Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES test:

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 both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.050, 0.158, 0.501, 1.582 and 5 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 chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro mammalian chromosomal aberration study:

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 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test.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 endoreduplication) 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. Based on the observations made, the test chemical is at the highest tested concentration of 0.028 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

In vitro Mammalian Cell Gene Mutation Test was carried out in compliance with the OECD Guideline No. 476, adopted by the council on 29 July 2016. The test chemical was evaluated in mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells. Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of the test chemical,the CHO cells were exposed to the test item in duplicate cultures at the doses of 1.953125, 3.90625, 7.8125 and 15.62 µg/mL of

culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction prepared from, sodium phenobarbitone and b- naphthoflavone-induced Wistar rats was used in the study. Dimethyl sulphoxide was used as a vehicle. The target cells were exposed to the test compound for 3 hours at 37 ± 1oC, with approximately 5% CO2supply. The cells were sub-cultured immediately to determine cytotoxicity as relative survival (RS) and to initiate the phenotypic expression period. The culture flasks were incubated at 37 ± 1oC with approximately 5% CO2supply during experimental periods. Concurrent vehicle and positive control groups were also included in the experiment, as specified by the test guideline. The cultures were sub-cultured during the expression period at suitable intervals. After phenotypic expression, the plates were incubated at 37 ± 1oC with approximately 5% CO2 supply for 11 days. After incubation, the medium was discarded, and the plates were stained and observed for the clones. Relative survival and mutant frequency were calculated for all treatment, vehicle control, and positive control groups. The relative survival (RS) was used as the measure of treatment-related cytotoxicity. The RS (relative survival) for cultures treated with the test chemical with and without metabolic activation system (S9) indicated that the test chemical induces 10 to 20% relative survival at the concentration of 15.625 µg/mL. The % relative survival (% RS) for the cloned cultures ranged from 12.4% to 84.4% for cultures treated without metabolic activation system and 14.8% to 79.8% for cultures treated with metabolic activation system at test item concentrations of 1.953125, 3.90625, 7.8125 and 15.625 µg/mL in the treatment medium. The results of the present study indicate that there was no significant difference in the mutant frequencies of cultures treated with the test chemical as compared to the vehicle control cultures, in the absence or presence of metabolic activation. An increase in the mutant frequency of concurrent positive controls demonstrated the sensitivity of the assay in the absence and presence of metabolic activation. Under the test conditions described in the study, it is concluded that the test chemical is non-mutagenic in 'In Vitro Mammalian cell gene mutation test' using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells 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
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 muta¬tions 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 operon
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:
Type and composition of metabolic activation system:
- source of S9 : Aroclor 1254 induced S9 metabolic activation system was procured from Defence Research and Developement Establishment
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution which contains D-glucose-6-phosphate 0.8 g, β-NADP 1.75 g, MgCl2 1.0 g, KCl 1.35g, Na2HPO4 6.4 g, NaH2PO4.H2O 1.4 g in 500 ml of distilled water
- concentration or volume of S9 mix and S9 in the final culture medium : 10% v/v
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability) : No data available
Test concentrations with justification for top dose:
0.0 (N.C), 0.0 (V.C), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate. Based on the results of pre-experiment, the doses were selected.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

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

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

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

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

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

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

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

A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0.002, 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 a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.001 – 2.5 mg/plate based on the solubility and precipitation test. There was no reduction in colony count but reduction in background lawn was observed in treated concentrations 2.5 mg/plate (T8), 0.791 mg/plate (T7) and no reduction in colony count as well as in background lawn in treated concentrations (0.250 (T6) mg/plate – 0.001 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: (0.050, 0.158, 0.501, 1.582 and 5 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: No data

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: No data
- Indication whether binucleate or mononucleate where appropriate: No data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Please refer the table in remark section.
- Negative (solvent/vehicle) historical control data: Please refer the section in remark section

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

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

3

11

23

110

236

R2

4

10

22

118

248

R3

4

12

24

112

250

VC

(0.00)

R1

5

15

28

120

264

R2

6

16

26

129

288

R3

8

14

27

124

274

T1

(0.050)

R1

4

12

24

123

240

R2

4

13

25

124

248

R3

5

13

23

122

252

T2

(0.158)

R1

5

14

25

123

260

R2

4

13

24

125

256

R3

4

13

24

123

248

T3

(0.501)

R1

5

14

26

124

254

R2

6

13

25

125

266

R3

4

14

23

124

274

T4

(1.582)

R1

6

15

26

125

280

R2

6

14

24

123

268

R3

5

13

25

126

272

T5

(5)

R1

7

14

27

126

284

R2

6

15

26

125

276

R3

5

15

26

127

264

PC

R1

176

632

1128

1592

1944

R2

164

608

1088

1608

1832

R3

182

666

1264

1678

2116

 

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

22

98

220

R2

5

12

20

108

234

R3

3

10

25

114

248

VC

(0.00)

R1

6

14

27

112

252

R2

5

14

23

120

268

R3

7

16

25

124

282

T1

(0.050)

R1

5

12

24

112

228

R2

4

14

21

114

236

R3

4

13

24

106

244

T2

(0.158)

R1

5

13

23

115

240

R2

5

12

23

108

254

R3

4

12

24

117

238

T3

(0.501)

R1

6

14

24

118

250

R2

5

13

25

114

242

R3

4

12

23

120

260

T4

(1.582)

R1

6

14

25

121

262

R2

5

15

23

122

244

R3

5

12

26

122

258

T5

(5)

R1

6

14

26

123

268

R2

6

16

25

124

274

R3

6

15

25

124

262

PC

R1

180

1308

788

1304

1692

R2

194

1368

816

1432

1832

R3

202

1434

942

1460

1868

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

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

3

11

18

114

242

R2

4

12

20

110

238

R3

3

11

24

107

256

VC

(0.00)

R1

6

16

24

128

292

R2

7

15

25

126

288

R3

5

16

29

132

290

T1

(0.050)

R1

3

11

23

120

252

R2

5

12

22

118

256

R3

4

14

24

99

262

T2

(0.158)

R1

4

13

25

109

256

R2

4

12

26

119

264

R3

5

14

21

112

268

T3

(0.501)

R1

6

14

24

119

258

R2

3

15

25

120

258

R3

5

13

24

113

262

T4

(1.582)

R1

5

14

24

121

276

R2

7

14

26

123

264

R3

4

15

25

119

280

T5

(5)

R1

6

16

28

120

284

R2

7

14

27

124

274

R3

5

15

28

118

268

PC

R1

198

496

1524

1976

1808

R2

206

526

1478

1884

1872

R3

178

570

1596

1964

1944

 

 

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

23

98

246

R2

4

12

22

108

244

R3

5

13

20

112

230

VC

(0.00)

R1

6

16

27

122

280

R2

7

15

29

130

296

R3

7

14

30

136

304

T1

(0.050)

R1

4

13

24

112

234

R2

5

12

23

100

248

R3

4

14

23

119

241

T2

(0.158)

R1

5

13

25

116

246

R2

4

14

24

118

238

R3

5

13

23

107

252

T3

(0.501)

R1

5

15

26

128

242

R2

4

14

23

116

256

R3

6

13

25

108

262

T4

(1.582)

R1

7

15

24

120

266

R2

5

16

26

128

258

R3

5

13

25

116

240

T5

(5)

R1

6

14

27

128

254

R2

7

15

26

121

266

R3

6

16

28

121

274

PC

R1

176

1540

920

1696

1824

R2

163

1572

984

1740

1768

R3

181

1612

1016

1704

1920

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 [10
μg/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)

3.67

0.58

11.00

1.00

23.00

1.00

113.33

4.16

244.67

7.57

VC

(0.00)

6.33

1.53

15.00

1.00

27.00

1.00

124.33

4.51

275.33

12.06

T1

(0.050)

4.33

0.58

12.67

0.58

24.00

1.00

123.00

1.00

246.67

6.11

T2

(0.158)

4.33

0.58

13.33

0.58

24.33

0.58

123.67

1.15

254.67

6.11

T3

(0.501)

5.00

1.00

13.67

0.58

24.67

1.53

124.33

0.58

264.67

10.07

T4

(1.582)

5.67

0.58

14.00

1.00

25.00

1.00

124.67

1.53

273.33

6.11

T5

(5)

6.00

1.00

14.67

0.58

26.33

0.58

126.00

1.00

274.67

10.07

PC

174.00

9.17

635.33

29.14

1160.00

92.26

1626.00

45.74

1964.00

143.05

 

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)

4.00

1.00

10.67

1.15

22.33

2.52

106.67

8.08

234.00

14.00

VC

(0.00)

6.00

1.00

14.67

1.15

25.00

2.00

118.67

6.11

267.33

15.01

T1

(0.050)

4.33

0.58

13.00

1.00

23.00

1.73

110.67

4.16

236.00

8.00

T2

(0.158)

4.67

0.58

12.33

0.58

23.33

0.58

113.33

4.73

244.00

8.72

T3

(0.501)

5.00

1.00

13.00

1.00

24.00

1.00

117.33

3.06

250.67

9.02

T4

(1.582)

5.33

0.58

13.67

1.53

24.67

1.53

121.67

0.58

254.67

9.45

T5

(5)

6.00

0.00

15.00

1.00

25.33

0.58

123.67

0.58

268.00

6.00

PC

192.00

11.14

1370.00

63.02

848.67

82.03

1398.67

83.17

1797.33

92.98

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

PC= Positive control

2-Aminoanthracene [2.5μg/plate]: TA 1537, TA 1535, TA 98, TA 100                  Methyl methanesulfonate [4μl/plate]: TA 102

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

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

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

 

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-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)

3.33

0.58

11.33

0.58

20.67

3.06

110.33

3.51

245.33

9.45

VC

(0.00)

6.00

1.00

15.67

0.58

26.00

2.65

128.67

3.06

290.00

2.00

T1

(0.050)

4.00

1.00

12.33

1.53

23.00

1.00

112.33

11.59

256.67

5.03

T2

(0.158)

4.33

0.58

13.00

1.00

24.00

2.65

113.33

5.13

262.67

6.11

T3

(0.501)

4.67

1.53

14.00

1.00

24.33

0.58

117.33

3.79

259.33

2.31

T4

(1.582)

5.33

1.53

14.33

0.58

25.00

1.00

121.00

2.00

273.33

8.33

T5

(5)

6.00

1.00

15.00

1.00

27.67

0.58

120.67

3.06

275.33

8.08

PC

194.00

14.42

530.67

37.22

1532.67

59.48

1941.33

50.01

1874.67

68.04

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.33

0.58

11.67

1.53

21.67

1.53

106.00

7.21

240.00

8.72

VC

(0.00)

6.67

0.58

15.00

1.00

28.67

1.53

129.33

7.02

293.33

12.22

T1

(0.050)

4.33

0.58

13.00

1.00

23.33

0.58

110.33

9.61

241.00

7.00

T2

(0.158)

4.67

0.58

13.33

0.58

24.00

1.00

113.67

5.86

245.33

7.02

T3

(0.501)

5.00

1.00

14.00

1.00

24.67

1.53

117.33

10.07

253.33

10.26

T4

(1.582)

5.67

1.15

14.67

1.53

25.00

1.00

121.33

6.11

254.67

13.32

T5

(5)

6.33

0.58

15.00

1.00

27.00

1.00

123.33

4.04

264.67

10.07

PC

173.33

9.29

1574.67

36.07

973.33

48.88

1713.33

23.44

1837.33

76.87

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 both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment.

Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

Trial I:

Trial I was performed with five concentration of test item along with the negative, vehicle and concurrent positive contros with the remaining three strains i.e TA 1535, TA 1537 and TA 102 by the plate incorporation method . For TA 98 and TA 100 revertant colony counts were directly incorporated in the trial I from the pre experiment upto the required five concentrations. [T4 (0.050 mg/plate) to T8 (5 mg/plate)].

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 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
Study period:
27 June 2018 to 08 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal 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:
Not applicable
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Human Blood
- Suitability of cells: No data

For lymphocytes:
- Sex, age and number of blood donors: Between 27 to 28 years of age range
- Whether whole blood or separated lymphocytes were used: Seperated Lymphocytes
- Whether blood from different donors were pooled or not: No data
- Mitogen used for lymphocytes:

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
- 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
Cytokinesis block (if used):
No data available
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 was procured from Defence Research and Development Establishment
- method of preparation of S9 mix : Appropriate quantity of S9 supernatant was mixed with S9 cofactor solution, which contains 0.80 g of D-glucose-6-phosphate, 1.00 g of MgCl2, 1.35 g of KCl, 6.40 g of Na2HPO4, 1.40 g of NaH2PO4.H2O, 1.75 g of β-NADP in 500 mL of RO water. During the experiment, S9 mix was freshly prepared.
- concentration or volume of S9 mix and S9 in the final culture medium : 1 % v/v for Phase I of experiment and 2 % v/v for Phase II of experiment.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data available
Test concentrations with justification for top dose:
0.007, 0.014 and 0.028 mg/mL on the basis of cytotoxicity study the doses were selected. (Both in presence and in absence of metabolic activation system)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
Untreated negative controls:
yes
Remarks:
Phosphate Buffer Saline
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
other: Cyclophosphamide Monohydrate ( with metabolic activation)
Details on test system and experimental conditions:
FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): indicate the identity of mitotic spindle inhibitor used (e.g., colchicine), its concentration and, duration and period of cell exposure. : Colcemide (0.3 µg/mL) was added 3 hour prior to harvesting and kept under incubation at 37 ± 2 °C

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays): 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. All slides, including those of positive, vehicle and negative controls, were independently coded before microscopic analysis

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored): 1000 cells per slides

- Criteria for scoring chromosome aberrations (selection of analysable cells and aberration identification): Chromosomal and chromatid breaks, acentric fragments, deletions, exchanges, pulverization, polyploidy (including endoreduplication) 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. To describe a cytotoxic effect the mitotic index (% cells in mitosis) were determined.

- Determination of polyploidy: Yes
- Determination of endoreplication: Yes


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; mitotic index (MI); relative population doubling (RPD); relative increase in cell count (RICC); replication index; cytokinesis-block proliferation index; cloning efficiency; relative total growth (RTG); relative survival (RS); other: Mitotic Index

- Any supplementary information relevant to cytotoxicity: To evaluate the toxicity of the test item a cytotoxicity assay was performed both in the presence and absence of metabolic activation system. Three test concentrations (0.007, 0.014 and 0.028 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.


- OTHER: No data
Rationale for test conditions:
No data available
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 negative 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:
Remarks:
Human peripheral blood lymphocytes
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
- Data on pH: The pH of test item in culture medium was assessed at 0 h and 4 h after incubation at 37 ± 2 °C. No significant change in pH was observed at 0 h and 4 h when compared with negative controls.
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: Slight precipitation was observed at 0.028 mg/mL, it was taken as highest concentration for the treatment in cytotoxic experiment.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): To evaluate the toxicity of test item cytotoxicity was performed both in presence and in absence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL of culture media. Cytotoxicity was not observed in the treated concentrations both, in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 9.89 (VC), 9.65 (T1), 9.59 (T2), 9.60 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 9.74 (T1), 9.60 (T2), 9.65 (T3) and 8.59 (PC). In the cytotoxicity experiment, the highest test concentration 0.028 (T3) mg/ mL of culture media did not show more than 50% reduction the mitotic index whe compared to the respective vehicle control both in the presence or absence of metaboli activation. Hence these concentrations [0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL] were selected for the main study.
Hence, 0.028 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.


Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytres in primary cultures: mitotic index (MI) : In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 9.89 (VC), 9.65 (T1), 9.59 (T2), 9.60 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 9.74 (T1), 9.60 (T2), 9.65 (T3) and 8.59 (PC).
o For cell lines: relative population doubling (RPD), relative Increase in cell count (RICC), number of cells treated and cells harvested for each culture, information on cell cycle length, doubling time or proliferation index. : No data

- Genotoxicity results (for both cell lines and lymphocytes): Genotoxicity negative

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Please reffer the table in remark section
- Negative (solvent/vehicle) historical control data: Please reffer the table in remarks section.
Remarks on result:
other: No mutagenic potential

Cytotoxicity result:

Cytotoxicity was assessed at the concentrations of 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL of culture media. Cytotoxicity was not observed in the treated concentrations both, in the absence and in the presence of metabolic activation (1%).

In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 9.89 (VC), 9.65 (T1), 9.59 (T2), 9.60 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 9.74 (T1), 9.60 (T2), 9.65 (T3) and 8.59 (PC). In the cytotoxicity experiment, the highest test concentration0.028 (T3) mg/ mLof culture media did not show more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence these concentrations [0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL] were selected for the main study. Hence, 0.028 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;

Phase I :

In the experiment, the cultures were exposed to 12H-phthaloperin-12-one (CAS No. 6925-69-5) 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), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.007 (T1), 0.014 (T2) and 0.028 (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 Cyclophosphamide monohydrate at the concentration of30 µg/mL in the presence of metabolic activation (1%) caused significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increase was biologically significant.

During the treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 9.98, 9.74, 9.60, 9.40, 9.50 and 8.45 and in the presence of metabolic activation were 10.13, 9.85, 9.75, 9.54, 9.65 and 8.70 for NC, VC, T1, T2, T3 and PC concentrations respectively.

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 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL culture both in presence and in absence 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), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.007 (T1), 0.014 (T2) and 0.028 (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 of 30 µg/mL in the presence of metabolic activation (2%) caused significant 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 no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 10.07, 9.80, 9.48, 9.55, 9.44 and 8.44 and in the presence of metabolic activation were 9.94, 9.95, 9.65, 9.70, 9.60 and 8.60 for NC, VC, T1, T2, T3 and PC concentrations respectively.

Conclusions:
The test chemical was not found to be mutagenic for chromosomal aberration in human peripheral blood lymphocyte both in presence (1% and 2%) and in the absence of metabolic activation under the specified conditions.
Hence it is not likely to be classified 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 test chemical in human peripheral blood lymphocyte cultures. The method 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 obtained 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. Based on solubility and precipitation test, Dimethyl Sulfoxide (DMSO) was selected as the vehicle for treatment. 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. There was slight precipitation observed at 0.028 mg/mL concentration. Hence 0.028 mg/mL was selected as the highest concentration for cytotoxicity test. Cytotoxicity of test chemical was evaluated both in absence and in presence of metabolic activation system. (1%) . Cytotoxicity was assessed at the concentrations of 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL of culture media. Cytotoxicity was not observed in the treated concentrations both, in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 9.89 (VC), 9.65 (T1), 9.59 (T2), 9.60 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 9.74 (T1), 9.60 (T2), 9.65 (T3) and 8.59 (PC). In the cytotoxicity experiment, the highest test concentration 0.028 (T3) mg/ mLof culture media did not show more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence these concentrations [0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL] were selected for the main study both in the presence and in the absence of metabolic activation. The main study was performed in two independent phases; In the phase 1 experiment, the cultures were exposed to 12H-phthaloperin-12-one 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), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.007 (T1), 0.014 (T2) and 0.028 (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 Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increase was biologically significant. During the treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 9.98, 9.74, 9.60, 9.40, 9.50 and 8.45 and in the presence of metabolic activation were 10.13, 9.85, 9.75, 9.54, 9.65 and 8.70 for NC, VC, T1, T2, T3 and PC concentrations respectively. 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 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL culture both in presence and in absence 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), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.007 (T1), 0.014 (T2) and 0.028 (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 Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant 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 no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.07, 9.80, 9.48, 9.55, 9.44 and 8.44 and in the presence of metabolic activation were 9.94, 9.95, 9.65, 9.70, 9.60 and 8.60 for NC, VC, T1, T2, T3 and PC concentrations.

From the above data obtained from results it can be concluded that the test chemical  was not found to be mutagenic for chromosomal aberration in human peripheral blood lymphocyte both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. Hence it is not likely to be classifed 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:
key study
Study period:
17 March 2020 to 28 April 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from experimental study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)
Principles of method if other than guideline:
The test chemical was evaluated in, in-vitro mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT locus
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells: Chinese Hamster Ovary Cell Line (CHO-K1) cell was obtained from National Centre for Cell Sciences, Pune, India
- Suitability of cells: Chinese hamster ovary (CHO) cell line was selected for this study because it is one of the most widely used cell type for a mammalian cell gene mutation test, and it is suggested by the current OECD test guideline 476.
- Normal cell cycle time (negative control): No data

For cell lines:
- Absence of Mycoplasma contamination: Mycoplasma contamination-free cell line was used for the study
- Number of passages if applicable: In-house Passage No. 4 and 5
- Methods for maintenance in cell culture:
- Cell cycle length, doubling time or proliferation index : No data
- Modal number of chromosomes: No data
- Periodically checked for karyotype stability: No data
- Periodically ‘cleansed’ of spontaneous mutants: Yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable: Hamʹs F-12K with L-glutamine containing 10% FBS and Penicillin-Streptomycin 1X with Trypsin-EDTA (0.25%) as the splitting agent, Dulbeccos PBS saline without calcium and magnesium as the cell refreshing agent. The cell line was stored at 37 ±1 deg C, ~ 5% CO2 in air, 85 to 95% humidity
Additional strain / cell type characteristics:
other: The cell line is hemizygous for the HPRT gene, which is located on the X chromosome. Two dimensional (attached) colonies are formed from single cells grown in a normal liquid medium.
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Mammalian liver Fraction (S9) was prepared in-house using liver of Wistar rats induced with sodium phenobarbitone and beta - naphthoflavone as inducing agents. S9 mix was prepared by mixing Sterile analytical grade water, 0.2 M phosphate buffer having pH 7.4, 0.1 M NADP, 1 M glucose-6- phosphate, 0.4 M MgCl2 and 1.65 M of KCl salt solution and Rat liver S9.
- source of S9 : Wistar rats, In-house bred at the test facility
- method of preparation of S9 mix : Mammalian liver Fraction (S9) was prepared in-house using liver of Wistar rats induced with sodium phenobarbitone and beta - naphthoflavone as inducing agents.
- concentration or volume of S9 mix and S9 in the final culture medium : The S9 mix was prepared in cold condition immediately before its use in the experiment. The in-house batch of liver S9 fraction with the protein concentration of 33 mg/mL was used in the study. The microsomal enzyme reaction mixture contained the following components for 10 mL.
a. Sterile analytical grade water 3.35 mL
b. 0.2 M phosphate buffer, pH 7.4 5.00 mL
c. 0.1 M NADP 0.40 mL
d. 1 M glucose-6- phosphate 0.05 mL
e. 0.4 M MgCl2, 1.65 M KCl salt solution 0.20 mL
f. Rat liver S9 1.00 mL
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): No data
Test concentrations with justification for top dose:
0, 1.953125, 3.90625, 7.8125 and 15.625 μg/mL were selected as the doses for the main study on the basis of results obtained from the preliminary cytotoxicity test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: The test chemical formed uniform suspension in dimethyl sulphoxide at the concentration of 200 mg/ml. Hence, dimethyl sulphoxide was chosen as a vehicle.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : Triplicate
- Number of independent experiments : Single experiment was conducted

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 20000000
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: No data
- Exposure duration/duration of treatment: 3 hours

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 11 days
- Selection time (if incubation with a selective agent): 11 days
- Fixation time (start of exposure up to fixation or harvest of cells): Not specified
- Method used: agar or microwell plates for the mouse lymphoma assay.
- If a selective agent is used (e.g., 6-thioguanine or trifluorothymidine), indicate its identity, its concentration and, duration and period of cell exposure.: 6 Thiogaunine
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: 2.1 to 2.2 million cells per concentration
- Criteria for small (slow growing) and large (fast growing) colonies: Not specified

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method.: % relative survival (RS)
- Any supplementary information relevant to cytotoxicity: The medium from each flask was removed, and the cells were trypsinised. After detachment of the cells from the surface of the flask, fresh complete medium was added to neutralize the trypsin effect. The cells from the duplicate cultures of each test concentration along with concurrent controls were collected in a centrifuge tube, and then cultures were centrifuged at 1600-2000 rpm. The supernatants were discarded, and the pellets were resuspended in fresh medium, then the cell density of each culture was measured by using a hemocytometer. The cells were diluted twice to achieve appropriate cell concentration culture medium, then were seeded in duplicates; at a density of 150 cells / 60 mm dish 5 ml culture medium. The plates were incubated at 37 ± 1 degree Celcious, with approximately 5% CO2 supply for 9 days. After incubation, the medium was discarded, and the clones were stained by using crystal violet and observed. Cytotoxicity to CHO cultures was expressed as % relative survival.

METHODS FOR MEASUREMENTS OF GENOTOXICIY

- OTHER:
EXPRESSION OF THE MUTANT PHENOTYPE
The cells from the duplicate cultures of each test concentration, along with concurrent controls,were sub-cultured in 5 ml of culture medium, at density of 2.1 to 2.2 million cells per concentration. Plates were incubated at 37 ± 1 degree Celcious and sub-cultured at suitable intervals (Day 4 and day 7) for the remaining 11 days expression period.

PHENOTYPIC EXPRESSION OF MUTANT CELLS IN SELECTIVE MEDIUM
Medium from each flask was removed, and the cells were trypsinised. After detachment of the cells from the surface of the flask, 1-3 ml of fresh medium was added to neutralize the trypsin effect. The cells from the duplicate cultures of each test concentration, along with concurrent controls, were collected in a centrifuge tube and centrifuged at 1600-2000 rpm.

The supernatant from each tube was discarded, and the pellet was resuspended in fresh complete medium, and the cell density of each culture was measured using a hemocytometer. The cells were diluted to appropriate cell concentration with culture medium, and cells were plated in triplicates; (60 mm dish); at a density of about 0.2 million on cells / 60mm dish in 5 ml culture medium containing 10% FBS and 6-Thiogaunine as a selective agent.
For cloning efficiency determinations, 150 cells were plated in duplicate in 60 mm dish,containing 5 ml of F12 nutrient mix medium with 10% FBS.
For phenotypic expression, the plates were incubated at 37 ± 1 degree celsius, with approximately 5% CO2 supply for 11 days. After incubation, the medium was discarded, and the plates were stained by crystal violet and observed for the numbers of clones.
Rationale for test conditions:
No data
Evaluation criteria:
Please refer additional information on materials and methods
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
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
- Data on pH: Volumes of 50 µl were drawn from stock formulations of the test item, prepared in dimethyl sulphoxide, and having concentrations of 2000, 1000, 500, 250, 125, 62.5 and 31.25 µg/ml (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/ml), were added into 4.95 ml of F-12 nutrient mix medium in 15 ml culture tubes. These preparations (designated as 0 h) were immediately checked for pH. The tubes were then placed in an incubator for 3 hours at a temperature of 37 ± 1 degree Celsius and were thereafter again re-checked for pH. The pH of test concentrations 62.5 and 31.25 µg/ml was found to be within the desired pH range of vehicle control medium.
- Data on osmolality: No data
- Possibility of evaporation from medium: No data
- Water solubility: No data
- Precipitation and time of the determination: Volumes of 50 µl were drawn from stock formulations of the test item, prepared in dimethyl sulphoxide, and having concentrations of 2000, 1000, 500, 250, 125, 62.5 and 31.25 µg/ml (2, 1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/ml), were added into 4.95 ml of F-12 nutrient mix medium in 15 ml culture tubes. These preparations (designated as 0 h) were immediately checked with unaided eyes for any precipitation. The tubes were then placed in an incubator for 3 hours at a temperature of 37 ± 1 degree Celsius and were thereafter again re-checked for precipitation. Heavy precipitation was observed from concentration 2000 to 125 µg/ml. Slight precipitation was observed at concentration of 62.5 µg/ml and no precipitation was observed at the concentration of 31.25 µg/ml.
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES (if applicable): For assessing the cytotoxicity of the test item, CHO-K1 cells were exposed to four concentrations of test chemical viz. 62.5, 31.25, 15.625 and 7.8125 µg/ml in treatment medium. Cytotoxicity was determined by the relative survival (RS) after a 3 hour of treatment in the presence and absence of a metabolic activation system (S9). Less than 10% relative survival was observed at the concentrations of 62.5 and 31.25 µg/ml, 10-20% relative survival was observed at 15.625 µg/ml and more than 20% relative survival was observed at 7.8125 µg/ml. The relative survival (RS) was observed to be 1.4, 4.3, 12.6, and 40.4% in the absence of metabolic activation system and 4.2, 7.9, 14.7 and 33.5% in the presence of metabolic activation system at the concentrations of 62.5, 31.25, 15.625 and 7.8125 µg/ml respectively. The concentration of 15.625 µg/ml was therefore selected as the highest concentration both in the presence and absence of a metabolic activation system, for the definitive study.


STUDY RESULTS
- Concurrent vehicle negative and positive control data : The results of positive controls are considered acceptable as per the acceptance criteria. Concurrent positive controls produced a statistically significant increase in mutant frequency in the presence and absence of metabolic activation when compared with the concurrent vehicle control.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency : Cytotoxicity was determined by the relative survival (RS) following a 3-hour treatment both in the presence and absence of a metabolic activation system (S9). Less than 10% relative survival was observed at the concentrations 62.5 and 31.25 µg/mL. 10-20% relative survival was observed at concentration of 15.625 µg/mL and more than 20% relative survival was observed at the concentration 7.8125 µg/mL. The relative survival (RS) was observed to be 1.4, 4.3, 12.6 and 40.4% in absence of metabolic activation system and 4.2, 7.9, 14.7 and 33.5% in presence of metabolic activation system at the concentrations of 62.5, 31.25, 15.625 and 7.8125 µg/ml respectively.

- Genotoxicity results:
o Number of cells treated and sub-cultures for each cultures : 20000000
o Number of cells plated in selective and non-selective medium : 2.1 to 2.2 million cells per concentration
o Number of colonies in non-selective medium and number of resistant colonies in selective medium, and related mutant frequency
o When using the thymidine kinase gene on L5178Y cells: colony sizing for the negative and positive controls and if the test chemical is positive, and related mutant frequency. For the MLA, the GEF evaluation.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: Refer table 2 as specified in any other information on results including tables
- Negative (solvent/vehicle) historical control data: Refer table 2 as specified in any other information on results including tables
Remarks on result:
other: Non-mutagenic in vitro

SUMMARY OF MUTANT FREQUENCY

Experiments

Without S9

With S9

Treatment

3 hours

3 hours

Test Group

Test

Concentration

(µg/ml)

Mutant Frequency

Mutation Factor

Mutant Frequency

Mutation Factor

VC

0

16.15

1.00

11.36

1.00

F1

15.625

6.67

0.41

4.61

0.41

F2

7.8125

12.98

0.80

13.87

1.22

F3

3.90625

10.13

0.63

11.55

1.02

F4

1.953125

20.03

1.24

18.05

1.59

PC

Ethylmethane

Sulfonate

(6 µg/ml)

67.42

4.17

-

-

PC

Benzopyrene

(200 µg/ml)

-

-

62.65

5.51

Mutant Frequencyis expressed asMutants / 106Cells

SUMMARY

IN VITRO MAMMALIAN CELL GENE MUTATION TEST (CHO/HPRT)

LABORATORY HISTORICAL CONTROL DATA -VEHICLE CONTROL

Mutant Frequency / 106survival cells -Without S9

 

Mutant Frequency / 106survival cells- With S9

Vehicle

Mean

S.D.

2 S.D.

Lower

Upper

Mean

S.D.

2 S.D.

Lower

Upper

DMSO

10.77

3.88

7.76

3.01

18.53

10.52

4.15

8.30

2.22

18.82

LABORATORY HISTORICAL CONTROL DATA –POSITIVE CONTROL

 

 

 

 

Mutant Frequency / 106survival cells -Without S9

 

Mutant Frequency / 106survival cells- With S9

EMS (200 µg/ml)

Benzopyrene (6 µg/ml)

Mean

93.33

92.46

S.D.

67.70

69.85

2 S.D.

135.40

139.71

Lower range

42.06

47.25

Upper range

228.73

232.17

3 Relative Survival

  Experiment No. 1                                                                                   Without S9 Mix

Dose Level

Concentration  (µg/ml in final medium)

Cloning Efficiency

 

%

Relative

Survival

Individual Plate Count

Average

colony counting

CE

Adjusted Cloning

efficiency

Plate 1

Plate 2

Plate 3

VC

0

148

142

120

136.7

0.91

1.24

100.0

F1

15.625

23

41

22

28.7

0.19

0.15

12.4

F2

7.8125

86

86

64

78.7

0.52

0.50

40.4

F3

3.90625

94

76

80

83.3

0.56

0.65

52.3

F4

1.953125

126

124

120

123.3

0.82

1.05

84.4

PC

6

126

130

142

132.7

0.88

0.76

61.0

PC- Ethylmethanesulfonate (For without S9 treatment)

Key:CE - Cloning Efficiency

4 Relative Survival

  Experiment No. 1                                                                                              With S9 Mix

Dose Level

Concentration  (µg/ml in final medium)

Cloning Efficiency

%

Relative

Survival

Individual Plate Count

Average

colony counting

CE

Adjusted Cloning

efficiency

Plate 1

Plate 2

Plate 3

VC

0

146

122

150

139.3

0.93

1.18

100.0

F1

15.625

40

28

27

31.7

0.21

0.18

15.1

F2

7.8125

58

54

50

54.0

0.36

0.39

32.8

F3

3.90625

88

112

92

97.3

0.65

0.77

65.0

F4

1.953125

124

114

104

114.0

0.76

0.94

79.8

PC

200

80

136

146

120.7

0.80

0.75

63.6

PC- Benzo(a)pyrene (For with S9 treatment)

Key:CE - Cloning Efficiency

5 PARALLEL CLONING EFFICIENCY (% CEAND % RCE)

Experiment  1                                                                      Without S9 Mix

ID No.

Dose Levels#

Concentration  (µg/ml in final medium)

Plate 1

Plate 2

Plate 3

Average

CE

% CE

 % RCE

VC

0

126

130

142

132.7

0.88

88.44

100

F1

15.625

56

64

22

47.3

0.32

31.56

35.68

F2

7.8125

50

38

68

52.0

0.35

34.67

39.20

F3

3.90625

68

92

40

66.7

0.44

44.44

50.25

F4

1.953125

74

74

66

71.3

0.48

47.56

53.77

PC

6

112

124

94

110.0

0.73

73.33

82.91

PC- Ethylmethanesulfonate (For without S9 treatment)

Key:CE - Cloning Efficiency, RCE - Relative Cloning Efficiency.

# Cloning Efficiency of pooled culture

6 PARALLEL CLONING EFFICIENCY (% CE AND % RCE)

  Experiment No. 1                         With S9 Mix

ID No.

Dose Levels #

Concentration  (µg/ml in final medium)

Plate 1

Plate 2

Plate 3

Average

CE

% CE

% RCE

VC

0

104

114

124

114.0

0.76

76.00

100

F1

15.625

34

72

48

51.3

0.34

34.22

45.03

F2

7.8125

52

62

32

48.7

0.32

32.44

42.69

F3

3.90625

62

56

86

68.0

0.45

45.33

59.65

F4

1.953125

52

54

30

45.3

0.30

30.22

39.77

PC

200

120

92

126

112.7

0.75

75.11

98.83

PC- Benzo(a)pyrene (For with S9 treatment)

Key:CE - Cloning Efficiency, RCE - Relative Cloning Efficiency.

# Cloning Efficiency of pooled culture

7 INDIVIDUAL MUTANT COLONY COUNT AND MUTANT FREQUENCY

Experiment No. 1                                                              Without S9 Mix

ID No.              Dose Levels

#

Concentration  (µg/ml in final medium)

Mutant frequency

Mutation Factor

Number of culture per 96 well plate

Mutant

 Frequency per 106survival cells

Plate 1

Plate 2

Plate 3

Total

VC

0

6

9

15

30.00

16.15

1.00

F1

15.625

0

2

2

4.00

6.67

0.41

F2

7.8125

1

6

2

9.00

12.98

0.80

F3

3.90625

5

1

3

9.00

10.13

0.63

F4

1.953125

10

3

7

20.00

20.03

1.24

PC

6

22

44

23

89.00

67.42

4.17

PC- Ethylmethanesulfonate (For without S9 treatment)

Key:# Mutant Frequency of pooled culture

8 INDIVIDUAL MUTANT COLONY COUNT AND MUTANT FREQUENCY

Experiment No. 1                                                                           With S9 Mix

ID No.              Dose Levels

#

Concentration  (µg/ml in final medium)

Mutant frequency

Mutation Factor

Number of culture per 96 well plate

Mutant Frequency per 106survival cells

Plate 1

Plate 2

Plate 3

Total

VC

0

5

3

11

19

11.36

1.00

F1

15.625

0

2

1

3

4.61

0.41

F2

7.8125

1

5

3

9

13.87

1.22

F3

3.90625

1

4

6

11

11.55

1.02

F4

1.953125

1

3

8

12

18.05

1.59

PC

200

28

27

25

80

62.65

5.51

PC- Benzo(a)pyrene (For with S9 treatment)

Key:# Mutant Frequency of pooled culture

Conclusions:
In Vitro Mammalian Cell Gene Mutation Test of the test chemical was carried out in compliance with the OECD Guideline No. 476 (29 July 2016). Under the conditions described in this study, it is concluded that the test chemical is non-mutagenic when tested in an In Vitro Mammalian cell gene mutation test using the Hprt gene in the presence and absence of S9 metabolic activation system and hence is likely to be non-mutagenic in vitro.
Executive summary:

In vitro Mammalian Cell Gene Mutation Test was carried out in compliance with the OECD Guideline No. 476, adopted by the council on 29 July 2016. The test chemical was evaluated in mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells.

Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of the test chemical, the CHO cells were exposed to the test item in duplicate cultures at the doses of 1.953, 3.90625, 7.8125 and 15.625 µg/mL of culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction prepared from, sodium phenobarbitone and b- naphthoflavone-induced Wistar rats was used in the study.

Dimethyl sulphoxide was used as a vehicle. The target cells were exposed to the test compound for 3 hours at 37 ± 1oC, with approximately 5% CO2 supply. The cells were sub-cultured immediately to determine cytotoxicity as relative survival (RS) and to initiate the phenotypic expression period. The culture flasks were incubated at 37 ± 1oC with approximately 5% CO2 supply during experimental periods. Concurrent vehicle and positive control groups were also included in the experiment, as specified by the test guideline. The cultures were sub-cultured during the expression period at suitable intervals. After phenotypic expression, the plates were incubated at 37 ± 1oC with approximately 5% CO2 supply for 11 days. After incubation, the medium was discarded, and the plates were stained and observed for the clones. Relative survivaland mutant frequency were calculated for all treatment, vehicle control, and positive control groups. 

The relative survival (RS) was used as the measure of treatment-related cytotoxicity. The RS (relative survival) for cultures treated with the test chemical with and without metabolic activation system (S9) indicated that the test chemical induces 10 to 20% relative survival at the concentration of 15.625 µg/mL. The % relative survival (% RS) for the cloned cultures ranged from 12.4% to 84.4% for cultures treated without metabolic activation system and 14.8% to 79.8% for cultures treated with metabolic activation system at test item concentrations of 1.953125, 3.90625, 7.8125, and 15.625 µg/mL in the treatment medium.

The results of the present study indicate that there was no significant difference in the mutant frequencies of cultures treated with the test chemical as compared to the vehicle control cultures, in the absence or presence of metabolic activation. An increase in the mutant frequency of concurrent positive controls demonstrated the sensitivity of the assay in the absence and presence of metabolic activation.

Under the test conditions described in the study, it is concluded that the test chemical is non-mutagenic in 'In Vitro Mammalian cell gene mutation test' using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells 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

Experimental data available for the target chemical was reviewed to determine the mutagenic nature of 12H-phthaloperin-12-one (CAS No. 6925-69-5). The studies are as mentioned below:

Ames test:

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 both with and without liver microsomal activation. Each concentration, including the negative, vehicle and positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.050, 0.158, 0.501, 1.582 and 5 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 chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro Mammalian chromosomal aberration study

This study was conducted to determine the chromosomal aberration induction potential of test chemical in human peripheral blood lymphocyte cultures. The method followed were as per OECD guideline No. 473, adopted on 29thJuly 2016.In VitroMammalian Chromosome Aberration Test. The experiment was conducted using human peripheral blood lymphocytes. Blood was obtained 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. Based on solubility and precipitation test, Dimethyl Sulfoxide (DMSO) was selected as the vehicle for treatment. 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. There was slight precipitation observed at 0.028 mg/mL concentration. Hence 0.028 mg/mL was selected as the highest concentration for cytotoxicity test. Cytotoxicity of test chemical was evaluated both in absence and in presence of metabolic activation system. (1%) . Cytotoxicity was assessed at the concentrations of 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL of culture media. Cytotoxicity was not observed in the treated concentrations both, in the absence and in the presence of metabolic activation (1%). In the absence of S9 mix, the mean mitotic index observed was 10.08 (NC), 9.89 (VC), 9.65 (T1), 9.59 (T2), 9.60 (T3) and 8.40 (PC). In the presence of S9 mix, the mean mitotic index observed was 10.03 (NC), 9.84 (VC), 9.74 (T1), 9.60 (T2), 9.65 (T3) and 8.59 (PC). In the cytotoxicity experiment, the highest test concentration 0.028 (T3) mg/ mLof culture media did not show more than 50% reduction the mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation. Hence these concentrations [0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL] were selected for the main study both in the presence and in the absence of metabolic activation. The main study was performed in two independent phases; In the phase 1 experiment, the cultures were exposed to 12H-phthaloperin-12-one 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), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.333 (T1), 0.333 (T2), 0.667 (T3) and 10.000 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.007 (T1), 0.014 (T2) and 0.028 (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 Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (1%) caused significant increase in percent aberrant cells. Even though the analysis did not reveal any statistical significance, the increase was biologically significant. During the treatment with test item in the absence and presence of S9 mix, there was no reduction in mitotic index observed at the tested concentrations. The observed mean mitotic index in the absence of metabolic activation were 9.98, 9.74, 9.60, 9.40, 9.50 and 8.45 and in the presence of metabolic activation were 10.13, 9.85, 9.75, 9.54, 9.65 and 8.70 for NC, VC, T1, T2, T3 and PC concentrations respectively. 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 0.007 (T1), 0.014 (T2) and 0.028 (T3) mg/mL culture both in presence and in absence 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), 0.333 (VC), 0.333 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 0.333 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.007 (T1), 0.014 (T2) and 0.028 (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 Cyclophosphamide monohydrate at the concentration of 30 µg/mL in the presence of metabolic activation (2%) caused significant 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 no reduction in mitotic index was observed at the tested concentrations. The observed mean mitotic indexin the absence of metabolic activation were 10.07, 9.80, 9.48, 9.55, 9.44 and 8.44 and in the presence of metabolic activation were 9.94, 9.95, 9.65, 9.70, 9.60 and 8.60 for NC, VC, T1, T2, T3 and PC concentrations.

The test chemical was found to be not mutagenic in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. Hence it is not likely to be classifed as a gene mutant as per the criteria mentioned in CLP regulation.

In vitro Mammalian cell gene mutation assay

In vitro Mammalian Cell Gene Mutation Test was carried out in compliance with the OECD Guideline No. 476, adopted by the council on 29 July 2016. The test chemical was evaluated in mammalian cell gene mutation assay to determine its ability to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells.

Based upon the preliminary tests conducted to assess the solubility/precipitation and cytotoxicity of the test chemical,the CHO cells were exposed to the test item in duplicate cultures at the doses of 1.953125, 3.90625, 7.8125 and 15.625 µg/mL of culture medium, in the absence and presence of metabolic activation system (S9). Liver S9 fraction prepared from, sodium phenobarbitone and b- naphthoflavone-induced Wistar rats was used in the study. Dimethyl sulphoxide was used as a vehicle. The target cells were exposed to the test compound for 3 hours at 37 ± 1oC, with approximately 5% CO2supply. The cells were sub-cultured immediately to determine cytotoxicity as relative survival (RS) and to initiate the phenotypic expression period. The culture flasks were incubated at 37 ± 1oC with approximately 5% CO2supply during experimental periods. Concurrent vehicle and positive control groups were also included in the experiment, as specified by the test guideline. The cultures were sub-cultured during the expression period at suitable intervals. After phenotypic expression, the plates were incubated at 37 ± 1oC with approximately 5% CO2 supply for 11 days. After incubation, the medium was discarded, and the plates were stained and observed for the clones. Relative survivaland mutant frequency were calculated for all treatment, vehicle control, and positive control groups. 

The relative survival (RS) was used as the measure of treatment-related cytotoxicity. The RS (relative survival) for cultures treated with the test chemical with and without metabolic activation system (S9) indicated that the test chemical induces 10 to 20% relative survival at the concentration of 15.625 µg/mL. The % relative survival (% RS) for the cloned cultures ranged from 12.4% to 84.4% for cultures treated without metabolic activation system and 14.8% to 79.8% for cultures treated with metabolic activation system at test item concentrations of 15.625, 7.8125, 3.90625 and 1.953125 µg/mL in the treatment medium. The results of the present study indicate that there was no significant difference in the mutant frequencies of cultures treated withthe test chemical as compared to the vehicle control cultures, in the absence or presence of metabolic activation. An increase in the mutant frequency of concurrent positive controls demonstrated the sensitivity of the assay in the absence and presence of metabolic activation. Under the test conditions described in the study, it is concluded that the test chemical is non-mutagenic in 'In Vitro Mammalian cell gene mutation test' using the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosome in the genome of Chinese hamster ovary (CHO) cells and hence it is not likely to classify as a "Gene mutant in vitro".

Justification for classification or non-classification

Based on the available experimental data summarized on the target chemical, the test chemical did not induce gene mutation in vitro. Hence it is not likely to be mutagenic in vitro as per the criteria mentioned in CLP regulation.