Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

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 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, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, 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 chromosome 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. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. 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 ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 mg/mL 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 (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} 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 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. Based on the observations made, the test chemical is at the highest tested concentration of 0.016 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 a gene toxicity test, Chinese Hamster Ovary (CHO) cells were exposed to the test chemical in the concentration of 0, 0.5, 1, 5 or 10 mM without S9-induced metabolic activation for 3 hours. The results showed that there was no evidence of cytotoxicity when CHO cells were treated with the test chemical. Independently of treatment concentration, the results showed no evidence of gene toxicity when exposed to the test chemical. Therefore, it is considered that the test chemical does not cause genetic mutation(s) in the absence of metabolic activation.

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 mutations in comparison to vehicle control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer. The protein concentration in the S9 fraction was 36.2 mg/mL.
- source of S9 : Aroclor 1254-induced S9 was procured from Defence Research and Development Organization and stored at -60°C to -80°C inside the deep freezer.
- method of preparation of S9 mix : An appropriate quantity of S9 supernatant is thawed and mixed with S9 cofactor solution to result in a final concentration of approximately 10 % v/v in the S9 mix. Cofactor solution contains the following quantity of chemicals in 500 mL of Distilled Water.
D-glucose-6-phosphate= 0.8 g
β-NADP = 1.75 g
MgCl2 = 1.0 g
KCl = 1.35 g
Na2HPO4 = 6.4 g
NaH2PO4.H2O = 1.4 g
During the experiment, the S9 mix was prepared freshly.
- concentration or volume of S9 mix and S9 in the final culture medium : The protein concentration in the S9 fraction was 36.2 mg/mL. Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability):Each batch of S9 mix was tested with 2-Aminoanthracene as well as benzo (a) pyrene for its efficiency. Thus, requirements of Ames were fulfilled, and the results of efficiency testing were archived at RCC Laboratories India Private Limited.
Test concentrations with justification for top dose:
0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used [none; no data; acetone; arachis oil; beeswax; carbowaxe; castor oil; cetosteryl alcohol; cetyl alcohol; CMC (carboxymethyl cellulose); coconut oil; corn oil; cotton seed oil; DMSO; ethanol; glycerol ester; glycolester; hydrogenated vegetable oil; lecithin; macrogel ester; maize oil; olive oil; paraffin oil; peanut oil; petrolatum; physiol. saline; poloxamer; polyethylene glycol; propylene glycol; silicone oil; sorbitan derivative; soya oil; theobroma oil; vegetable oil; aqueous solvents (water or saline or culture medium)]: DMSO
- Justification for choice of solvent/vehicle: The test chemical was solulble in DMSO
Untreated negative controls:
yes
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
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No data
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations (0, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plate ) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test).

Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test.
There was no reduction in colony count but reduction in bacterial background lawn was observed in treated concentrations 5.0 mg/plate (T8), 1.582 mg/plate (T7) and no reduction in colony count as well as in bacterial background lawn in treated concentrations (0.501 (T6) mg/plate – 0.002 (T1) mg/plate) both in absence and in the presence of metabolic activation. Based on the results of pre-experiment following doses were selected for the main study trials: 0, 0.005, 0.016, 0.050, 0.158 and 0.501 mg/plate , both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

STUDY RESULTS
- Concurrent vehicle negative and positive control data : The spontaneous reversions in the solvent, negative and positive controls were within the range of the inhouse historical control data

Ames test:
- Mean number of revertant colonies per plate and standard deviation: No substantial increase in the number of revertant colonies of any tester strains were observed following treatment with the test chemical at any dose level in both confirmatory tests neither in the presence nor in the absence of metabolic activation system. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Remarks on result:
other: No mutagenic potential

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Without metabolic activation (-S9)

With metabolic activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

108

20

110

22

R2

116

17

114

16

R3

110

22

117

17

VC

(0.00)

R1

128

27

135

29

R2

130

24

132

28

R3

122

28

128

26

T1

(0.002)

R1

110

18

112

19

R2

111

19

114

17

R3

115

20

117

18

T2

(0.005)

R1

109

21

115

21

R2

113

20

119

20

R3

114

19

120

20

T3

(0.016)

R1

119

22

122

22

R2

117

22

120

21

R3

116

21

123

24

T4

(0.050)

R1

119

18

124

23

R2

120

21

124

22

R3

118

24

125

25

T5

(0.158)

R1

121

22

126

24

R2

123

23

127

25

R3

119

24

125

24

T6

(0.501)

R1

124

25

128

25

R2

125

24

130

26

R3

122

24

129

27

T7

(1.582)

R1

123

25

129

24

R2

125

24

127

26

R3

126

26

131

27

T8

(5)

R1

125

27

130

28

R2

127

25

129

27

R3

126

26

126

25

PC

R1

1320

956

1376

1544

R2

1236

972

1392

1608

R3

1448

1012

1352

1428

NC           =     Negative control

VC           =   Vehicle Control

PC            =     Positive control             

R              =     Replicate

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

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

Sodium azide [10μg/plate]: TA 100,

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

 

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

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

4

9

22

110

244

R2

4

11

16

114

229

R3

3

11

17

117

237

VC

(0.00)

R1

7

16

29

135

294

R2

7

15

28

132

278

R3

8

15

26

128

294

T1

(0.005)

R1

4

12

21

115

235

R2

4

11

20

119

236

R3

4

10

20

120

240

T2

(0.016)

R1

5

13

22

122

241

R2

4

11

21

120

248

R3

4

12

24

123

237

T3

(0.050)

R1

4

13

23

124

248

R2

5

13

22

124

254

R3

5

14

25

125

261

T4

(0.158)

R1

6

14

24

126

265

R2

5

13

25

127

269

R3

6

15

24

125

275

T5

(0.501)

R1

5

14

25

128

268

R2

8

16

26

130

283

R3

7

15

27

129

274

PC

R1

148

502

1544

1376

1590

R2

164

428

1608

1392

1648

R3

167

474

1428

1352

1674

 

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

108

237

R2

5

9

17

116

228

R3

4

12

22

110

240

VC

(0.00)

R1

8

16

27

128

290

R2

7

14

24

130

264

R3

6

15

28

122

282

T1

(0.005)

R1

5

11

21

109

234

R2

4

10

20

113

236

R3

5

11

19

114

240

T2

(0.016)

R1

6

10

22

119

244

R2

5

11

22

117

241

R3

4

12

21

116

247

T3

(0.050)

R1

5

13

18

119

260

R2

6

12

21

120

251

R3

5

11

24

118

247

T4

(0.158)

R1

6

13

22

121

271

R2

6

12

23

123

259

R3

5

14

24

119

263

T5

(0.501)

R1

6

13

25

124

274

R2

8

15

24

125

269

R3

6

14

24

122

277

PC

R1

158

1016

956

1320

1672

R2

172

1200

972

1236

1548

R3

164

1158

1012

1448

1624

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

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

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

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

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

10.33

1.15

18.33

3.21

113.67

3.51

236.67

7.51

VC

(0.00)

7.33

0.58

15.33

0.58

27.67

1.53

131.67

3.51

288.67

9.24

T1

(0.005)

4.00

0.00

11.00

1.00

20.33

0.58

118.00

2.65

237.00

2.65

T2

(0.016)

4.33

0.58

12.00

1.00

22.33

1.53

121.67

1.53

242.00

5.57

T3

(0.050)

4.67

0.58

13.33

0.58

23.33

1.53

124.33

0.58

254.33

6.51

T4

(0.158)

5.67

0.58

14.00

1.00

24.33

0.58

126.00

1.00

269.67

5.03

T5

(0.501)

6.67

1.53

15.00

1.00

26.00

1.00

129.00

1.00

275.00

7.55

PC

159.67

10.21

468.00

37.36

1526.67

91.24

1373.33

20.13

1637.33

43.00

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.33

0.58

10.33

1.53

19.67

2.52

111.33

4.16

235.00

6.24

VC

(0.00)

7.00

1.00

15.00

1.00

26.33

2.08

126.67

4.16

278.67

13.32

T1

(0.005)

4.67

0.58

10.67

0.58

20.00

1.00

112.00

2.65

236.67

3.06

T2

(0.016)

5.00

1.00

11.00

1.00

21.67

0.58

117.33

1.53

244.00

3.00

T3

(0.050)

5.33

0.58

12.00

1.00

21.00

3.00

119.00

1.00

252.67

6.66

T4

(0.158)

5.67

0.58

13.00

1.00

23.00

1.00

121.00

2.00

264.33

6.11

T5

(0.501)

6.67

1.15

14.00

1.00

24.33

0.58

123.67

1.53

273.33

4.04

PC

164.67

7.02

1124.67

96.42

980.00

28.84

1334.67

106.76

1614.67

62.52

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

PC= Positive control

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

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

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

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

 

 


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

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

4.00

1.00

10.33

0.58

15.67

0.58

90.33

5.51

243.00

7.00

VC

(0.00)

7.33

0.58

15.00

1.00

27.67

0.58

114.67

3.51

279.67

4.73

T1

(0.005)

4.67

1.15

10.67

1.15

16.00

1.00

93.67

7.57

247.33

6.11

T2

(0.016)

5.00

0.00

12.00

1.00

16.67

1.53

100.00

2.65

254.67

6.11

T3

(0.050)

5.67

0.58

11.67

0.58

19.00

2.65

104.67

6.03

262.33

6.11

T4

(0.158)

6.67

0.58

13.00

1.00

23.00

2.00

107.33

4.51

271.33

5.51

T5

(0.501)

7.00

0.00

14.67

0.58

26.33

0.58

109.67

7.02

278.00

2.65

PC

155.33

5.69

390.67

28.10

1118.00

24.98

1382.67

42.39

1426.00

36.17

 

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

0.00

10.67

1.53

15.33

1.53

90.00

6.00

236.33

4.04

VC

(0.00)

7.33

0.58

15.33

0.58

26.33

1.53

115.00

5.57

281.00

7.00

T1

(0.005)

4.33

0.58

11.00

1.00

15.67

0.58

94.67

5.51

239.00

5.00

T2

(0.016)

4.67

0.58

12.00

1.00

16.33

1.53

99.33

2.52

247.00

6.00

T3

(0.050)

5.33

0.58

13.00

1.00

20.00

1.00

104.00

1.00

252.00

9.54

T4

(0.158)

6.33

0.58

13.67

1.53

23.00

1.00

107.00

3.00

264.67

11.50

T5

(0.501)

7.00

1.00

15.00

1.00

26.00

1.00

110.67

4.04

279.67

5.13

PC

162.67

7.77

1424.00

44.54

801.33

28.38

1274.67

33.31

1593.33

46.01

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, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, 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.

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.

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
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: 27-32 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:
Type and composition of metabolic activation system: S9 metabolic activation system
Test concentrations with justification for top dose:
0.00, 0.004, 0.008 and 0.016 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
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 22-25 hrs (without metabolic activation system)
- Expression time: 16-21 hrs (with and without metabolic activation system- Phase I and II)
- Selection time (if incubation with a selection agent):No data
- Fixation time (start of exposure up to fixation or harvest of cells): 21-25 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: 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 per cytotoxicity experiment were selected. Since there was cytotoxicity observed in the experiment I (0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL) and II (0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL), experiment III is conducted with further lower concentrations (0.004 (T7), 0.008 (T8) and 0.016 (T9) 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 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
Rationale for test conditions:
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
Key result
Species / strain:
lymphocytes: Human perpheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item.
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 slight precipitation observed at 1 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. Cytotoxicity was assessed at the concentrations of 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL at initial cytotoxicity experiment (Experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (Experiment II) was conducted with 0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (Experiment III) was conducted with further lower concentrations of 0.004 (T7), 0.008 (T8) and 0.016 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.04 (NC), 9.83 (VC), 8.40 (T7), 6.43 (T8), 5.19 (T9) and 8.53 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.98 (NC), 9.94 (VC), 8.24 (T7), 6.63 (T8), 5.10 (T9) and 8.54 (PC).

In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.016 was selected as highest concentration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.

Hence the concentrations selected for the main study are 0.004, 0.008 and 0.016 mg/mL. The main study was performed in two independent phases

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

Cytotoxicity results:

                

Before conducting the chromosomal aberration study, the test chemical was evaluated for cytotoxicity both in the absence and presence of metabolic activation system (1%). Cytotoxicity was assessed at the concentrations of 0.25 (T1), 0.5 (T2) and 1.0 (T3) mg/mL at initial cytotoxicity experiment (Experiment I). All the tested concentrations at intial cytotoxicity experiment were cytotoxic. A second cytotoxicity experiment (Experiment II) was conducted with 0.031 (T4), 0.0625 (T5) and 0.125 (T6) mg/mL of culture media. In second cytotoxicity experiment all tested concentrations were cytotoxic.

Hence one more cytotoxicity experiment (Experiment III) was conducted with further lower concentrations of 0.004 (T7), 0.008 (T8) and 0.016 (T9) mg/mL of culture media. In the absence of S9 mix, the mean mitotic index observed was 10.04 (NC), 9.83 (VC), 8.40 (T7), 6.43 (T8), 5.19 (T9) and 8.53 (PC). In the presence of S9 mix, the mean mitotic index observed was 9.98 (NC), 9.94 (VC), 8.24 (T7), 6.63 (T8), 5.10 (T9) and 8.54 (PC).

In the cytotoxicity experiment III the highest test concentration 0.016 (T9) mg/ mL of culture media show 47.16 % reduction in absence of metabolic activation and 48.69% in the presence of metabolic activation indicates slight cytotoxicity of test item. Hence 0.016 was selected as highest concentration for main study considering the selection of test concentrations upto cytotoxicity. The mitotic index when compared to the respective vehicle control both in the presence or absence of metabolic activation.

Hence the concentrations selected for the main study are 0.004, 0.008 and 0.016 mg/mL. The main study was performed in two independent phases

Phase 1 results:           

In the experiment, the cultures were exposed to the test chemical 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) 0.667 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the absence of metabolic activation and 0.667 (NC), 0.333 (VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.004 (T1), 0.008 (T2) and 0.016(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 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 noreduction in mitotic index observed at the tested concentrations.The observed mean mitotic index in the absence of metabolic activation were 10.03, 9.95, 8.75, 6.05, 5.50 and 8.63 and in the presence of metabolic activation were 10.13, 9.89, 8.34, 6.39, 5.04 and 8.64 for NC, VC, T1, T2, T3 and PC concentrations respectively.

Phase 2 results:

            

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 0.004 (T1), 0.008 (T2) and 0.016 (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), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 10.000 (PC) in the absence of metabolic activation and 0.333 (NC), 0.333(VC), 0.667 (T1), 0.667 (T2), 0.667 (T3) and 10.333 (PC) in the presence of metabolic activation at the concentration of 0.00 (NC), 0.00 (VC), 0.004 (T1), 0.008 (T2) and 0.016 (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 index in the absence of metabolic activation were 10.11, 9.95, 8.84, 6.44, 5.05 and 8.44 and in the presence of metabolic activation were 10.06, 9.94, 8.55, 6.73, 5.14 and 8.59 for NC, T1, T2, T3 and PC concentrations respectively.

Conclusions:
The test chemical is not mutagenic at the highest tested concentration of 0.016 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions.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 29th July 2016 “In Vitro Mammalian Chromosome Aberration Test.Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. 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 ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 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 (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} 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 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. The test chemical is not mutagenic at the highest tested concentration of 0.016 mg/ml both in the presence (1% and 2%) and in the absence of metabolic activation under the specified conditions. 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:
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 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Mammalian cell gene mutation study was performed to determine the mutagenic nature of the test chemical
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-guanine phosphoribosyl transferase (HGPRT)
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Cell line used: Chinese Hamster Ovary (CHO) cells- Type and identity of media: Ham's F-12K (Kaighn's) Medium containing 2 mM L-Glutamine supplemented with 10% Fetal Bovine Serum and 1% Penicillin-Streptomycin (10,000 U/mL).- Properly maintained: Yes- Periodically checked for Mycoplasma contamination: Not applicable- Periodically checked for karyotype stability: Not applicable
Additional strain / cell type characteristics:
other: Hypodiploid, modal No. 20
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
: S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany).
- method of preparation of S9 mix
- concentration or volume of S9 mix and S9 in the final culture medium
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
Test concentrations with justification for top dose:
0, 0.5, 1, 5 or 10 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Ethanol
Justification for choice of solvent/ vehicle:
Justification of Ethanol as a solvent and vehicle:
1) The test chemical was not soluble in water or PBS, and therefore was ethanol chosen as a solvent instead.
2) When neither water nor PBS can be used, ethanol is a commonly used instead in biological applications as a solvent to dissolve test chemicals. Previous studies has shown that ethanol is neither genotoxic nor cytotoxic when used at low to moderate treatment concentrations. Since we use a limit concentration of maximum 1% of ethanol in our studies, ethanol is regarded to be non-toxic, non-genotoxic and thus does not affect our results in the study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9,10-dimethylbenzanthracene
other: N-ethyl-N-nitrosourea (ENU) (2.5 mM) was used as positive control substance in experiments without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: In medium with pre-incubation

DURATION
- Pre-incubation: One week involving 3 days of incubation with Hypoxanthine-aminopterin-thymidine (HAT) in medium as a mutant cleansing stage, followed by overnight incubation with hypoxanthine-thymidine (HT) in medium prior to a 3-4 days incubation in regular cell medium. After seeding and prior to treatment, the mutant-free cells were incubated for an additional of 24 hours.
- Exposure duration: 3 hours
- Expression time: 7 days
- Selection time: 14 days
- Fixation time: 7 days (harvest of cells)

SELECTION AGENT (mutation assays): 6-thioguanine (TG)
6-Thioguanine (TG; Sigma-Aldrich Sweden AB, Stockholm, Sweden) was dissolved in 0.5% Na2CO3 solution to a stock solution of 2 mM. The solution is then sterile filtered and stored at -20°C.

STAIN (for cytogenetic assays): Crystal violet

NUMBER OF REPLICATIONS: A minimum of 2 replicates per dose concentration including negative and positive control.

NUMBER OF CELLS EVALUATED: 5 x 10 E5 cells were plated 7 days after treatment and whatever cells left, after 14 days of incubation with the selection medium, were evaluated.

DETERMINATION OF CYTOTOXICITY

Cells (0.5 x 10-5 per well) were seeded in fresh medium (2 ml per well) in 6 well-plates and were incubated overnight at 37°C, 5% CO2. On the day of exposure, the test chemical added to each applicable well to give a final concentrations of 0, 0.5, 1.0, 5.0 or 10.0 mM and in the absence or 4% (80 microliter) presence of S9-induced metabolic activation. Negative controls (cell medium), solvent/vehicle controls (PBS) and positive control substances were also included in each experiment. ENU was used as positive control substance in experiments without metabolic activation, while 7,12-dimethylbenz(a) anthracene was used in experiments with metabolic activation. During the exposure period, treated cultures were incubated at 37°C, 5% CO2.
After the 3 hour exposure period, cells were washed several times with sterile PBS to remove the test chemical. Cells were then trypsinized and re-seeded in duplicates from two parallel duplicate cultures into new 6-well plates, containing fresh medium, in the concentration of 0.5 x 10-5 cells per well. The cultures were then incubated for 24 and 48 hrs at 37°C, 5% CO2. The relative total growth and cytotoxicity was evaluated 24 and 48 hrs after seeding by counting in a haemocytometer.
A minimum of 2 replicates per dose concentration, including negative and positive control, were performed for each experiment.

OTHER: PRELIMINARY DOSE-FINDING/TOXICITY TEST

Initial tests of dose-finding was completed without S9-induced metabolic activation. A range of test concentrations (0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 or 1.0 mM) was applied at a volume of 20 microliter per applicable well 24 hrs after seeding to single cultures (4 x 104 cells per well) in fresh medium (180 microliter per well) in 96-well plates. The cell population (control and treated cells) were assessed 24 and 48 hrs after treatment using the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and the bicinchoninic acid (BCA) assay to assess cell viability and protein concentration, respectively.
In the MTT assay, 20 microliter of MTT solution was added to each well and the 96-well plates were returned to the incubator for 1 hour. After 1 hour, the MTT solution was removed by gently pouring out the fluid from the plate onto a paper cloth. Each well was washed gently with two times of 100 microliter of PBS. The blue formazan product was dissolved by addition of 100 microliter of 100% DMSO per well. The plates were swirled gently for 10 min. to dissolve the precipitate. Absorbance was monitored at 540 nm using a SpectraMax M2 Microplate Reader and the software SoftMax Pro® (Molecular Devices, Sunnyvale, CA, USA).
After 24 and 48 hrs of treatment, the medium of the 96-well plates destined for analyzing protein content was removed by gently pouring out the fluid from the plate onto a paper cloth. Each well was washed gently with two times of 100 microliters of PBS before 50 microliter of Mammalian protein extraction reagent (M-PER) solution was added to each well. The plates were swirled gently overnight before 25 microliteres of the cell lysate were transferred to a new 96-well plate together with 25 l bovine serum albumin (25 microliter of 5-750 microgram/ml in M-PER) as a standard and with 25 microliterof M-PER as a blank. A solution, containing 50 part of BCA Reagent A (containing sodium carbonate, sodium bicarbonate, bicinchoninic acid and sodium tartrate in 0.1 M sodium hydroxide) and 1 part of BCA Reagent B (containing 4% cupric sulfate), was added to each well and the plates were mixed thoroughly on a plate shaker for 30 sec. The plates were then covered and incubated for 30 min. at 37°C. After cooling the plate to RT, absorbance was measured at 562 nm using a Multiskan™ FC Microplate Photometer and the software ScanIt™ (Thermo Fisher Scientific, Waltham, MA, USA.
A minimum of 6 replicates per dose concentration from 3 independent cultures was performed for each experiment. Results obtained by the two cytotoxicity assays were expressed as percentage of controls. From the basis of the results from the MTT and BCA assays, test concentrations of the chemical was chosen to be included in the gene toxicity test
Rationale for test conditions:
No data available
Evaluation criteria:
The number of mutant colonies were counted and compared to the negative and positive controls. The mutation frequency was calculated.
Statistics:
The Standard deviation (± SD) was calculated.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
PRELIMINARY DOSE-FINDING/TOXICITY TEST

All tests of the preliminary dose-finding study was completed without S9-induced metabolic activation. The test chemical was added to the cells in the test concentrations (0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 or 1.0 mM) and the MTT and BCA assays were used to evaluate the chemical effects on cell viability and total protein concentration, respectively. Based on the results from the MTT and BCA assays, revised test concentrations of the chemical may be chosen to be included in the gene toxicity test. pH and osmolality was not determined in the preliminary dose-finding/toxicity test.
At 24 and 48 hrs, the test chemical showed no evidence of cytotoxic effects at any of the tested concentrations. Similar results were seen for the BCA assay. Since no cytotoxicity was observed in the MTT and BCA assay after 24 and 48 hrs of treatment in this preliminary dose-finding test, further testing concentrations were revised and adapted to have a maximum test concentration of 10 mM when cells should be exposed to the test chemical for 3 hrs. In addition, since the test chemical could only be dissolved in ethanol, higher test concentrations of the test chemical than 10 mM could result in a toxic effect of ethanol.

Cytotoxicity test
In similarity to the Gene mutation test, the test chemical was added to each applicable well, and in the absence or presence of S9 liver microsomal fraction, to give a final concentration of 0, 0.5, 1.0, 5.0 or 10.0 mM. Negative controls, solvent/vehicle controls and positive control substance(s) were also included in each experiment. pH and osmolality was not determined in the gene mutation test.
The test chemical, in the above mentioned concentrations, did not cause any cytotoxic effects when CHO cells were exposed to the test chemical for 3 hrs. Thus, we conclude that the test chemical does not induce cytotoxic effects at concentrations ≤ 10.0 mM.


STUDY RESULTS
- Concurrent vehicle negative and positive control data: The positive control 7,12-dimethylbenz(a) anthracene showed occurrence of genetric mutation as well as ENU (as a positive control in the absence of S9 liver microsomal fraction) gave a clear indication of occurring gene mutations. When the mutation frequency was determined,a frequency of 5.23 x 10-4 was shown after a 3 hour exposure of 7,12-dimethylbenz(a) anthracene in the presence of 4% S9 liver microsomal fraction, and 3.28 x 10-4 for ENU as the positive control and in the absence of S9 liver microsomal fraction
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: The test chemical in the above mentioned concentrations, did not cause any cytotoxic effects when CHO cells were exposed to the test chemical for 3 hrs.
o Relative total growth (RTG) or relative survival (RS) and cloning efficiency

- Genotoxicity results:No genotoxicity was observed at 0.5, 1.0, 5.0 or 10.0 mM. Since no tested concentration of the test chemical in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that the test chemical does not give rise to gene mutations when CHO cells were exposed in vitro to the test chemical at 0, 0.5, 1.0, 5.0 or 10.0 mM for 3 hrs.
Remarks on result:
other: No mutagenic potential

GENE MUTATION TEST

 

Table 1A.

Effect of test chemical exposure on gene toxicity in CHO cells. After being exposed to the test chemical for 3 hrs, cells was washed with sterile PBS and then incubated for 7 days at 37°C, 5% CO2. After 7 days, cells were re-seeded in new 6-well plates in the absence or presence of 10mM TG as a selection agent and returned to the incubator for 14 days at 37°C, 5% CO2. On day 15, all 6-well plates were stained with crystal violet and the number of colonies were counted manually. The results are presented as the total number of colonies found in the number of independent wells analyzed (e.g. 0 colonies in 4 wells will give 0/4) (n = 2 samples from 2 independent cultures).

 

 

With S9

Without S9

 

with TG

without TG

with TG

without TG

Neg. control

0/4

229/4

0/4

258/4

Pos. control

1/4

241/4

17/4

330/4

0.5 mM

0/4

267/4

0/4

253/4

1.0 mM

0/4

232/4

0/4

249/4

5.0 mM

0/4

225/4

0/4

224/4

10.0 mM

0/4

210/4

0/4

244/4

 

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to the test chemical in the absence or presence of 4% S9 liver microsomal fraction. N/A, no colonies present in the samples selected with TG, i.e. no mutation frequency could be determined.

 

 

With S9

Without S9

Neg. control

N/A

N/A

Pos. control

-3.38 x10-4

3.28x10-4

0.5 mM

N/A

N/A

1.0 mM

N/A

N/A

5.0 mM

N/A

N/A

10.0 mM

N/A

N/A

PRELIMINARY DOSE-FINDING/TOXICITY TEST - SEEDING OF CELLS

Methods: MTT & BCA

Cell line: CHO

Passage: 15

Day of seeding: July 08, 2014

 

 

Flask 1

Flask 2

Flask 3

Number of cells in cell culture flask (cells/ml)

 

0.693 x 106

 

0.555 x 106

0.563 x 106

Total number of cells in cell culture flask

 

13.8 x 106

 

11.1 x 106

11.3 x 106

Number of cells to be seeded per well (in 96-well plate)

 

3200

 

3200

3200

Dilution(s) made to create a cell suspension with 3200 cells per 180ml

 

2.6 ml cell suspension + 77.4 ml cell medium

 

3.2 ml cell suspension + 76.8 ml cell medium

3.2 ml cell suspension + 76.8 ml cell medium

 

PRELIMINARY DOSE-FINDING/TOXICITY TEST - EXPOSURE

Methods: MTT & BCA

Concentrations tested: 0 (vehicle control), 0.005, 0.01, 0.025, 0.05, 0.1, 0.25, 0.5 or 1.0 mM

Volume added to each well: 20ml

Day of treatment: July 09, 2014

 

Tested substance was added according to the following treatment schedule (with all concentrations in mM):

 

 

1

2

3

4

5

6

7

8

9

10

11

12

A

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

B

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

C

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

D

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

E

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

F

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

G

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

H

Medium only

0

0

0.005

0.01

0.025

0.05

0.1

0.25

0.5

1

Medium only

 

 

MAIN TEST/GENE TOXICITY TEST – SEEDING OF CELLS

 

CYTOTOXICITY & GENE TOXICITY

Method: OECD 476

Application: Cytotoxicity & Gene toxicity

Cell line: CHO

Passage at experiment initiation: 12

Concentrations tested: 0 (vehicle control), 0.5, 1.0, 5.0 and 10.0 mM

Day of seeding for HPRT mutation cleansing: September 29, 2014

Day of seeding prior chemical exposure: October 07, 2014

Day of chemical exposure: October 08, 2014

 

 

Flask 1

Flask 2

Number of cells in cell culture flask (cells/ml)

 

4.18 x 106

 

3.48 x 106

Total number of cells in cell culture flask

 

18.81 x 106

 

15.66 x 106

Number of cells to be seeded per well (for cytotoxicity)

 

5.0 x 104

 

5.0 x 105

Dilution(s) made to create a cell suspension with 2.0 x 105cells per 2 ml for cytotoxicity

 

165ml cell suspension + 13.55 ml cell medium

 

198ml cell suspension + 13.56 ml cell medium

Number of cells to be seeded per well (for gene toxicity)

 

5.0 x 104

 

5.0 x 104

Dilution(s) made to create a cell suspension with 5.0 x 104cells per 2 ml for gene toxicity

 

165ml cell suspension + 13.55 ml cell medium

 

 

198ml cell suspension + 13.56 ml cell medium

 

MAIN TEST/GENE TOXICITY TEST – CYTOTOXICITY DATA

 

RE-SEEDING AFTER EXPOSURE

Method: OECD 476

Application: Cytotoxicity

Passage when re-seeding cells for cytotoxicity: 14

Concentrations tested: 0 (vehicle control), 0.5, 1.0, 5.0 and 10.0 mM

Day of re-seeding cells for cytotoxicity testing: October 08, 2014

Number of cells to be re-seeded per well (24 h and 48 h): 5.0 x 104

 

Total number of cells (x 106) per well after 3 hrs of 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol exposure without or with metabolic activation (S9) and prior to re-seeding. A haemocytometer was used for identifying the total number of cells (x 106) per well:

 

 

Flask 1

Flask 2

 

Without S9

With S9

Without S9

With S9

Neg. control

0.093

0.199

0.097

0.173

Pos. control

0.079

0.150

0.073

0.150

0.5 mM

0.066a

0.136

0.097

0.143

1.0 mM

0.066b

0.160

0.047c

0.079

5.0 mM

0.073

0.203

0.057d

0.126

10.0 mM

0.073

0.146

0.057e

0.129

 

a)Since the number of cells was low in this sample, 750ml (corresponding to 16650 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.

b)Since the number of cells was low in this sample, 750ml (corresponding to 16650 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.

c)Since the number of cells was low in this sample, 750ml (corresponding to 11625 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.

d)Since the number of cells was low in this sample, 750ml (corresponding to 14100 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.

e)Since the number of cells was low in this sample, 750ml (corresponding to 14100 cells) was re-seeded to each well applicable for the upcoming 24 and 48 hour analysis.

 

 

 

CYTOTOXICTY AFTER 24 & 48 HOURS

Study number: SSS/LR/01

Study plan number: SSS/08-1 & SSS/08-2

Method: OECD 476

Application: Cytotoxicity

Tested substance: 3-(5,5,6-trimethylbicyclo[2.2.1]hept-2-yl)cyclohexan-1-ol

Concentrations tested: 0 (vehicle control), 0.5, 1.0, 5.0 and 10.0 mM

Day for 24 h analysis of cytotoxicity: October 09, 2014

Day for 48 h analysis of cytotoxicity: October 10, 2014

 

Raw data of cytotoxicity at 24 hours after chemical exposure and re-seeding using haemocytometer for identifying the total number of cells (x 106) per well (please see overleaf):

 

Without metabolic activation

 

Flask 1

Flask 2

 

Sample 1

Sample 2

Sample 1

Sample 2

Neg. control

0.0689

0.0533

0.0444

0.0400

Pos. control

0.0467

0.0422

0.0267

0.0222

0.5 mM

0.0020

0.0222

0.0356

0.0267

1.0 mM

0.0489

0.0422

0.0400

0.0289

5.0 mM

0.0311

0.0311

0.0222

0.0178

10.0 mM

0.0244

0.0244

0.0222

0.0178

With metabolic activation

 

Flask 1

Flask 2

 

Sample 1

Sample 2

Sample 1

Sample 2

Neg. control

0.0356

0.0289

0.0178

0.0222

Pos. control

0.0311

0.0267

0.0267

0.0200

0.5 mM

0.0156

0.0111

0.0089

0.0067

1.0 mM

0.0244

0.0222

0.0178

0.0133

5.0 mM

0.0244

0.0222

0.0200

0.0178

10.0 mM

0.0244

0.0178

0.0333

0.0289

 

 

Raw data of cytotoxicity at 48 hours after chemical exposure and re-seeding using haemocytometer for identifying the number of cells per well:

 

Without metabolic activation

 

Flask 1

Flask 2

 

Sample 1

Sample 2

Sample 1

Sample 2

Neg. control

0.0956

0.1111

0.1222

0.1089

Pos. control

0.0933

0.0844

0.0600

0.0756

0.5 mM

0.0422

0.0467

0.0689

0.0756

1.0 mM

0.1133

0.1133

0.0756

0.0756

5.0 mM

0.0911

0.0889

0.1066

0.0933

10.0 mM

0.0978

0.0911

0.0511

0.0511

 

With metabolic activation

 

Flask 1

Flask 2

 

Sample 1

Sample 2

Sample 1

Sample 2

Neg. control

0.0978

0.0956

0.0400

0.0400

Pos. control

0.0778

0.0911

0.0422

0.0422

0.5 mM

0.0177

0.0156

0.0156

0.0133

1.0 mM

0.0533

0.0467

0.0222

0.0311

5.0 mM

0.0333

0.0356

0.0556

0.0578

10.0 mM

0.0398

0.0422

0.0911

0.0822

Conclusions:
The test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 10.0 mM for 3 hrs or more, and the test chemical does not induce cytotoxic effects at concentrations ≤ 10.0 mM.Hence, the test chemical can be considered to be non mutagenic to CHO cells.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. The study was performed according to OECD 476 Guidelines. A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the bicinchoninic acid(BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test.In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.The results showed an indication of gene mutation in the positive control 7,12-dimethylbenz(a) anthracene (in the presence of S9 liver microsomal fraction) as well as in the positive control ENU (in the absence of S9 liver microsomal fraction).The test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 10.0 mM for 3 hrs or more, and the test chemical does not induce cytotoxic effects at concentrations ≤ 10.0 mM. Hence, the test chemical can be considered to be non mutagenic to CHO cells in the presence and absence of metabolic activation.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data available for the target chemical was reviewed to determine the mutagenic nature of the 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 theSalmonella typhimuriumstrains 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, 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5.0 mg/plates were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0, 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, 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 chromosome 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. Blood samples were obtained by vein puncture using syringe from healthy donor (non smoker, non alcoholic) not receiving medication for at least 3 months and being in the range of 27-32 years age. Samples were collected in heparinized vials. 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 ethanol and used at dose level of 0, 0.004, 0.008 and 0.016 mg/mL 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 (0.004, 0.008 and 0.016 mg/mL {cytotoxicity experiment 3} 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 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. Based on the observations made, the test chemical is at the highest tested concentration of 0.016 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 non-mutagenic nature of the test chemical is further supported by the in vitro mammalian cell gene mutation assay. These include experimental studies performed on CHO cell lines and Mouse lymphoma cell line for the test chemical.

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the test chemical when administered to Chinese Hamster Ovary (CHO) cells. The study was performed according to OECD 476 Guidelines.A preliminary dose-finding study was conducted prior to the main study. A range of different concentrations of the test chemical were tested in 96-well plates and analyzed by two commonly used assays, i.e. the colorimetric assay of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)and the bicinchoninic acid(BCA) assay to assess cell viability and protein concentration, respectively. From the basis of the results from the MTT and BCA assays, test concentrations of the test chemical was chosen to be included in the gene toxicity test. In the genotoxicity test, the test chemical was administered to CHO cells for 3 hrs at the dose levels of 0, 0.5, 1.0, 5.0 or 10.0 mM and in the absence or presence of exogenous metabolic activation. CHO cells representing the negative controls were exposed to the vehicle. Positive controls, such as N-ethyl-N-nitrosourea (ENU) experiments without metabolic activation and 7,12-dimethylbenz(a) anthracene in experiments with metabolic activation, were also included in each test.The results showed an indication of gene mutation in the positive control 7,12-dimethylbenz(a) anthracene (in the presence of S9 liver microsomal fraction) as well as in the positive control ENU (in the absence of S9 liver microsomal fraction). The test chemical does not give rise to gene mutations when exposed to the test chemical at ≤ 10.0 mM for 3 hrs or more, and the test chemical does not induce cytotoxic effects at concentrations ≤ 10.0 mM.Hence, the test chemical can be considered to be non mutagenic to CHO cells in the presence and absence of metabolic activation.

This result is supported by a study performed using L5178Y mouse lymphoma cells to evaluate the mutagenicity of the test chemical.L5178Y mouse lymphoma cells, clone 3.7.2C, were obtained directly from Dr. D. Clive. S9 was prepared from the livers of Aroclor 1254-induced male Fischer 344 rats.Replicate positive control cultures and vehicle (solvent) control cultures were included with each experiment. In experiments without S9 mix (designated non-activation), the positive control was 5 nl/ml of MMS.MCA requires activation by S9 mix to become mutagenic to L5178Y cells, and this chemical was used at 2.5 microgram/ml in all activation experiments employing S9 from Aroclor-induced rats. Preliminary studies of test chemical solubility and cytotoxicity were conducted prior to performing the first mutation. But, unless otherwise noted, no pH adjustments were made. Test chemical toxicity to 24-hr cell suspension growth was determined for 4-hr treatments with a range of doses up to a maximum of 5,000 microgram/ml for soluble chemicals.Each mutation experiment usually consisted of four solvent controls, three positive controls, and five or six test chemical concentrations in duplicate or triplicate cultures. The test chemical was tested without S9 mix (non-activation trials), and, if no clear mutagenic response was obtained, the chemical was retested in the presence of S9 mix. At least two experiments (trials) were performed with and/or without S9 mix, and additional trials were performed to help resolve conflicting or indeterminate responses.The test chemical was usually lethal at 200 microgram/ml in the absence or presence of S9 mix, and 150 microgram/ml caused average RTG values that ranged from 24% to 27% without S9 and 52% to 94% with S9. Thus S9 tended to reduce the toxicity of non-lethal concentrations of the test chemical. No evidence for mutagenicity was obtained. In non-activation experiment Trial 2, a highly toxic (10% RTG) treatment of one culture with 200 microgram/ml caused no change in the MF. Based on all these observations, the test chemical was evaluated to be not mutagenic to L5178Y mouse lymphoma cells.

The above mouse lymphoma result is further supported by a similar assay performed to determine the mutagenic potential of the test chemical.          

L5178Y TK +/- mouse lymphoma cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Company, Research Triangle Park, NC. The cells were grown in Fischer's medium for leukemic cells of mice supplemented with 10% horse serum, antibiotics (50 U penicillin/mi and 50 /~g streptomycin/ml), and 0.02% Pluronic F-68. All serum lots were pre-screened for their ability to support optimal growth. The cells were checked for the presence of mycoplasma by agar block isolation and Hoechst staining before and after cryopreservation.The toxicity of each chemical was first determined both with and without S9 prepared from Aroclor-1254-induced male Fischer 344 rats. S9 mix was prepared according to the procedure of Clive et al. (1979). Cells at a concentration of 6 × 105 microgram/ml were exposed for 4 h to a range of concentrations from 0.0 to 10000 microgram/ml or the limit of solubility. The cells were then washed, resuspended in growth medium, and incubated at 37 °C for 48 h. The rate of cell growth was determined for each of the treated cultures and compared to the rate of growth of the solvent controls. The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity. For each assay there was a solvent control, a positive control for the test without metabolic activation and for the test with metabolic activation. The maximum solvent concentration was 1% for organic solvents and 10% for water. These levels had no effects on cell growth or spontaneous mutation frequency.The mutagenicity assay was performed according to the procedure described by Clive and Spector (1975). Cells in duplicate cultures were exposed to the test chemical, positive control, and solvent control for 4 h at 37°C; washed twice with growth medium; and maintained at 37 °C for 48 h in log phase growth to allow recovery and mutant expression. The cultures were adjusted to 0.3 × 106 cells/ml at 24-h intervals. They were then cloned in soft agar medium containing Fischer's medium, 20% horse serum, 2 mM sodium pyruvate, 0.02% Pluronic F-68 and 0.35% Noble agar. Resistance to trifluorothymidine (TFT) was determined by adding 3 microgram/ml TFT to one set of plates. The 100 × stock solution of TFT in saline was stored at -70°C and thawed immediately before use. Plates were incubated at 37°C in 5% CO 2 in air for 12 days, and then counted with an automatic colony counter. Mutant frequencies were expressed as mutants per 104 surviving cells. In general, a response was considered positive if there was a dose-related increase in the mutant frequency above the spontaneous control frequency, with a 2-fold increase at more than 1 dose and relative total growth greater than 10%.The results in all the assays with test chemical were negative. Hence, the test chemical can be considered to be non mutagenic to L5178Y TK +/- mouse lymphoma cells.

The results in all the assays with test chemical were negative. Hence, the test chemical can be considered to be non mutagenic to when tested in vitro in mammalian cells.

Justification for classification or non-classification

Based on the data available, the test chemical does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.