 µl of test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final concentration of 2 mg/ml in the medium. Changes in the pH were measured at 0 and 4th hours.

- Preparation of the test item solution:200.3 mg of test item was dissolved in 1 ml of DMSO to achieve a final concentration of 200 mg/ml. From this stock, subsequent serial dilutions with DMSO were made using spacing factor 2 to obtain concentrations of 1, 0.5 and 0.25 mg/ml.

- Preliminary cytotoxicity assay:Cytotoxicity was assessed at concentrations of 0, 0.125, 0.5, 1 and 2 mg/ml in both presence and absence of metabolic activation using triplicate cultures.
Evaluation criteria:
Criteria of acceptance of the test:
- The concurrent negative control is considered acceptable for addition to the literature negative control database.
- Concurrent positive controls induce responses compatible with those generated in the historical positive control database and produce a statistically significant increase compared to the concurrent negative control.
- Two experimental conditions (i.e. with and without metabolic activation) were tested unless one resulted in positive results.
- Adequate number of cells and concentrations are analyzable.
- The criteria for the selection of top concentration are consistent.
- The spontaneous mutant frequency of vehicle control should be between 5 and 20 x10-6.

Evaluation criteria
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) the increase is concentration-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the literature negative control data.
Providing that all acceptability criteria are fulfilled, a test chemical is considered negative if, in all experimental conditions examined:
a) none of the test concentrations exhibits a significant increase compared with the concurrent negative control,
b) all results are inside the distribution of the literature negative control data.

The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.
Statistics:
Fisher´s exact test (NCSS statistical software) was used to assess the dose-dependency upon comparing the mutation frequencies of the test-item-treated and control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 2 mg/ml 76.64% (-S9), 73.86% (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:To determine the changes in the pH of the medium, 50 µl of the Test Item (200 mg/ml) was added to 4.950 ml of complete medium, resulting in a final Test Item concentration of 2 mg/ml in the medium. Changes in the pH are as mentioned in any other information on materials and methods
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: The chemical was soluble in distilled water (200 mg/mL)
- Precipitation and time of the determination: Precipitation of Test Item was performed at the start of the experiment by adding 50 µl of the Test Item (200 mg/ml) to 4.950 ml of culture media to attain 2 mg/ml. No precipitation was observed at a tested concentration of 2 mg/ml.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Preliminary cytotoxicity test:
Based on solubility and precipitation test, the preliminary cytotoxicity testing was performed with concentrations of 0 (VC), 0.125, 0.25, 0.5, 1 and 2 mg/ml and both in the presence (1 % v/v S9 mix) and absence of S9 metabolic activation system. Cytotoxicity was assessed by the relative survival (RS, that is, cloning efficiency (CE) of cells plated immediately after treatment adjusted by any loss of cells during treatment as compared with cloning efficiency in negative controls) values. No cytotoxicity (<60% RS ) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation.

Main study:
In the main study, CHO cells were exposed to the test item at concentrations of 0 (VC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. There was no significant reduction in RS (cytotoxicity) or increase of mutation frequency at any concentrations tested neither in the presence nor in the absence of S9 metabolic activation.No significant reduction in RS (cytotoxicity) and no increase in MF was observed in vehicle control (distilled water) neither in the presence nor absence of S9 metabolic activation. The positive controls (Ethylmethanesulfonate (-S9) and Beno[a]pyrene (+S9) produced statistically significant increases in mutation frequency as compared to the vehicle control.
STUDY RESULTS
- Concurrent vehicle negative and positive control data:
There was no significant reduction in RS (cytotoxicity), and no increase in MF was observed in vehicle control (distilled water) neither in the presence nor absence of metabolic activation.The positive controls (Ethylmethanesulfonate and Beno[a]pyrene in absence and presence of metabolic activation, respectively) used in the study produced statistically significant increases in mutation frequency (236.11x10-6 [Ethylmethanesulfonate], 229.52x10-6 [Benzo(a)pyrene] in the absence and presence of metabolic activation, respectively) indicating the sensitivity of the test system to specific mutagens, and confirmed that the test conditions were appropriate and that the metabolic activation system functioned properly.

Table 1: Relative Survival – Preliminary Cytotoxicity Assay:Absence of metabolic activation

 

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

VC

Distilled water

20000000

23800000

241

234

234

236.33

100

2.363

2.812

100.00

T1

0.125 mg/ml

20000000

23500000

234

224

228

228.67

100

2.287

2.687

95.54

T2

0.25 mg/ml

20000000

23100000

238

234

229

233.67

100

2.337

2.699

95.96

T3

0.5 mg/ml

20000000

23200000

228

220

224

224.00

100

2.240

2.598

92.39

T4

1 mg/ml

20000000

22700000

198

204

218

206.67

100

2.067

2.346

83.41

T5

2 mg/ml

20000000

22400000

186

198

204

196.00

100

1.960

2.195

78.06

Key:VC = Vehicle Control, T5- T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.

 

Table 2: Relative Survival – Preliminary Cytotoxicity Assay:Presenceof metabolic activation

 

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

VC

Distilled water

20000000

23700000

238

244

232

238.00

100

2.380

2.820

100.00

T1

0.125 mg/ml

20000000

23400000

224

231

239

231.33

100

2.313

2.707

95.97

T2

0.25 mg/ml

20000000

23200000

226

233

234

231.00

100

2.310

2.680

95.01

T3

0.5 mg/ml

20000000

22800000

221

219

227

222.33

100

2.223

2.535

89.87

T4

1 mg/ml

20000000

22500000

194

204

218

205.33

100

2.053

2.310

81.91

T5

2 mg/ml

20000000

22200000

181

194

194

189.67

100

1.897

2.105

74.65

 

Key: VC = Vehicle Control, T5- T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, ml = milliliter.

Table 3: Relative Survival –Main Study: Absence of metabolic activation

 

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

VC

Distilled water

20000000

23480000

218

224

228

223.33

100

2.233

2.622

100

T1

0.25 mg/ml

20000000

23240000

218

213

220

217.00

100

2.170

2.522

96.17

T2

0.5 mg/ml

20000000

23160000

211

217

217

215.00

100

2.150

2.490

94.96

T3

1 mg/ml

20000000

22440000

187

197

190

191.33

100

1.913

2.147

81.88

T4

2 mg/ml

20000000

21920000

182

189

179

183.33

100

1.833

2.009

76.64

PC

400 µg/ml

20000000

22840000

180

181

189

183.33

100

1.833

2.094

79.85

 

Key: VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.

  

Table 4: Relative Survival –Main Study:Presenceof metabolic activation

 

Dose

 level

Concentration

No. of Cells

No. of colonies

Mean Colony count

No. of

cells seeded

CE

Adjusted

 CE

RS

Before

After

R1

R2

R3

VC

Distilled water

20000000

23820000

224

219

226

223.00

100

2.230

2.656

100

T1

0.25 mg/ml

20000000

23400000

220

217

221

219.33

100

2.193

2.566

96.62

T2

0.5 mg/ml

20000000

23240000

214

211

208

211.00

100

2.110

2.452

92.31

T3

1 mg/ml

20000000

22620000

194

191

187

190.67

100

1.907

2.156

81.19

T4

2 mg/ml

20000000

21440000

183

179

187

183.00

100

1.830

1.962

73.86

PC

30 µg/ml

20000000

22460000

177

179

179

178.33

100

1.783

2.003

75.40

 

Key: VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.

 

Table 5: Cloning Efficiency(Non selective medium)Main Study: Absence of metabolic activation

 

Dose level

Non Selective medium

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

VC

Distilled water

100

204

194

211

203

2.03

T1

0.25 mg/ml

100

185

194

188

189

1.89

T2

0.5 mg/ml

100

178

184

184

182

1.82

T3

1 mg/ml

100

172

168

173

171

1.71

T4

2 mg/ml

100

164

161

174

166

1.66

PC

400 µg/ml

100

174

162

168

168

1.68

 

Key: VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.

 

Table 6: Cloning Efficiency(Non selective medium)Main Study: Presenceof metabolic activation

 

Dose level

Non Selective medium

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

VC

Distilled water

100

213

214

210

212

2.12

T1

0.25 mg/ml

100

191

187

197

192

1.92

T2

0.5 mg/ml

100

182

187

193

187

1.87

T3

1 mg/ml

100

174

182

171

176

1.76

T4

2 mg/ml

100

168

171

162

167

1.67

PC

30 µg/ml

100

170

174

181

175

1.75

 

Key: VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.

Table 7: Cloning Efficiency(Selective medium): Absenceof metabolic activation

 

Dose level

Selective medium

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

VC

Distilled water

200000

4

2

4

3.33

0.00001667

T1

0.25 mg/ml

200000

2

3

3

2.67

0.00001333

T2

0.5 mg/ml

200000

4

2

3

3.00

0.00001500

T3

1 mg/ml

200000

3

2

3

2.67

0.00001333

T4

2 mg/ml

200000

2

3

3

2.67

0.00001333

PC

400 µg/ml

200000

76

79

83

79.33

0.00039667

 

Key: VC = Vehicle Control, PC = Positive Control (Ethylmethanesulfonate), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.

 

 

Table 8: Cloning Efficiency(Selective medium)Phase I: Presence of metabolic activation

 

Dose level

Selective medium

 

Concentration

No. of cells

 seeded

No. of colonies

Mean No. of colonies

CE

R1

R2

R3

VC

Distilled water

200000

4

2

3

3.00

0.00001500

T1

0.25 mg/ml

200000

4

3

3

3.33

0.00001667

T2

0.5 mg/ml

200000

2

4

3

3.00

0.00001500

T3

1 mg/ml

200000

3

3

4

3.33

0.00001667

T4

2 mg/ml

200000

3

4

4

3.67

0.00001833

PC

30 µg/ml

200000

75

84

82

80.33

0.00040167

 

Key: VC = Vehicle Control, PC = Positive Control (Benzo[a]pyrene), T4-T1= Test Item concentration from higher to lower, R = Replicate, CE = Cloning Efficiency, RS = Relative Survival, mg = milligram, µg = microgram, ml = milliliter.


Table9: Mutation Frequency: Absence of metabolic activation

 

Dose level

Absence of metabolic activation

Concentration

Mutation Frequency

MF x 10-6

VC

Distilled water

0.00000821

8.21

T1

0.25 mg/ml

0.00000705

7.05

T2

0.5 mg/ml

0.00000824

8.24

T3

1 mg/ml

0.00000780

7.80

T4

2 mg/ml

0.00000802

8.02

PC

400 µg/ml

0.00023611

236.11

 

Dose level

Presence of metabolic activation

Concentration

Mutation Frequency

MF x 10-6

VC

Distilled water

0.00000706

7.06

T1

0.25 mg/ml

0.00000870

8.70

T2

0.5 mg/ml

0.00000801

8.01

T3

1 mg/ml

0.00000949

9.49

T4

2 mg/ml

0.00001098

10.98

PC

30 µg/ml

0.00022952

229.52

Key: VC = Vehicle Control, PC = Positive Control (Absence of metabolic activation- Ethylmethanesulfonate, Presence of metabolic activation- Benzo[a]pyrene), T4-T1= Test item concentration from higher to lower, MF = Mutation Frequency, mg = milligram, µg = microgram, ml = milliliter

Conclusions:
The registered substance, Sodium 3-nitrobenzene-1-sulfonate (CAS 127-68-4) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.

Executive summary:

A GLP compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, Sodium 3-nitrobenzene-1-sulfonate (CAS 127 -68 -4) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control. Results: No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, the relative survival values were 100% (vehicle control), 96.17% (at 0.25 mg/ml), 94.96% (at 0.5 mg/ml), 81.88% (at 1 mg/ml) and 76.64% (at 2 mg/ml). In the presence of metabolic activation, the relative survival values were 100.00% (vehicle control), 96.62% (at 0.25 mg/ml), 92.31% (at 0.5 mg/ml), 81.19% (at 1 mg/ml) and 73.86% (at 2 mg/ml). No significant increase in the mutation frequency (MF) either in absence(7.05x10-6, 8.24 x10-6, 7.80 x10-6 and 8.02 x10-6 at 0.25, 0.5, 1 and 2 mg/ml, respectively) or presence of metabolic activation (8.70 x10-6, 8.01x10-6, 9.49x10-6 and 10.98x10-6 at 0.25, 0.5, 1 and 2 mg/ml, respectively) was observed when compared to vehicle control (8.21 x10-6 and 7.06 x 10-6, absence and presence, respectively).No significant reduction in RS values (cytotoxicity) and no increase in mutation frequency was observed in vehicle control (Distilled water). The positive controls i.e Ethylmethanesulfonate (-S9) and Benzo(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS: 236.11x10-6; Beno[a]pyrene 229.52x10-6) as compared to the vehicle control (8.21 x 10-6, -S9; 7.06 x 10-6, +S9). Conclusion: The registered substance, Sodium 3-nitrobenzene-1-sulfonate (CAS 127-68-4) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.

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

Genetic toxicity in vivo

Description of key information

Gene mutation toxicity study was performed by to determine the mutagenic nature of test chemical on mouse by micronucleus test. In vivo Gene mutation study was conducted in male and female mouse NMRI mouse. The test substance was exposed at the concentration of 0, 1500, 3000, 6000 mg/kg by oral gavage route of exposure.The erythrocytes containing micronuclei was observed. There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (6000 mg/kg, 3000 mg/kg and 1500 mg/kg) or between the various sacrifice intervals (16, 24 and 48 hours).  Thus, under the experimental conditions chosen, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis. Hence the test chemical is not likely to classify as a gene mutant in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
To evaluate the mutagenic potential of the test chemical in mouse by micronucleus assay.
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
The investigations were carried out in healthy male and female NMRI mice. Animals with a mean weight of about 26 g were used for the study.
For the duration of about one week the animals were housed in Makrolon cages, type M III, in groups of 5 separately according to sex in fully air-conditioned rooms in which central air conditioning guaranteed a range of 20 - 24°C for temperature and a range of 30 - 70% for relative humidity. Before the start of the treatment, the animals were transferred to Makrolon cages, type MI, and housed individually under the same conditions until the end of the test.
The animals were identified using cage cards.
The day/night rhythm was 12 hours (12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours).
Standardized pelleted feed and drinking water from bottles were available ad libitum.
Route of administration:
oral: gavage
Vehicle:
aqua dest.
Details on exposure:
single exposure
Frequency of treatment:
single exposure
Remarks:
Doses / Concentrations:
0, 1500, 3000, 6000 mg/kg
Basis:
no data
No. of animals per sex per dose:
5
Control animals:
yes
Positive control(s):
cyclophosphamide; vincristine
Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
In a pretest for the determination of the acute oral toxicity the highest applicable amount of 6000 mg/kg body weight was survived by all animals and led only to irregular respiration 30 minutes after treatment of the animals. Doses > 6000 mg/kg body weight could not be administered due to technical reasons (no complete mixture of aqua dest. and test substance, i.e. formation of 2 phases). Therefore, a dose of 6000 mg/kg body weight was selected
as the highest dose in the present cytogenetic study. 3000 mg/kg and 1500 mg/kg body weight were administered as further doses.
All test substance formulations were prepared immediately before the single oral administration. For control purposes, male and female animals were given merely the solvent aqua dest. by the same route, or, as positive control, cyclophosphamid or vincristine in amounts as indicated above once orally or intraperitoneally, respectively.
Evaluation criteria:
Polychromatic to normochromatic erythrocytes was observed .
Statistics:
No data
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential
Additional information on results:
The single oral administration of Ludigol Granulat in doses of 6000 mg/kg, 3000 mg/kg and 1500 mg/kg body weight did not lead to any increase in the number of polychromatic erythrocytes containing either small or large micronuclei. The rate of micronuclei was always in the same range as that of the negative control in all dose groups and at all sacrifice intervals. No inhibition of erythropoiesis determined from the ratio of polychromatic to normochromatic erythrocytes was detected.
Conclusions:
There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (6000 mg/kg, 3000 mg/kg and 1500 mg/kg) or between the various sacrifice intervals (16, 24 and 48 hours).  Thus, under the experimental conditions chosen, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis.
Executive summary:

Gene mutation toxicity study was performed by to determine the mutagenic nature of test chemical on mouse by micronucleus test. In vivo Gene mutation study was conducted in male and female mouse NMRI mouse. The test substance was exposed at the concentration of 0, 1500, 3000, 6000 mg/kg by oral gavage route of exposure.The erythrocytes containing micronuclei was observed. There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (6000 mg/kg, 3000 mg/kg and 1500 mg/kg) or between the various sacrifice intervals (16, 24 and 48 hours).  Thus, under the experimental conditions chosen, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis. Hence the test chemical is not likely to classify as a gene mutant in vivo.

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

Additional information

Genetic toxicity In-vitro:

Various experimental studies for the target chemical and its read across chemicals was reviewed to determine the mutagenic nature of target substance. The studies are as mentioned below:

Ames assay:

Ames assay as per the OECD 471 guideline was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation(S9 mix). Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC),  0.002, 0.005, 0.016, 0.050 and 0.158 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, vehicle and positive controls were within the range of 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 frameshifts in the genome of Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 used.

Genetic toxicity in vitro study was also assessed for the test chemical. For this purpose AMES test was performed.The test material was exposed to Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA in the presence and absence of metabolic activation S9. The test chemical was dissolved in distilled water and used at dose level of 0, 156, 313, 625, 1250, 2500, 5000µg/plate. Concurrent solvent and positive control plates were also included in the study. No mutagenic effects were observed in all strains, in the presence and absence of metabolic activation. The test chemical did not induce gene mutation in Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli 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 vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was performed to evaluate the mutagenic potential of the test chemical in Chinese hamster lung (CHL) cells by chromosomal aberration test. The test material was exposed to Chinese hamster lung-derived fiibroblast cell line (CHL) in the presence and absence of metabolic activation S9. The test chemical was dissolved in physiological saline and used at dose level of 0, 625, 1250, 2500, 5000 µg/plate in the short term treatment (6 hrs) and continuous treatment method (24 and 48 hrs). The doses for the main study were based on preliminary dose range finding study conducted. Two hours before the end of culturing, colcemid was added to a final concentration of 0.2 μg / mL. Cells were detached by trypsin treatment and cells were collected by centrifugation. After hypotonic treatment with a 75 mM potassium chloride aqueous solution, the cells were fixed with a cooled methanol-acetic acid (3: 1) mixed solution prepared at the time of use. Chromosome specimens were prepared by air drying method and then stained with 1.4% Giemsa solution for about 15 minutes. Three slide specimens were prepared for each dish. Mid-metaphase images of 100 cells per dish, that is, 200 cells of two dishes per concentration, were observed under a microscope with a total magnification of 600 times. The number of cells observed, the type and number of structural abnormalities and the number of ploidy cells were counted. For cells with structural abnormality, the case where cells having a gap only were included (+ g) and not included (- g) was recorded separately. Based on the observations made, the test chemical did not induce chromosomal aberration in Chinese hamster lung-derived fibroblast cell line (CHL) in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Supporting the above study and classification, In vitro mammalian chromosome aberration test was also performed to determine the mutagenic nature of the test chemical. The study was performed using Chinese hamster lung (CHL) cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in suitable solvent and used at dose level with top dose being 5 mg/mL. The study was commenced by short term treatment method for 6 hrs in the presence of S9 and continuous treatment method for 24 and 48 hrs in the absence of S9 followed by a recovery period of 18 hrs. Methods for measuring cytotoxicity, as relative cell growth, were an estimation of monolayer confluence using a monocellater or other equipment, or survival cell counts. Structural aberrations and polyploidy were evaluated independently in 100 or 200 metaphases per concentration. Each experiment was classified as (a) positive (+):≥10% cells with CAs;(b) equivocal (?): ≥5–10% cells with CAs, or (c) negative (−): less than 5% cells with CAs. Based on the observations made, the test chemical did not induce gene mutation inChinese hamster lung (CHL) cells 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 vitro mammalian cell gene mutation assay:

A GLP compliant mammalian cell gene mutation assay (OECD TG 476) was conducted to assess the ability of the registered substance, Sodium 3-nitrobenzene-1-sulfonate (CAS 127 -68 -4) to induce gene mutation at the hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus of Chinese Hamster Ovary (CHO) cells. The test was performed both in the presence and absence of an exogenous metabolic activation system (liver microsomal S9 fraction was obtained from phenobarbital and β-naphthoflavone-injected rats). Test concentrations were selected from preliminary results on cytotoxicity, solubility, and precipitation tests. In the initial cytotoxicity test, CHO cells were exposed to the substance at concentrations of 0 (NC), 0.125, 0.5, 1 and 2 mg/ml, both in the presence and absence of S9 metabolic activation. Cytotoxicity was determined by relative survival (RS), i.e., cloning efficiency measured immediately after treatment and adjusted for any cell loss during treatment as compared to the negative control.

Results:No cytotoxicity (<60% RS) or limiting precipitation was observed up to the highest recommended concentration (2 mg/ml) neither in the presence nor in the absence of S9 metabolic activation. In the gene mutation study, CHO cells were treated for 4 hours at concentrations of 0 (NC), 0.25, 0.5, 1 or 2 mg/ml with and without S9 metabolic activation. Reference mutagens were also included in the test i.e., Ethylmethanesulfonate (EMS, -S9) and Beno(a)pyrene (+S9). In the absence of metabolic activation, the relative survival values were 100% (vehicle control), 96.17% (at 0.25 mg/ml), 94.96% (at 0.5 mg/ml), 81.88% (at 1 mg/ml) and 76.64% (at 2 mg/ml). In the presence of metabolic activation, the relative survival values were 100.00% (vehicle control), 96.62% (at 0.25 mg/ml), 92.31% (at 0.5 mg/ml), 81.19% (at 1 mg/ml) and 73.86% (at 2 mg/ml). No significant increase in the mutation frequency (MF) either in absence(7.05x10-6, 8.24 x10-6, 7.80 x10-6and 8.02 x10-6at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) or presence of metabolic activation (8.70 x10-6, 8.01x10-6, 9.49x10-6and 10.98x10-6at 0.25 mg/ml, 0.5 mg/ml, 1 mg/ml and 2 mg/ml, respectively) was observed when compared to vehicle control (8.21 x10-6, 7.06 x 10-6, absence and presence, respectively).

No significant reduction in RS values (cytotoxicity) and no increase in mutation frequency was observed in vehicle control (Distilled water). The positive controls i.e Ethylmethanesulfonate (-S9) and Benzo(a)pyrene (+S9) produced statistically significant increases in mutation frequency (EMS: 236.11x10-6; Beno[a]pyrene 229.52x10-6) as compared to the vehicle control (8.21 x 10-6, -S9; 7.06 x 10-6, +S9).

Conclusion: The registered substance, Sodium 3-nitrobenzene-1-sulfonate (CAS 127-68-4) tested non-mutagenic in a mammalian cell gene mutation assay which was performed according to OECD TG 476 when CHO cells were exposed up to 2 mg/ml with and without S9 metabolic activation system.

Genetic toxicity In-vivo:

Experimental study were reviewed to determine the mutagenic nature of target substance.The study is as mentioned below:

Gene mutation toxicity study was performed by to determine the mutagenic nature of test chemical on mouse by micronucleus test. In vivo Gene mutation study was conducted in male and female mouse NMRI mouse. The test substance was exposed at the concentration of 0, 1500, 3000, 6000 mg/kg by oral gavage route of exposure.The erythrocytes containing micronuclei was observed. There were no biologically relevant, significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the 3 dose groups (6000 mg/kg, 3000 mg/kg and 1500 mg/kg) or between the various sacrifice intervals (16, 24 and 48 hours).  Thus, under the experimental conditions chosen, the test substance has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis. Hence the test chemical is not likely to classify as a gene mutant in vivo.

Based on the experimental data for the target chemical and available supporting data from various closely related test chemicals, the target chemical does not induce gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo.

Justification for classification or non-classification

Based on the experimental data for the target chemical and available supporting data from various closely related test chemicals, the target chemical does not induce gene mutation in vitro and in vivo. Hence the test chemical is not likely to classify as a gene mutant in vitro and in vivo.