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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:
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

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 Sodium 2-hydroxybenzoate (CAS No. 54-21-7) to induce gene muta¬tions 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report):sodium salicylate
- Molecular formula :C7H6O3.Na
- Molecular weight : 160.1035 g/mol
- Substance type: organic
- Physical state: solid
- Smilies notation: c1(c(cccc1)O)C(=O)[O-].[Na+]
- InChI: 1S/C7H6O3.Na/c8-6-4-2-1-3-5(6)7(9)10;/h1-4,8H,(H,9,10);/q;+1/p-1

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), 0.002, 0.005, 0.016, 0.050 and 0.158 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:
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 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

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

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

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 Sodium 2-hydroxybenzoate (CAS No. 54-21-7) 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 Sodium 2-hydroxybenzoate (CAS No. 54-21-7) 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 item Sodium 2-hydroxybenzoate (CAS No. 54-21-7) did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.