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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
27-02-2018 - 26-03-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from study report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
This study was performed to investigate the potential of the test chemical to induce gene mutations in comparison to 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:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: Sunset Yellow FCF
- IUPAC name: disodium 6-hydroxy-5-[(4-sulfonatophenyl)diazenyl]naphthalene-2-sulfonate
- Molecular formula: C16H12N2O7S2.2Na
- Molecular weight: 454.38 g/mol
- Substance type: organic
- Physical state: solid

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other:
Cytokinesis block (if used):
No data
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254 induced S9 metabolic activation system
Test concentrations with justification for top dose:
0.0 (NC), 0.005, 0.016, 0.050, 0.158, 0.501 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RO water
- Justification for choice of solvent/vehicle: The test chemical was soluble in RO water
Controls
Untreated negative controls:
not specified
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

Results and discussion

Test results
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No 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 5.0 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 mg/plate based on the solubility and precipitation test. There was no reduction in colony count as well as in background lawn in treated concentrations 5 (T8) 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: 0.050, 0.158, 0.501, 1.582 and 5 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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

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

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 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

Any other information on results incl. tables

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

110

26

124

28

R2

106

24

122

26

R3

104

25

126

24

T1

(0.002)

R1

91

14

86

16

R2

84

13

82

18

R3

87

13

88

16

T2

(0.005)

R1

86

14

90

19

R2

92

17

88

21

R3

85

18

86

17

T3

(0.016)

R1

90

16

102

19

R2

96

14

98

20

R3

84

16

106

21

T4

(0.050)

R1

90

18

106

21

R2

88

16

98

23

R3

98

16

90

19

T5

(0.158)

R1

95

18

104

20

R2

88

19

100

18

R3

96

18

108

22

T6

(0.501)

R1

94

20

108

18

R2

101

21

110

20

R3

98

19

114

24

T7

(1.582)

R1

96

20

112

18

R2

104

23

116

22

R3

98

20

118

24

T8

(5)

R1

102

23

118

23

R2

98

25

114

25

R3

100

24

120

24

PC

R1

1232

896

1458

1242

R2

1280

944

1432

1180

R3

1296

936

1488

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

8

15

28

124

284

R2

8

14

26

122

278

R3

6

16

24

126

284

T1

(0.005)

R1

6

10

19

90

228

R2

5

11

21

88

238

R3

5

12

17

86

232

T2

(0.016)

R1

7

12

19

102

227

R2

5

11

20

98

244

R3

6

12

21

106

236

T3

(0.050)

R1

6

13

21

106

242

R2

7

12

23

98

254

R3

6

12

19

90

236

T4

(0.158)

R1

6

14

20

104

254

R2

5

13

18

100

260

R3

5

12

22

108

262

T5

(0.501)

R1

7

14

18

108

268

R2

5

15

20

110

274

R3

6

15

24

114

270

PC

R1

172

398

1242

1458

1336

R2

186

408

1180

1432

1384

R3

190

440

1208

1488

1344

 

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

26

110

272

R2

6

15

24

106

296

R3

8

14

25

104

268

T1

(0.005)

R1

4

11

14

86

226

R2

5

10

17

92

231

R3

5

11

18

85

218

T2

(0.016)

R1

5

13

16

90

226

R2

6

12

14

96

234

R3

5

10

16

84

232

T3

(0.050)

R1

6

14

18

90

242

R2

6

13

16

88

238

R3

6

11

16

98

250

T4

(0.158)

R1

6

14

18

95

244

R2

6

15

19

88

252

R3

5

13

18

96

260

T5

(0.501)

R1

7

14

20

94

268

R2

7

16

21

101

274

R3

6

14

19

98

284

PC

R1

168

1218

896

1232

1624

R2

183

1230

944

1280

1688

R3

175

1170

936

1296

1680

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

17

28

121

278

R2

6

16

27

119

262

R3

7

16

27

120

272

T1

(0.005)

R1

5

12

19

104

234

R2

5

12

21

98

240

R3

4

11

22

102

230

T2

(0.016)

R1

5

13

23

106

240

R2

6

13

22

110

236

R3

5

12

24

104

242

T3

(0.050)

R1

5

12

20

112

250

R2

6

15

21

108

240

R3

4

13

23

112

258

T4

(0.158)

R1

5

14

23

114

266

R2

6

13

23

108

260

R3

6

14

24

112

258

T5

(0.501)

R1

7

15

26

117

260

R2

7

14

25

115

272

R3

6

14

23

118

258

PC

R1

179

398

1360

1518

1736

R2

158

418

1384

1448

1704

R3

184

376

1328

1480

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

16

28

114

264

R2

7

15

26

112

256

R3

6

13

26

118

274

T1

(0.005)

R1

5

10

20

102

229

R2

4

10

22

108

237

R3

4

11

18

110

240

T2

(0.016)

R1

6

12

21

104

242

R2

4

11

23

110

246

R3

5

14

22

108

256

T3

(0.050)

R1

5

13

19

106

260

R2

4

13

23

110

252

R3

5

12

22

111

254

T4

(0.158)

R1

5

11

25

116

262

R2

5

12

20

112

250

R3

6

13

23

108

260

T5

(0.501)

R1

6

13

26

116

266

R2

6

14

23

112

252

R3

7

12

21

114

258

PC

R1

187

1208

928

1086

1608

R2

178

1160

894

1164

1656

R3

182

1224

952

1128

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)

7.33

1.15

15.00

1.00

26.00

2.00

124.00

2.00

282.00

3.46

T1

(0.005)

5.33

0.58

11.00

1.00

19.00

2.00

88.00

2.00

232.67

5.03

T2

(0.016)

6.00

1.00

11.67

0.58

20.00

1.00

102.00

4.00

235.67

8.50

T3

(0.050)

6.33

0.58

12.33

0.58

21.00

2.00

98.00

8.00

244.00

9.17

T4

(0.158)

5.33

0.58

13.00

1.00

20.00

2.00

104.00

4.00

258.67

4.16

T5

(0501)

6.00

1.00

14.67

0.58

20.67

3.06

110.67

3.06

270.67

3.06

PC

182.67

9.45

415.33

21.94

1210.00

31.05

1459.33

28.02

1354.67

25.72

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

7.00

1.00

15.00

1.00

25.00

1.00

106.67

3.06

278.67

15.14

T1

(0.005)

4.67

0.58

10.67

0.58

16.33

2.08

87.67

3.79

225.00

6.56

T2

(0.016)

5.33

0.58

11.67

1.53

15.33

1.15

90.00

6.00

230.67

4.16

T3

(0.050)

6.00

0.00

12.67

1.53

16.67

1.15

92.00

5.29

243.33

6.11

T4

(0.158)

5.67

0.58

14.00

1.00

18.33

0.58

93.00

4.36

252.00

8.00

T5

(0501)

6.67

0.58

14.67

1.15

20.00

1.00

97.67

3.51

275.33

8.08

PC

175.33

7.51

1206.00

31.75

925.33

25.72

1269.33

33.31

1664.00

34.87

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-INCUBATIONMETHOD
(TRIAL II)

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

7.00

1.00

16.33

0.58

27.33

0.58

120.00

1.00

270.67

8.08

T1

(0.005)

4.67

0.58

11.67

0.58

20.67

1.53

101.33

3.06

234.67

5.03

T2

(0.016)

5.33

0.58

12.67

0.58

23.00

1.00

106.67

3.06

239.33

3.06

T3

(0.050)

5.00

1.00

13.33

1.53

21.33

1.53

110.67

2.31

249.33

9.02

T4

(0.158)

5.67

0.58

13.67

0.58

23.33

0.58

111.33

3.06

261.33

4.16

T5

(0501)

6.67

0.58

14.33

0.58

24.67

1.53

116.67

1.53

263.33

7.57

PC

173.67

13.80

397.33

21.01

1357.33

28.10

1482.00

35.04

1717.33

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

14.67

1.53

26.67

1.15

114.67

3.06

264.67

9.02

T1

(0.005)

4.33

0.58

10.33

0.58

20.00

2.00

106.67

4.16

235.33

5.69

T2

(0.016)

5.00

1.00

12.33

1.53

22.00

1.00

107.33

3.06

248.00

7.21

T3

(0.050)

4.67

0.58

12.67

0.58

21.33

2.08

109.00

2.65

255.33

4.16

T4

(0.158)

5.33

0.58

12.00

1.00

22.67

2.52

112.00

4.00

257.33

6.43

T5

(0501)

6.33

0.58

13.00

1.00

23.33

2.52

114.00

2.00

258.67

7.02

PC

182.33

4.51

1197.33

33.31

924.67

29.14

1126.00

39.04

1610.67

44.06

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

Applicant's summary and conclusion

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 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 5.0 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), 0.005, 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, 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.