Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

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.

In vitro mammalian chromosome aberration study:

The test chemical is non-clastogenic at the highest tested concentration of 0.125 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro mammalian cell gene mutation assay:

The test chemical did not induce gene mutation in SIK cultures/ TCDD induced keratinocytes and hence it is not likely to classify as a gene mutant in vitro.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13-12-2018 - 21-12-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of 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 (incl. certificate)
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:
not specified
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), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

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

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

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

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

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

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

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

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
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: Precipitation was checked as insolubility to assess precipitation in the final mixture under the actual test conditions and evident to the unaided eye. Test item dissolved in DMSO at 50 mg/mL concentration was checked for precipitation. Different amounts of formulated test item preparation (50 mg/mL) were added to overlay agar (top agar) in test tubes to give various test item concentration of (maximum 5 mg/plate) and plated on minimal glucose agar (MGA) plates. Precipitation was noticed at 5 mg/plate, 3.75 mg/plate and 2.5 mg/plate concentration which were assumed to interfere with the scoring. At treatment concentration 1.25 mg/plate slight precipitation was observed which was assumed to be non-interfering with the scoring. Therefore 1.25 mg/plate was selected as highest concentration for 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), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 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 the pre-experiment, the concentration range of the test item was 0.000 – 1.25 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100 there was no reduction in colony count but reduction in background lawn was observed in treated concentration 1.25 (T8) mg/plate and no reduction in colony count as well as in background lawn in treated concentrations 0.396 (T7) mg/plate – 0.0003 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0.0 (NC), 0.0(VC), 0.004, 0.013, 0.040, 0.125 and 0.396 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9). The concentrations used in the experiment (pre-experiment, Trial-I, Trial-II) were placed with (√10) half log interval.

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

106

20

113

20

R2

110

19

116

18

R3

109

18

121

21

VC

(0.00)

R1

116

24

125

24

R2

120

23

121

27

R3

117

25

123

26

T1

(0.0003)

R1

102

20

110

20

R2

101

20

105

21

R3

108

21

116

18

T2

(0.001)

R1

110

18

119

16

R2

104

22

120

22

R3

102

17

117

20

T3

(0.004)

R1

106

21

119

19

R2

112

23

115

22

R3

110

17

118

21

T4

(0.013)

R1

112

16

119

19

R2

116

24

125

23

R3

116

25

118

25

T5

(0.040)

R1

104

21

122

23

R2

117

22

120

25

R3

110

24

117

21

T6

(0.125)

R1

110

20

120

25

R2

115

18

123

23

R3

114

22

122

22

T7

(0.396)

R1

116

24

117

20

R2

106

23

122

24

R3

115

18

124

21

T8

(1.25)

R1

119

21

123

27

R2

118

18

120

26

R3

115

17

123

24

PC

R1

1608

904

1600

1280

R2

1592

912

1528

1248

R3

1616

1008

1544

1328

NC          =    Negative control

VC          =  Vehicle Control

PC          =    Positive control

R             =    Replicate

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

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

Sodium azide [10μg/plate]: TA 100,

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

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

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

11

20

106

256

R2

4

9

19

110

224

R3

5

10

18

109

220

VC

(0.00)

R1

8

15

24

116

256

R2

5

16

23

120

260

R3

8

14

25

117

272

T1

(0.004)

R1

6

12

21

106

232

R2

5

11

23

112

236

R3

5

9

17

110

246

T2

(0.013)

R1

7

10

16

112

240

R2

5

13

24

116

234

R3

5

11

25

116

238

T3

(0.040)

R1

6

13

21

104

242

R2

7

11

22

117

236

R3

5

12

24

110

250

T4

(0.125)

R1

7

14

20

110

256

R2

6

15

18

115

238

R3

6

13

22

114

252

T5

(0.396)

R1

8

14

24

116

240

R2

6

13

23

106

250

R3

6

12

18

115

262

PC

R1

180

1188

904

1608

1808

R2

188

1240

912

1592

1760

R3

156

1232

1008

1616

1860

 

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

11

20

113

250

R2

5

11

18

116

232

R3

4

10

21

121

220

VC

(0.00)

R1

6

15

24

125

276

R2

7

14

27

121

260

R3

8

17

26

123

258

T1

(0.004)

R1

5

12

19

119

242

R2

6

11

22

115

240

R3

6

13

21

118

238

T2

(0.013)

R1

7

13

19

119

222

R2

6

12

23

125

244

R3

5

10

25

118

246

T3

(0.040)

R1

6

11

23

122

250

R2

5

15

25

120

246

R3

6

14

21

117

252

T4

(0.125)

R1

7

15

25

120

244

R2

6

14

23

123

238

R3

6

15

22

122

246

T5

(0.396)

R1

6

16

20

117

262

R2

7

14

24

122

256

R3

7

12

21

124

252

PC

R1

172

448

1280

1600

1344

R2

180

420

1248

1528

1352

R3

 190

388

1328

1544

1312

 

NC= Negative Control,VC= Vehicle Control,T =Test concentrati388on (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 Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

4

10

20

110

232

R2

5

12

17

112

224

R3

5

13

20

113

240

VC

(0.00)

R1

6

12

25

125

276

R2

7

14

26

128

268

R3

8

13

23

125

250

T1

(0.004)

R1

5

12

21

110

236

R2

5

14

20

116

222

R3

6

10

22

118

240

T2

(0.013)

R1

5

12

23

119

242

R2

4

14

20

122

248

R3

6

12

22

120

230

T3

(0.040)

R1

7

13

20

119

236

R2

5

12

24

116

238

R3

5

15

25

116

234

T4

(0.125)

R1

7

14

21

117

268

R2

5

13

22

118

242

R3

6

11

21

120

244

T5

(0.396)

R1

6

12

24

121

260

R2

6

13

25

122

256

R3

7

12

23

123

258

PC

R1

188

1120

956

1088

1680

R2

200

1232

940

1272

1632

R3

192

1256

912

1128

1568

 

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

5

12

22

115

242

R2

5

11

17

107

230

R3

5

13

20

109

244

VC

(0.00)

R1

6

16

25

123

270

R2

7

13

23

136

264

R3

7

14

25

125

264

T1

(0.004)

R1

4

12

22

107

246

R2

5

12

21

105

242

R3

7

13

20

123

242

T2

(0.013)

R1

6

12

22

124

236

R2

5

13

20

120

250

R3

7

15

24

119

248

T3

(0.040)

R1

4

16

23

121

254

R2

6

13

24

124

252

R3

7

12

21

126

248

T4

(0.125)

R1

4

14

20

117

258

R2

5

13

26

117

262

R3

6

15

25

123

258

T5

(0.396)

R1

7

16

24

129

250

R2

6

11

21

124

248

R3

6

12

20

120

262

PC

R1

200

380

1280

1520

1440

R2

186

432

1344

1472

1448

R3

212

448

1360

1488

1520

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

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

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

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

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.33

1.53

10.00

1.00

19.00

1.00

108.33

2.08

233.33

19.73

VC

(0.00)

7.00

1.73

15.00

1.00

24.00

1.00

117.67

2.08

262.67

8.33

T1

(0.004)

5.33

0.58

10.67

1.53

20.33

3.06

109.33

3.06

238.00

7.21

T2

(0.013)

5.67

1.15

11.33

1.53

21.67

4.93

114.67

2.31

237.33

3.06

T3

(0.040)

6.00

1.00

12.00

1.00

22.33

1.53

110.33

6.51

242.67

7.02

T4

(0.125)

6.33

0.58

14.00

1.00

20.00

2.00

113.00

2.65

248.67

9.45

T5

(0.396)

6.67

1.15

13.00

1.00

21.67

3.21

112.33

5.51

250.67

11.02

PC

174.67

16.65

1220.00

28.00

941.33

57.87

1605.33

12.22

1809.33

50.01

 

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.00

1.00

10.67

0.58

19.67

1.53

116.67

4.04

234.00

15.10

VC

(0.00)

7.00

1.00

15.33

1.53

25.67

1.53

123.00

2.00

264.67

9.87

T1

(0.004)

5.67

0.58

12.00

1.00

20.67

1.53

117.33

2.08

240.00

2.00

T2

(0.013)

6.00

1.00

11.67

1.53

22.33

3.06

120.67

3.79

237.33

13.32

T3

(0.040)

5.67

0.58

13.33

2.08

23.00

2.00

119.67

2.52

249.33

3.06

T4

(0.125)

6.33

0.58

14.67

0.58

23.33

1.53

121.67

1.53

242.67

4.16

T5

(0.396)

6.67

0.58

14.00

2.00

21.67

2.08

121.00

3.61

256.67

5.03

PC

180.67

9.02

418.67

30.02

1285.33

40.27

1557.33

37.81

1336.00

21.17

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

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  

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

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

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

Methyl methanesulfonate [4μl/plate]: TA 102

 

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

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.67

0.58

11.67

1.53

19.00

1.73

111.67

1.53

232.00

8.00

VC

(0.00)

7.00

1.00

13.00

1.00

24.67

1.53

126.00

1.73

264.67

13.32

T1

(0.004)

5.33

0.58

12.00

2.00

21.00

1.00

114.67

4.16

232.67

9.45

T2

(0.013)

5.00

1.00

12.67

1.15

21.67

1.53

120.33

1.53

240.00

9.17

T3

(0.040)

5.67

1.15

13.33

1.53

23.00

2.65

117.00

1.73

236.00

2.00

T4

(0.125)

6.00

1.00

12.67

1.53

21.33

0.58

118.33

1.53

251.33

14.47

T5

(0.396)

6.33

0.58

12.33

0.58

24.00

1.00

122.00

1.00

258.00

2.00

PC

193.33

6.11

1202.67

72.59

936.00

22.27

1162.67

96.77

1626.67

56.19

 

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

5.00

0.00

12.00

1.00

19.67

2.52

110.33

4.16

238.67

7.57

VC

(0.00)

6.67

0.58

14.33

1.53

24.33

1.15

128.00

7.00

266.00

3.46

T1

(0.004)

5.33

1.53

12.33

0.58

21.00

1.00

111.67

9.87

243.33

2.31

T2

(0.013)

6.00

1.00

13.33

1.53

22.00

2.00

121.00

2.65

244.67

7.57

T3

(0.040)

5.67

1.53

13.67

2.08

22.67

1.53

123.67

2.52

251.33

3.06

T4

(0.125)

5.00

1.00

14.00

1.00

23.67

3.21

119.00

3.46

259.33

2.31

T5

(0.396)

6.33

0.58

13.00

2.65

21.67

2.08

124.33

4.51

253.33

7.57

PC

199.33

13.01

420.00

35.55

1328.00

42.33

1493.33

24.44

1469.33

44.06

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

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100

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

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

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

Methyl methanesulfonate: [4μl/plate]: TA 102

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

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.

The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 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), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 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 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 methodsi.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 item with the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

Endpoint:
in vitro cytogenicity / chromosome aberration 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:
Data is from study report
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Principles of method if other than guideline:
This in vitro assay was performed to assess the potential of the test chemical to induce structural / numerical chromosomal aberrations in one experiment (phase I). The induction of cytogenetic damage in human lymphocytes was assessed with and without metabolic activation. Due to the negative result in phase I, a second experiment (phase II) was performed.
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Human blood
- Suitability of cells: No data
- Cell cycle length, doubling time or proliferation index:
- Sex, age and number of blood donors if applicable:Age: 25-30 years age
- Whether whole blood or separated lymphocytes were used if applicable: Separated lymphocytes were used
- 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: Blood cultures were set up in medium containing RPMI-1640, Fetal Bovine Serum, Phytohaemagglutinin, Heparin solution, Whole Blood and Antibiotic Solution
- 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):
No data
Metabolic activation:
with and without
Metabolic activation system:
S9 metabolic activation system
Test concentrations with justification for top dose:
0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mL
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:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks

DURATION
- Preincubation period: No data
- Exposure duration: Phase 1: 4 hrs (with and without metabolic activation system)
Phase 2: 4 hrs (with metabolic activation system) and 24 hrs (without metabolic activation system)
- Expression time (cells in growth medium): Phase 1: 20 hrs (with and without metabolic activation system)
Phase 2: 20 hrs (with metabolic activation system)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): Giemsa stain in phosphate buffer

NUMBER OF REPLICATIONS: No data

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The cultures were incubated at 37 ± 2 °C for duration (exposure period) as mentioned. For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The labelled slides were dried over a slide warmer at 50°C and labelled. At least one slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant.

NUMBER OF CELLS EVALUATED: A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: Mitotic index
- Any supplementary information relevant to cytotoxicity: Cytotoxicity was assessed at the concentrations of 0.062, 0.125 and 0.250 mg/mL of culture media

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

- OTHER: No data
Evaluation criteria:
A test item can be classified as clastogenic if:
 At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent vehicle control
 If the increase is dose-related
 Any of the results are outside the historical negative control range
A test item can be classified as non – clastogenic if:
 None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control
 If there is no dose-related increase
 All results are within the historical negative control range
Statistical significance was confirmed by means of the non-parametric Mann Whitney Test. However, both biological and statistical significance should be considered together.

If the above mentioned criteria for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Statistics:
Statistical significance at the p < 0.05 was evaluated by means of the non-parametric Mann-Whitney test
Species / strain:
lymphocytes: Human perpheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
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: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. Significant change in pH was not observed at 0 h and 4 h when compared with negative controls.
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: There was no precipitation observed at 0.0625 mg/mL concentration
- 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 cytotoxicity assay was performed both in the presence and absence of metabolic activation system. 3 test concentrations (0.062, 0.125 and 0.250 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with vehicle control. The procedure for conducting cytotoxicity was the same as main experiment phase I up to the scoring of the mitotic index, except slide coding.

On the basis of solubility, precipitation and effect on media pH of the test item, the maximum concentration 0.250 mg/mL was selected as the highest concentration for the treatment in cytotoxicity experiment.

An experiment was performed to assess the effect of test item on the pH of media at 0 h and 4 h exposure. The pH of test item in culture medium was assessed after incubation at 37 °C.

No Significant change in pH was observed at 0 h and 4 h when compared with negative controls. Based on this the highest dose was selected for cytotoxicity experiment.

Based on the results of cyto-toxicity experiment following doses were selected for the main study:
0.031, 0.062 and 0.125 mg/mL culture media.

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: Please refer table remarks section

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

1.1 Cytotoxic Experiment    

Before conducting the chromosomal aberration study,3,4,4’-Trichlorocarbanilide (CAS NO. 101-20-2)was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.0 (NC), 0.0 (VC), 0.062 (T1), 0.125 (T2) and 0.250 (T3) mg/mL of culture media. Cytotoxicity was observed in treated concentration of 0.250 (T3) mg/mL both in the absence and in the presence of metabolic activation (1%).

In the absence of S9 mix, the mean mitotic index observed was 10.06 (NC), 9.88 (VC), 8.75 (T1),  7.04 (T2), 4.74 (T3) and 8.58 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.23 (NC), 9.94 (VC), 8.74 (T1), 7.70 (T2), 4.94 (T3) and 8.65 (PC).

In the cytotoxicity experiment, the highest test concentration 0.250 (T3) mg/ mLof culture mediashowed more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation confirms the cytotoxicity effect. Hence these concentration were not selected for the main study.

Hence, 0.125 mg/mL of culture media was selected as the highest concentration for main study both in the presence and in the absence of metabolic activation.

The main study was performed in two independent phases;

1.1.1 Phase I

In the experiment, the cultures were exposed to 3,4,4’-Trichlorocarbanilide (CAS NO. 101-20-2) for a short period of time (4 h) both in the absence and in the presence of metabolic activation system (1%).The mean percentage of aberrant cells was 0.333 (NC), 0.667 (VC), 0.333 (T1), 0.333 (T2), 0.333 (T3) and 9.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.333 (T2), 0.333 (T3) and 10.000 (PC)in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mL and positive controls, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) causedsignificant increase in percent aberrant cells.Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During thetreatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index observed at the tested concentrations.The observed mean mitotic indexin the absence of metabolic activation were 9.97, 9.95, 8.75, 8.95, 7.70 and 8.71 andin the presence ofmetabolic activation were 10.00, 10.01, 8.85, 9.10, 7.96 and 8.79 for(NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mLand 30 µg/mL(PC)concentrations respectively.

 

1.1.2 Phase II

The phase II experiment was performed to confirm the negative results obtained in the absence and in the presence of metabolic activation in Phase I. In the Phase II, test item concentrations used were   0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mLand 30µg/mL(PC)culture both in absence and presence of metabolic activation (2%). The duration of exposure to the test item in presence of metabolic activation system was 4 hours and in absence of metabolic activation the duration of exposure was 24 hours. The mean percent aberrant cells were 0.333 (NC), 0.667 (VC) 0.333 (T1), 0.333 (T2), 0.333 (T3) and 9.667 (PC) in the absence of metabolic activation and 0.333 (NC), 0.667 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 11.000 (PC) in the presence of metabolic activation at the concentration of 0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mL of culture and positive control, respectively.

Treatment with Ethyl methanesulfonate at the concentration of 600 µg/mL in the absence of metabolic activation and Cyclophosphamidemonohydrate at the concentration of30 µg/mL in the presence of metabolic activation (2%) causedsignificant increase in percent aberrant cells.Though the analysis did not reveal any statistical significance, the increase was biologically significant.

The increased frequency of aberrations observed in the concurrent positive control groups (Phase I and II) demonstrated the sensitivity of the test system, suitability of the methods and conditions employed in the experiment.

Treatment with test item in the absence and presence of S9 mix, there was noreduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 9.99, 10.03, 8.81, 9.10, 7.98 and 8.75 andin the presence ofmetabolic activation were 10.04, 10.05, 9.00, 9.10, 8.16 and 8.79 for0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) and 30 µg/mL(PC)concentrations respectively.

Note:NC: Negative control; VC: Vehicle control; T1: Test concentration 1; T2: Test concentration 2; T3: Test concentration 3; PC: Positive Control.

Conclusions:
The test chemical is non-clastogenic at the highest tested concentration of 0.125 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.
Executive summary:

This study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.062, 0.125 and 0.250 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endo-reduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is non-clastogenic at the highest tested concentration of 0.125 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 publication
Qualifier:
according to
Guideline:
other: Refer below priciple
Principles of method if other than guideline:
In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
mammalian cell line, other: SIK culture/ TCDD induced keratinocytes
Details on mammalian cell type (if applicable):
No data
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No data
Metabolic activation:
not specified
Metabolic activation system:
No data
Test concentrations with justification for top dose:
4 μM
Vehicle / solvent:
No data
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: o-aminoazotoluene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for gene mutation at HPRT locus
Statistics:
No data
Species / strain:
mammalian cell line, other: SIK cultures/ TCDD induced keratinocytes
Metabolic activation:
not specified
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Mutation data:

Agent

Concentration

Mut/Surv

FrequencyX10-6

Positive control

13

24/273000

88

Test chemical

4

0/375000

<3

Conclusions:
The test chemical did not induce gene mutation in SIK cultures/ TCDD induced keratinocytes and hence it is not likely to classify as a gene mutant in vitro.
Executive summary:

Modified hypoxanthine-guanine phophoribosyltransferase (HPRT) assay was performed to determine the mutagenic nature of the test chemical. The study was performed using SIK cultures/ TCDD induced keratinocytes. The test chemical was tested at dose level of 4 μM. o-aminoazotoluene was used as positive control chemical. The test chemical mutant colonies were below the limit of detection while the positive control clearly elicited mutations. Based on the observations made, the test chemical did not induce gene mutation in SIK cultures/ TCDD induced keratinocytes and hence it is not likely to classify as a gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene mutation in vitro:

Data available from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

Ames assay:

Ames assay was performed to investigate the potential of the test chemical to induce gene mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.0 (VC), 0.0003, 0.001, 0.004, 0.013, 0.040, 0.125, 0.396 and 1.25 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), 0.0 (VC), 0.004, 0.013, 0.040, 0.125 and 0.396 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 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 item with the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

 

In another study, Salmonella/microsome test in the absence of exogenous metabolic activation and in the presence of liver S-9 from Aroclor-induced male Sprague-Dawley rats and Syrian hamsters was performed to evaluate the mutagenic nature of test chemical using S. typhimurium tester strains TA1535, TA97, TA98 and TA100. The study was performed as per the preincubation assay and the preincubation time was 20 mins and the plates were incubated for 48 hrs. The test compound was dissolved in DMSO and was used at a dosage level of 0.0, 0.010, 0.030, 0.100, 0.300, 1.000, 3.000, 10.000, 33.000, 100.000 µg/plate in the preincubation assay of 48 hrs. Concurrent solvent and positive control chemicals were included in the study. The test chemical did not induce a reproducible, dose-related increase in his+ revertants over the corresponding solvent in the S. typhimurium tester strains TA100, TA97, TA1535 and TA98 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Along with these studies, the Ames assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium strain TA98, TA100, TA1535 or TA1537 in the presence and absence of 10% rat and hamster liver S9 metabolic activation system. The test chemical was dissolved in DMSO and used at dose level of 0.0, 0.01, 0.03, 0.10, 0.30, 1.0, 3.0, 10.0, 33.0, 100.0 ug/Plate. Concurrent solvent and negative control plates were also included in the study. Also, the positive control was used. The test chemical did not induce gene mutation in Salmonella typhimurium strain TA98 in the presence and absence of 10% rat and hamster liver S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

Above results are supported with another study performed to determine the mutagenic nature of the test chemical in vitro by Ames assay. The study was performed using Salmonella typhimurium strains TA100, TA98, TA1535, TA1537 and TA1538 in the presence and absence of S9 metabolic activation system. The test chemical was freshly prepared in DMSO.The test chemical did not induce gene mutation in Salmonella typhimurium strains TA100, TA98, TA1535, TA1537 and TA1538 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

 

All these studies are further supported with the Ames assay performed to determine the mutagenic nature of the test chemical in vitro. The study was performed using Salmonella typhimurium strainsTA100, TA98, TA1535, TA1537 in the presence and absence of S9 metabolic activation system. The test chemical was prepared in DMSO and used at dose level of 0, 8, 40, 200, 1000, 5000 ug/plate in test 1 and 0, 125, 250, 500, 1000, 2000, and 4000 ug/plate in test 2. Concurrent solvent and positive control plates were included in the study. Due to substance precipitation beginning at 2000 µg/plate, doses of 4000 µg and 5000 µg could not be used for assessment. In spite of the low doses used, the positive controls increased the mutant counts significantly over negative control levels, demonstrating the sensitivity of the test system. 2000 µg/plate resulted in no cytotoxic effect, however the test substance precipitated at 2000 µg and higher. Based on the findings, the test chemical did not induce gene mutation in Salmonella typhimurium strainsTA100, TA98, TA1535, TA1537 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:

The study was conducted to determine the chromosomal aberration induction potential of the test chemical in human peripheral blood lymphocyte cultures. The methods followed were as per OECD guideline No. 473, adopted on 29thJuly 2016 “ In Vitro Mammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was drawn from a healthy volunteer, by venous puncture using heparinised syringe. The experiment was performed both in the presence and in the absence of metabolic activation system after 48 h mitogenic stimulation. The test chemical was dissolved in DMSO and used at dose level of 0.0 (NC), 0.0 (VC) 0.031 (T1), 0.062 (T2) and 0.125 (T3) mg/mL in the presence and absence of S9 metabolic activation system in phase 1 and phase 2. Phase I of experiment was performed by short term treatment method both in the presence and absence of metabolic activation system (1%). Phase II of experiment was performed by short term treatment as well as long term treatment method. Long term treatment was performed in absence of metabolic activation to confirm the negative results obtained in the absence of metabolic activation in Phase I. Short term treatment method was performed with increased metabolic activation (2%) condition to confirm the negative results obtained in the presence of metabolic activation in Phase I. The doses for the main study were based on the cytotoxicity study conducted both in the presence and absence of metabolic activation system. 3 test concentrations (0.062, 0.125 and 0.250 mg/mL of culture media) based on the solubility, precipitation and pH test of the test item were tested. Cytotoxicity was determined by reduction in the mitotic index in comparison with negative control. The medium of the proliferating blood culture was removed by centrifugation at 1500 rpm for 10 minutes. The cells were suspended in plain medium (medium without serum) mixed with S9 mix (Phase I - 1 % and Phase II - 2 % v/v) and in complete media mixed with phosphate buffer for the treatment in presence and in absence of metabolic activation system respectively. A volume of 7.92 mL of proliferating culture was dispensed to individual sterile culture tubes/flasks. Each tube/flask according to treatment groups was identified. Negative control tubes were treated with 80 µL of RPMI media and treatment group were treated with 80 µL of respective test item stock solution. The cultures were incubated at 37 ± 2 °C for duration (exposure period). For Phase I, after incubation cells were spun down by gentle centrifugation at 1500 rpm for 10 minutes. The supernatant with the dissolved test item was discarded and the cells were re-suspended in Phosphate Buffer Saline (PBS). The washing procedure was repeated once again. After washing the cells were re-suspended in complete culture medium (RPMI-1640 with 10 % serum) and cultured at 37 ± 2 °C for 1.5 normal cell cycle lengths (22 - 25 hours). The cultures were harvested at the end of incubation of 24 hours after treatment. Before 3 hours of harvesting, 240 µL of colcemid (10 µg/mL) (final concentration: 0.3 µg/mL) was added to each of the culture tube, and kept under incubation at 37 ± 2 °C. The cultures were harvested 24 hours after beginning of treatment by centrifugation at 1500 rpm for 10 minutes. The supernatant was discarded and the cells were re-suspended in 7 mL of freshly prepared, pre-warmed (37 ± 2 °C) hypotonic solution of potassium chloride (0.075 M KCl). Then the cell suspension was allowed to stand at 37 ± 2 °C for 30 minutes in water bath. After hypotonic treatment, the culture was centrifuged and supernatant was removed. After that 5 mL of freshly prepared, chilled Carnoy’s fixative (3:1 methanol: acetic acid solution) was added and left for 5 min. The cells were collected by centrifugation and washed twice with Carnoy’s fixative. After the final centrifugation, the supernatant was removed completely, and the cell pellet resuspended in 0.5 mL of Carnoy’s fixative. The slides were prepared by dropping the cell suspension onto a clean ice-chilled microscope slide. The slides were dried over a slide warmer and labelled. At least two slide was made from each sample. The cells were stained with 5 % fresh Giemsa stain in phosphate buffer and mounted using DPX mountant. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. A minimum of 1000 cells were counted in different fields of slide per culture and the number of metaphases were recorded for mitotic index (MI) calculation. 300 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides. Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endo-reduplication) and disintegrations were recorded as structural chromosomal aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. Only metaphases with 46± 2 centromere regions were included in the analysis. The test chemical is non-clastogenic at the highest tested concentration of 0.125 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions and hence it is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

 

In another in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of test chemical by EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test. The study was performed using Chinese hamster ovary (i.e., CHO) cells, both in the presence and absence of an Aroclor 1254 -induced rat liver S9 activation system. The test chemical was dissolved in DMSO was tested at dose levels of 31.3, 62.5, 125, 250, 500, 1000, 1500 and 2000 μg/mL. Mitomycin C was used as the positive control in the non-activated study and cyclophosphamid as the positive control in the activated study. In the assay, CHO cells were treated for 4 and 20h in the non-activated cells and for 4h in the S9 activated test system. All cells were harvested 20 h after treatment initiation. The cytotoxic concentration was found to be > 3160 μg/mL in both the 4h activated and non-activated test systems and equal to 3160 μg/mL in the 20h non activated system. The percentage of cells with structural or numerical aberrations in the test groups was not significantly increased compared to the solvent control at any dose level in any of the test groups. Based on these findings, the test chemical did not induce gene mutation in Chinese Hamster Ovary (CHO) in the presence and absence of an Aroclor 1254-induced rat liver S9 activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay:

 

Modified hypoxanthine-guanine phophoribosyl transferase (HPRT) assay was performed to determine the mutagenic nature of the test chemical. The study was performed using SIK cultures/ TCDD induced keratinocytes. The test chemical was tested at dose level of 4 μM o-aminoazotoluene was used as positive control chemical. The test chemical mutant colonies were below the limit of detection while the positive control clearly elicited mutations. Based on the observations made, the test chemical did not induce gene mutation in SIK cultures/ TCDD induced keratinocytes and hence it is not likely to classify as a gene mutant in vitro.

 

In another study, In vitro mammalian cell gene mutation was performed to determine the mutagenic nature of the test chemical in vitro. The study was performed using Chinese hamster ovary (CHO-K1-BH4) cells in the presence and absence of S9 metabolic activation system prepared from adult male Sprague-Dawley rats pretreated with Aroclor 1254 at the concentrations of 0, 50, 100, 130, 160, 200 µg/ml. 1% argon-purged ethanol used as solvent. The plates were observed for 6-Thioguanine resistant mutants. CHO cell survival was decreased in a concentration-dependent manner by the given test chemical. Hence, the test chemical did not induce gene mutation in Chinese hamster ovary (CHO-K1-BH4) 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.

 

Along with these studies, In vitro mammalian cell gene mutation study was performed to determine the mutagenic nature of the test chemical in vitro. The study was performed using L5178Y mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was freshly prepared in DMSO and used at dose level of 5.0, 7.5, 10.0, 15.0, and 20.0 μg/ml without S9-mix, 3.5, 7.5, 10.0 and 15.0 μg/ml with S9-mix in experiment 1 and 2.5, 5.0, 7.5, 10.0 and 15.0 μg/ml without S9-mix, 1.0, 5.0, 7.5, 10.0 and 15.0 μg/ml with S9-mix in experiment 2. Test concentrations were based on the results of a preliminary toxicity test considering suspension growth. In the main test, cells were treated for 3 h in the absence or presence of S9-mix followed by an expression period of 48 h to fix the DNA damage into a stable tk mutation. Toxicity was measured in the main experiments as mean % survival relative to the solvent control cultures. Negative and positive controls were included. A more or less dose dependent increase in the mutant frequency was found in all experiments. However, the increase in mutant frequency was either less than 2-fold the background mutant frequency or for concentrations with a mutant frequency higher than the 2-fold back ground level the relative survival exceeded the 10%. Based on the observations made, the test chemical did not induce gene mutation in L5178Y mouse lymphoma 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.

 

Justification for classification or non-classification

Based on the data available, the given test chemical does not exhibit gene mutation in vitro by Ames assay, In vitro mammalian chromosome aberration study and In vitro mammalian cell gene mutation assay. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.