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Diss Factsheets

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
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
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:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium iodide
EC Number:
231-679-3
EC Name:
Sodium iodide
Cas Number:
7681-82-5
Molecular formula:
INa
IUPAC Name:
sodium iodide
Details on test material:
- Name of test material: Sodium iodide
- Molecular formula: INa
- Molecular weight: 149.89427
- Substance type: Inorganic
- 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), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was solulble in Distilled water
Controls
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
Distilled 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 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 (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 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.
In TA 98 and TA 100 there was no reduction in colony count as well as in background lawn was observed in treated concentration 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: 0.0 (NC), 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).

The concentrations used in the experiment (pre-experiment, Trial-1, Trial-2) were placed with (√10) half log interval.

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

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

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

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

Any other information on results incl. tables

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

25

124

25

R2

116

22

126

26

R3

124

23

120

24

T1

(0.002)

R1

98

15

108

18

R2

118

17

102

18

R3

102

14

104

16

T2

(0.005)

R1

104

16

108

22

R2

102

15

110

20

R3

106

15

106

18

T3

(0.016)

R1

104

20

112

20

R2

110

18

108

18

R3

108

20

110

22

T4

(0.050)

R1

112

17

112

21

R2

120

20

116

23

R3

114

22

110

20

T5

(0.158)

R1

118

22

120

21

R2

110

20

122

23

R3

106

23

118

22

T6

(0.501)

R1

110

22

112

24

R2

118

16

119

22

R3

112

17

122

21

T7

(1.582)

R1

118

22

120

23

R2

110

23

122

22

R3

122

19

118

20

T8

(5)

R1

120

22

120

23

R2

118

23

122

24

R3

123

22

124

24

PC

R1

1224

1016

1488

1224

R2

1240

984

1456

1176

R3

1256

992

1440

1160

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

14

25

124

255

R2

8

13

26

126

246

R3

7

15

24

120

252

T1

(0.050)

R1

5

11

21

112

246

R2

4

12

23

116

238

R3

7

10

20

110

242

T2

(0.158)

R1

6

10

21

120

230

R2

4

12

23

122

250

R3

7

13

22

118

222

T3

(0.501)

R1

6

10

24

112

238

R2

4

14

22

119

248

R3

5

9

21

122

229

T4

(1.582)

R1

7

10

23

120

240

R2

7

11

22

122

248

R3

5

13

20

118

250

T5

(5)

R1

6

12

23

120

232

R2

7

12

24

122

243

R3

5

14

24

124

244

PC

R1

186

548

1224

1488

1280

R2

190

480

1176

1456

1360

R3

171

458

1160

1440

1296

 

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

6

14

25

122

253

R2

7

15

22

116

268

R3

7

16

23

124

276

T1

(0.050)

R1

6

14

17

112

256

R2

4

10

20

120

255

R3

5

9

22

114

240

T2

(0.158)

R1

5

15

22

118

220

R2

6

12

20

110

232

R3

5

10

23

106

248

T3

(0.501)

R1

6

12

22

110

242

R2

6

12

16

118

244

R3

5

11

17

112

250

T4

(1.582)

R1

6

15

22

118

242

R2

4

10

23

110

258

R3

6

14

19

122

260

T5

(5)

R1

7

15

22

120

266

R2

5

14

23

118

250

R3

6

13

22

123

246

PC

R1

168

1240

1016

1224

1888

R2

181

1192

984

1240

1664

R3

166

1144

992

1256

1608

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

7

16

23

121

273

R2

6

15

25

119

253

R3

8

15

26

118

260

T1

(0.050)

R1

5

9

21

102

238

R2

6

12

19

105

226

R3

6

12

20

103

220

T2

(0.158)

R1

6

13

22

109

226

R2

5

12

25

110

242

R3

5

13

23

114

240

T3

(0.501)

R1

6

15

25

115

248

R2

4

16

23

116

246

R3

5

12

24

112

234

T4

(1.582)

R1

7

14

20

118

258

R2

5

11

23

117

255

R3

6

14

25

119

270

T5

(5)

R1

6

15

26

109

250

R2

6

15

24

119

260

R3

7

12

23

115

262

PC

R1

171

232

1336

1456

1430

R2

184

241

1360

1528

1446

R3

192

250

1284

1504

1520

 

 

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

6

17

25

122

255

R2

7

17

27

121

260

R3

7

14

25

116

278

T1

(0.050)

R1

6

10

22

104

225

R2

5

11

20

106

220

R3

5

13

19

102

218

T2

(0.158)

R1

6

12

18

113

218

R2

5

15

22

116

216

R3

4

12

22

115

210

T3

(0.501)

R1

6

13

23

106

220

R2

6

11

25

101

224

R3

5

12

22

117

232

T4

(1.582)

R1

7

13

22

118

232

R2

6

14

24

119

235

R3

5

13

20

121

240

T5

(5)

R1

7

16

24

120

252

R2

6

16

26

114

250

R3

6

12

26

112

246

PC

R1

184

1264

896

1128

1576

R2

178

1232

936

1086

1616

R3

166

1192

966

1170

1664

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

0.58

14.00

1.00

25.00

1.00

123.33

3.06

251.00

4.58

T1

(0.050)

5.33

1.53

11.00

1.00

21.33

1.53

112.67

3.06

242.00

4.00

T2

(0.158)

5.67

1.53

11.67

1.53

22.00

1.00

120.00

2.00

234.00

14.42

T3

(0.501)

5.00

1.00

11.00

2.65

22.33

1.53

117.67

5.13

238.33

9.50

T4

(1.582)

6.33

1.15

11.33

1.53

21.67

1.53

120.00

2.00

246.00

5.29

T5

(5)

6.00

1.00

12.67

1.15

23.67

0.58

122.00

2.00

239.67

6.66

PC

182.33

10.02

495.33

46.92

1186.67

33.31

1461.33

24.44

1312.00

42.33

 

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

15.00

1.00

23.33

1.53

120.67

4.16

265.67

11.68

T1

(0.050)

5.00

1.00

11.00

2.65

19.67

2.52

115.33

4.16

250.33

8.96

T2

(0.158)

5.33

0.58

12.33

2.52

21.67

1.53

111.33

6.11

233.33

14.05

T3

(0.501)

5.67

0.58

11.67

0.58

18.33

3.21

113.33

4.16

245.33

4.16

T4

(1.582)

5.33

1.15

13.00

2.65

21.33

2.08

116.67

6.11

253.33

9.87

T5

(5)

6.00

1.00

14.00

1.00

22.33

0.58

120.33

2.52

254.00

10.58

PC

171.67

8.14

1192.00

48.00

997.33

16.65

1240.00

16.00

1720.00

148.16

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

 

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

15.33

0.58

24.67

1.53

119.33

1.53

262.00

10.15

T1

(0.050)

5.67

0.58

11.00

1.73

20.00

1.00

103.33

1.53

228.00

9.17

T2

(0.158)

5.33

0.58

12.67

0.58

23.33

1.53

111.00

2.65

236.00

8.72

T3

(0.501)

5.00

1.00

14.33

2.08

24.00

1.00

114.33

2.08

242.67

7.57

T4

(1.582)

6.00

1.00

13.00

1.73

22.67

2.52

118.00

1.00

261.00

7.94

T5

(5)

6.33

0.58

14.00

1.73

24.33

1.53

114.33

5.03

257.33

6.43

PC

182.33

10.60

241.00

9.00

1326.67

38.85

1496.00

36.66

1465.33

48.01

 

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

16.00

1.73

25.67

1.15

119.67

3.21

264.33

12.10

T1

(0.050)

5.33

0.58

11.33

1.53

20.33

1.53

104.00

2.00

221.00

3.61

T2

(0.158)

5.00

1.00

13.00

1.73

20.67

2.31

114.67

1.53

214.67

4.16

T3

(0.501)

5.67

0.58

12.00

1.00

23.33

1.53

108.00

8.19

225.33

6.11

T4

(1.582)

6.00

1.00

13.33

0.58

22.00

2.00

119.33

1.53

235.67

4.04

T5

(5)

6.33

0.58

14.67

2.31

25.33

1.15

115.33

4.16

249.33

3.06

PC

176.00

9.17

1229.33

36.07

932.67

35.12

1128.00

42.00

1618.67

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 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 and 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), 0.050, 0.158, 0.501, 1.582 and 5 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9).

No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the test chemical (CAS no 7681-82-5) 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 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.

Conclusion

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.