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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria

Based on the available results and applying the weight of evidence approach the test chemical can be considered to be non-mutagenic to 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.

 

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17-04-2018 - 10-05-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report.
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
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. 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.
- source of S9
- 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 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 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)]: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
RO water
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-Nitro-o-phenylenediamine (TA 1537, TA 98, without S9); 2-Aminoanthracene (TA 1535, TA 1537, TA 98, TA 100 and TA 102, with S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation- Trial I); preincubation (Trial II)

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

SELECTION AGENT (mutation assays): No data

SPINDLE INHIBITOR (cytogenetic assays): No data

STAIN (for cytogenetic assays): No data

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

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Not applicable

NUMBER OF CELLS EVALUATED: No data

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

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: No data

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

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

- OTHER: No data
Rationale for test conditions:
No data
Evaluation criteria:
A test item is considered as a mutagen, if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100 and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding vehicle/solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative control and vehicle control such an increase is not considered biologically relevant.
Statistics:
No data
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 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. In TA 98 and TA 100, cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate (T7 to T8), moderate inhibition was observed in the treated concentrations 0.501 mg/plate (T6) and there was no reduction in colony count as well as background lawn in any of the following concentrations tested; 0.002, 0.005, 0.016, 0.050 and 0.158 ( T1 to T5) mg/plate both in absence and in the presence of metabolic activation, when compared to that of the negative control group. Based on the results of pre-experiment following doses were selected for the main study trials: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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

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

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No data
- Negative (solvent/vehicle) historical control data: No data

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

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Without metabolic activation (-S9)

With metabolic activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

122

24

126

25

R2

118

20

119

20

R3

124

19

121

22

T1

(0.002)

R1

106

17

108

19

R2

110

19

112

17

R3

111

17

106

17

T2

(0.005)

R1

108

19

112

21

R2

102

20

108

23

R3

104

18

110

20

T3

(0.016)

R1

114

20

108

18

R2

106

22

110

16

R3

102

19

114

18

T4

(0.050)

R1

116

21

106

21

R2

112

17

114

23

R3

118

18

108

19

T5

(0.158)

R1

102

18

100

17

R2

98

17

96

18

R3

100

15

104

17

T6

(0.501)

R1

36 (+ + +)

2 (+ + +)

42 ( + + + )

4 (+ + +)

R2

26 (+ + +)

5 (+ + +)

34 (+ + +)

3 (+ + +)

R3

24 (+ + +)

3 (+ + +)

30 ( + + +)

3 (+ + +)

T7

(1.582)

R1

0 (+)

0 (+)

0 (+)

0 (+)

R2

0 (+)

0 (+)

0 (+)

0 (+)

R3

0 (+)

0 (+)

0 (+)

0 (+)

T8

(5)

R1

0 (+)

0 (+)

0 (+)

0 (+)

R2

0 (+)

0 (+)

0 (+)

0 (+)

R3

0 (+)

0 (+)

0 (+)

0 (+)

PC

R1

1088

960

1584

1242

R2

1136

992

1616

1180

R3

1168

1008

1600

1208

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

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

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

Sodium azide [10μg/plate]: TA 100,

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

 

 

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

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

16

25

126

280

R2

6

15

20

119

272

R3

8

14

22

121

260

T1

(0.002)

R1

4

13

19

108

242

R2

5

11

17

112

238

R3

5

10

17

106

232

T2

(0.005)

R1

5

12

21

112

240

R2

4

13

23

108

248

R3

4

13

20

110

252

T3

(0.016)

R1

6

14

18

108

256

R2

5

12

16

110

248

R3

4

13

18

114

264

T4

((0.050)

R1

6

15

21

106

260

R2

5

12

23

114

254

R3

5

14

19

108

268

T5

(0.158)

R1

6

14

17

100

270

R2

6

15

18

96

266

R3

5

14

17

104

258

PC

R1

168

480

1242

1584

1384

R2

186

452

1180

1616

1336

R3

170

492

1208

1600

1312

 

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

16

24

122

276

R2

6

14

20

118

264

R3

7

13

19

124

258

T1

(0.002)

R1

5

12

17

106

232

R2

4

14

19

110

240

R3

5

11

17

111

236

T2

(0.005)

R1

6

13

19

108

242

R2

4

15

20

102

238

R3

5

13

18

104

246

T3

(0.016)

R1

5

14

20

114

240

R2

5

14

22

106

252

R3

6

15

19

102

236

T4

((0.050)

R1

6

14

21

116

254

R2

5

12

17

112

250

R3

6

13

18

118

262

T5

(0.158)

R1

6

15

18

102

256

R2

6

15

17

98

260

R3

6

14

15

100

252

PC

R1

180

1320

960

1088

1824

R2

174

1272

992

1136

1840

R3

170

1344

1008

1168

1888

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

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

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

 

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

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

8

16

28

128

272

R2

6

14

25

123

258

R3

7

13

23

124

260

T1

(0.002)

R1

5

11

20

120

230

R2

4

10

20

118

238

R3

5

13

19

121

244

T2

(0.005)

R1

5

13

21

123

250

R2

4

12

23

120

246

R3

4

12

24

124

240

T3

(0.016)

R1

6

10

19

122

238

R2

5

14

25

119

246

R3

5

15

26

125

254

T4

((0.050)

R1

6

13

25

123

248

R2

6

12

24

124

256

R3

5

15

23

124

250

T5

(0.158)

R1

7

15

26

125

258

R2

6

14

22

123

264

R3

6

14

25

123

260

PC

R1

162

380

1344

1440

1680

R2

174

440

1360

1472

1704

R3

180

420

1384

1504

1712

 

 

Dose

(mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

15

26

126

260

R2

6

13

23

123

252

R3

7

13

22

120

248

T1

(0.002)

R1

4

10

21

108

232

R2

5

13

23

102

228

R3

5

11

19

104

236

T2

(0.005)

R1

4

14

18

106

234

R2

4

11

20

112

230

R3

4

12

23

110

242

T3

(0.016)

R1

5

13

24

114

248

R2

6

14

22

108

240

R3

4

12

19

111

252

T4

((0.050)

R1

6

13

23

114

250

R2

5

13

24

116

254

R3

5

14

20

120

246

T5

(0.158)

R1

6

14

24

123

258

R2

6

14

23

120

248

R3

5

13

25

121

250

PC

R1

180

1168

890

1248

1552

R2

178

1184

924

1280

1520

R3

182

1216

916

1304

1568

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

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

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

 

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

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

7.00

1.00

15.00

1.00

22.33

2.52

122.00

3.61

270.67

10.07

T1

(0.002)

4.67

0.58

11.33

1.53

17.67

1.15

108.67

3.06

237.33

5.03

T2

(0.005)

4.33

0.58

12.67

0.58

21.33

1.53

110.00

2.00

246.67

6.11

T3

(0.016)

5.00

1.00

13.00

1.00

17.33

1.15

110.67

3.06

256.00

7.02

T4

(0.050)

5.33

0.58

13.67

1.53

21.00

2.00

109.33

4.16

260.67

8.00

T5

(0.158)

5.67

0.58

14.33

0.58

17.33

0.58

100.00

4.00

264.67

6.11

PC

174.67

9.87

474.67

20.53

1210.00

31.05

1600.00

16.00

1344.00

36.66

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

6.67

0.58

14.33

1.53

21.00

2.65

121.33

3.06

266.00

9.17

T1

(0.002)

4.67

0.58

12.33

1.53

17.67

1.15

109.00

2.65

236.00

4.00

T2

(0.005)

5.00

1.00

13.67

1.15

19.00

1.00

104.67

3.06

242.00

4.00

T3

(0.016)

5.33

0.58

14.33

0.58

20.33

1.53

107.33

6.11

242.67

8.33

T4

(0.050)

5.67

0.58

13.00

1.00

18.67

2.08

115.33

3.06

255.33

6.11

T5

(0.158)

6.00

0.00

14.67

0.58

16.67

1.53

100.00

2.00

256.00

4.00

PC

174.67

5.03

1312.00

36.66

986.67

24.44

1130.67

40.27

1850.67

33.31

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

PC= Positive control

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

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

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

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

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-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)

7.00

1.00

14.33

1.53

25.33

2.52

125.00

2.65

263.33

7.57

T1

(0.002)

4.67

0.58

11.33

1.53

19.67

0.58

119.67

1.53

237.33

7.02

T2

(0.005)

4.33

0.58

12.33

0.58

22.67

1.53

122.33

2.08

245.33

5.03

T3

(0.016)

5.33

0.58

13.00

2.65

23.33

3.79

122.00

3.00

246.00

8.00

T4

(0.050)

5.67

0.58

13.33

1.53

24.00

1.00

123.67

0.58

251.33

4.16

T5

(0.158)

6.33

0.58

14.33

0.58

24.33

2.08

123.67

1.15

260.67

3.06

PC

172.00

9.17

413.33

30.55

1362.67

20.13

1472.00

32.00

1698.67

16.65

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

6.67

0.58

13.67

1.15

23.67

2.08

123.00

3.00

253.33

6.11

T1

(0.002)

4.67

0.58

11.33

1.53

21.00

2.00

104.67

3.06

232.00

4.00

T2

(0.005)

4.00

0.00

12.33

1.53

20.33

2.52

109.33

3.06

235.33

6.11

T3

(0.016)

5.00

1.00

13.00

1.00

21.67

2.52

111.00

3.00

246.67

6.11

T4

(0.050)

5.33

0.58

13.33

0.58

22.33

2.08

116.67

3.06

250.00

4.00

T5

(0.158)

5.67

0.58

13.67

0.58

24.00

1.00

121.33

1.53

252.00

5.29

PC

180.00

2.00

1189.33

24.44

910.00

17.78

1277.33

28.10

1546.67

24.44

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

PC= Positive control

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

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

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

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

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

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

Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using 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, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

 

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from a peer reviewed journal.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
To evaluate the mutagenic potential of the test chemical in bacterial cell lines
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
No Data Available
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
No Data Available
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Aroclor 1254-induced rat- and hamster-liver S9.
Test concentrations with justification for top dose:
0.000, 3.300, 10.000, 33.300, 100.000, 333.300 ug/plate were used in this study. Dose levels were selected based on the results of a pre-experiment using the TA100 strain. In the pre-experiment, cytotoxicity, as defined by a decrease in the number of revertant colonies or a clearing in the density of the background lawn, was observed at ≥333.3 µg/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 was used as a vehicle


- Justification for choice of solvent/vehicle:

- Justification for percentage of solvent in the final culture medium:
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine and 2-aminoanthracene
Remarks:
Positive controls in the absence of S9 were sodium azide (TA1535 and TA 100), 9-aminoacridine (TA97 and TA 1537), and 4-nitro-o-phenylenediamine (TA98). The positive control in the presence of S9 was 2-aminoanthracene for all strains.
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate) : TRIPLICATE
- Number of independent experiments : SINGLE

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable):
- Test substance added in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk : Pre-incubation

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 37"C
- Exposure duration/duration of treatment: 20 minutes
- Harvest time after the end of treatment (sampling/recovery times):

- Other: The test chemical was tested for mutagenicity as per the the preincubation method. The test chemical, Salmonella culture, and S-9 mix or buffer were incubated at 37"C, without shaking, for 20 min. The top agar was added, and the contents of the tubes were mixed and poured onto the surface of petri dishes that contained Vogel- Bonner medium. The histidine-revertant (his') colonies arising on these plates were counted following 2 days incubation at 37°C. The plates were hand-counted when a precipitate was present; otherwise automatic colony counters were used. At least five doses of the chemical were tested in triplicate. Experiments were repeated at least 1 wk following the initial trial. Each chemical was tested initially at half-log doses up to a dose that elicited toxicity.


Evaluation criteria:
An individual trial was judged mutagenic (+) if a dose-related increase over the corresponding solvent control was seen, and it was judged weakly mutagenic C+W) if a low-level dose response was seen. A trial was considered questionable (?) if a dose related increase was judged insufficiently high to justify a call of " + W," if only a single dose was elevated over the control, or if a non-dose-related increase was seen. The distinctions between a weak mutagenic response and a mutagenic response, or between a weak mutagenic response and a questionable mutagenic response are highly subjective.
Statistics:
Mutagenic responses of Salmonella tester strains to the test chemical concentrations were reported (mean+/- SEM; three plates)
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
STUDY RESULTS
- Concurrent vehicle negative and positive control data : The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay.

Ames test:
- Signs of toxicity
- Individual plate counts : No trend of increased number of revertant colonies with increased dosing of the test chemical was observed. The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay.
- Mean number of revertant colonies per plate and standard deviation

Remarks on result:
other: not mutagenic
Conclusions:
The test chemical was concluded to be non-mutagenic in TA98, TA100, TA1535 and TA1537 in the presence and absence of metabolic activation.
Executive summary:

The chemical was tested in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 0 (solvent control), 1, 3.3, 10.0, 33.0, 100.0 and 333.3 µg/plate with and without metabolic activation (Aroclor 1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results of a pre-experiment using the TA100 strain. In the pre-experiment, cytotoxicity, as defined by a decrease in the number of revertant colonies or a clearing in the density of the background lawn, was observed at ≥333.3 µg/plate. Therefore, 333 µg/plate was selected as top dose in the main experiment. Sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine and 2-aminoanthracene served as positive controls. The test chemical failed to produce any significant increase in the number of revertant colonies. No trend of increased number of revertant colonies with increased dosing of the test chemical was observed. The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay. Based on the data, the test chemical was concluded to be non-mutagenic in TA98, TA100, TA1535 and TA1537 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

In vitro gene mutation study in bacteria

Various studies have been performed to evaluate the mutagenic potential of the test chemical. The results include in vitro studies performed on various bacterial strains for the test chemical which are mentioned as follows:

Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using 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, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

 

This is supported by another Salmonella Mutagenicity Tests carried out to determine the degree of mutation response obtained after bacterial strains were exposed to the test chemical.The chemical was tested in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 at 0 (solvent control), 1, 3.3, 10.0, 33.0, 100.0 and 333.3 µg/plate with and without metabolic activation (Aroclor 1254-induced rat- and hamster-liver S9). Dose levels were selected based on the results of a pre-experiment using the TA100 strain. In the pre-experiment, cytotoxicity, as defined by a decrease in the number of revertant colonies or a clearing in the density of the background lawn, was observed at ≥333.3 µg/plate. Therefore, 333 µg/plate was selected as top dose in the main experiment. Sodium azide, 9-aminoacridine, 4-nitro-o-phenylenediamine and 2-aminoanthracene served as positive controls. The test chemical failed to produce any significant increase in the number of revertant colonies.No trend of increased number of revertant colonies with increased dosing of the test chemical was observed. The positive controls produced distinct increases in the number of revertant colonies, thus confirming the validity of the assay. Based on the data, the test chemical was concluded to be non-mutagenic in TA98, TA100, TA1535 and TA1537 in the presence and absence of metabolic activation.

 

Based on the available results and applying the weight of evidence approach the test chemical can be considered to be non mutagenic to 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.

Justification for classification or non-classification

Based on the available results, the test chemical cannot be expected to produce mutagenic reactions to bacterial and mammalian cell lines when tested under in vitro conditions. Hence, the test chemical can be classified under the category "Not Classified' as per CLP Regulation.