Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

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 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, 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.

 

Mamalian cell gene mutation assay:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of Sodium salicylate (CAS No. 54-21-7) when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, sodium salicylate was administered to CHO cells for 3 hrs at the dose levels of 0.0625, 0.125, 0.25 or 0.5 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.

Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at the concentration 0.0625 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of sodium salicylate, i.e. these concentrations did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 3.08 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of sodium salicylate in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposedin vitroto the test chemical at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs.

Based on the results of the current study, it is concluded that sodium salicylate (CAS no 54 -21 -7) does not give rise to gene mutations when CHO cells are exposed to the test chemicalin vitroat 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs, in the presence or abscence 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
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:
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
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:
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
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
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

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 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.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
The study is from study report and was conducted in accordance with the Good Laboratory Practice Principles as Published by OECD in 1998, No 1 ENV/MC/CHEM(98)17
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
The purpose of this study was to assess toxic and genotoxic effects of Sodium salicylate on Chinese Hamster Ovary (CHO) cells by using several different in vitro-based assays, including genotoxicity tests based on the OECD Guideline No. 476 “In Vitro Mammalian Cell Gene Mutation Test”.
GLP compliance:
not specified
Type of assay:
mammalian cell gene mutation assay
Specific details on test material used for the study:
- Name of test material (IUPAC name): Sodium salicylate
- Molecular Formula: C7H5NaO3
- Molecular Weight: 160.10 g/mol
- Substance type: Organic
- Physical state: Solid
- Analytical purity: ≥ 99.5%
- Consistency: White powder/flakes
- Activity (Clinical Indication): Industrial Chemical
- Stability under test conditions: Stable
- Storage condition of test material: As per requirements mentioned in guidance for safe use
Target gene:
Cells deficient in hypoxanthine-guanine phosphoribosyl transferase (HPRT) due to the mutation HPRT+/- to HPRT-/- are resistant to cytotoxic effects of 6-thioguanine (TG). HPRT proficient cells are sensitive to TG (which causes inhibition of cellular metabolism and halts further cell division since HPRT enzyme activity is important for DNA synthesis), so mutant cells can proliferate in the presence of TG, while normal cells, containing hypoxanthine-guanine phosphoribosyl transferase cannot.

This in vitro test is an assay for the detection of forward gene mutations at the in hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus on the X chromosomes of hypodiploid, modal No. 20, CHO cells. Gene and chromosome mutations are considered as an initial step in the carcinogenic process.
The hypodiploid CHO cells are exposed to the test item with and without exogenous metabolic activation. Following an expression time the descendants of the treated cell population are monitored for the loss of functional HPRT enzyme.
HPRT catalyses the transformation of the purine analogues 6-thioguanine (TG) rendering them cytotoxic to normal cells. Hence, cells with mutations in the HPRT gene cannot phosphoribosylate the analogue and survive treatment with TG.

Therefore, mutated cells are able to proliferate in the presence of TG whereas the non-mutated cells die. However, the mutant phenotype requires a certain period of time before it is completely expressed. The phenotypic expression is achieved by allowing exponential growth of the cells for 7 days.
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:
S9 liver microsomal fraction obtained from Arcolor 1254-induced male Sprague-Dawley rats (Supplier: Molecular Toxicology Inc. via Trinova Biochem GmbH, Giessen, Germany)
Test concentrations with justification for top dose:
0, 0.0625, 0.125, 0.25 or 0.5 mM
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Phosphate-buffered saline (PBS)
Justification for choice of solvent/ vehicle: Sodium salicylate was easily dissolved in PBS.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Phosphate-buffered saline
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
N-ethyl-N-nitrosourea (ENU) was the positive control substance in the tests done without S9
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)

SPINDLE INHIBITOR (cytogenetic assays):
Not applicable

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
Cytotoxicity test
After being exposed to the test chemical for 3 hours, in the absence or presence of S9, cells were trypsinized and 0.5 x 10 E5 cells per well was seeded in duplicates from two parallel duplicate cultures into 6-well plates in fresh medium. The relative total growth and cytotoxicity was evaluated 24 and 48 hours after seeding.
Rationale for test conditions:
No data
Evaluation criteria:
The cell line was observed for gene mutation
Statistics:
No data
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
not valid
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No data
Remarks on result:
other: No mutagenic potential

Table 1A.Effect of sodium salicylate 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

184/4

0/4

208/4

Pos. control

0/4

221/4

12/4

143/4

0.0625 mM

2/4a

218/4

0/4

198/4

0.125 mM

0/4

226/4

0/4

185/4

0.25 mM

0/4

177/4

0/4

181/4

0.5 mM

0/4

185/4

0/4

198/4

 

a)2 very diffuse colonies were found in one single well.

 

 

Table 1B.Mutation frequency in CHO cells after 3 hrs of exposure to sodium salicylate 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

N/A

3.08x10-4

0.0625 mM

N/Aa

N/A

0.125 mM

N/A

N/A

0.25 mM

N/A

N/A

0.5 mM

N/A

N/A

 

a)Since only diffuse colonies were found in one single well (see Table 1A), these diffuse colonies were not regarded as reliable and true colonies since the cells seemed to be apoptotic.


 

Conclusions:
Sodium salicylate in the concentration of 0, 0.0625, 0.125, 0.25 or 0.5 mM did not show any evidence of gene toxicity when CHO cells were exposed to the test chemical, in the presence or abscence of metabolic activation.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of Sodium salicylate (CAS No. 54-21-7) when administered to Chinese Hamster Ovary (CHO) cells.

In the genotoxicity test, sodium salicylate was administered to CHO cells for 3 hrs at the dose levels of 0.0625, 0.125, 0.25 or 0.5 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.

Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at the concentration 0.0625 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of sodium salicylate, i.e. these concentrations did not show any evidence of diffuse or clear colonies present.

When the mutation frequency was determined, a frequency of 3.08 x 10-4was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of sodium salicylate in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs.

Conclusion

Based on the results of the current study, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs, in the presence or absence of metabolic activation.

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

Genetic toxicity in vivo

Description of key information

Gene mutation in vivo:

In vivo sister chromatid exchange was performed to determine the mutagenic nature of Sodium salicylate. The study was performed using Swiss albino male mice. The test chemical was dissolved in 2% gum acacia in ditilled water at dose level of 0 or 350 mg/Kg. Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. In the single-dose oral study, SS was gavaged with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half an hour after tablet implantation to different groups of 5 animals each. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases. No significant increase in SCE or RI was observed for SS for all the doses tested when compared with solvent control. Sodium salicylate did not induce sister chromatid exchange in the bone marrow cells of mice treated orally and hence it is not likely to classify as a gene mutant in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
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 peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vivo sister chromatid exchange by the oral route was performed to determine the mutagenic nature of Sodium salicylate
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Specific details on test material used for the study:
- Name of test material: Sodium salicylate
- IUPAC name: Sodium salicylate
- Molecular formula: C7H6O3.Na
- Molecular weight: 160.1035 g/mol
- Substance type: Organic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data
Species:
rat
Strain:
Swiss
Remarks:
albino
Details on species / strain selection:
No data
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Division of Laboratory animals, Central Drug Research Institute, Lucknow
- Age at study initiation: 10-12 week old
- Weight at study initiation: 30 g
- Assigned to test groups randomly: No data
- Fasting period before study: No data
- Housing: They were kept five per
cage with husk bedding
- Diet (e.g. ad libitum): Standard rodent
pellet diet (Gold Mohor, Lipton India Ltd., Chandigarb, India) ad libitum
- Water (e.g. ad libitum): Water ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28 ± 2°C
- Humidity (%): 60_+ 5%
- Air changes (per hr): No data
- Photoperiod (hrs dark / hrs light): 12 h light/12 h dark

IN-LIFE DATES: From: To: No data
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: distilled water in 2% gum acacia
- Justification for choice of solvent/vehicle: The test chemical was soluble in distilled water in 2% gum acacia
- Concentration of test material in vehicle: 0 or 350 mg/Kg
- Amount of vehicle (if gavage or dermal): 0.3mL/mouse
- Type and concentration of dispersant aid (if powder): No data
- Lot/batch no. (if required): No data
- Purity: No data
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test chemical was dissolved with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data
Duration of treatment / exposure:
No data
Frequency of treatment:
Once
Post exposure period:
No data
Remarks:
0 or 350 mg/Kg
No. of animals per sex per dose:
Total: 15 male mice
0 mg/Kg: 5 male mice
350 mg/Kg: 5 male mice
Positive control: 5 male mice
Control animals:
yes, concurrent vehicle
Positive control(s):
Mitomycin C
- Justification for choice of positive control(s): No data
- Route of administration: Oral
- Doses / concentrations: No data
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The oral dose for the test compound was approx. 1/3 of the oral LD50 of mice reported earlier. The other two lower doses were the serial dilutions of the highest doses selected for each chemical.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. In the single-dose oral study, SS was gavaged with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half an hour after tablet implantation to different groups of 5 animals each. For SCE analysis, colchicine (4
mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation.

DETAILS OF SLIDE PREPARATION: Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique
All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested.

METHOD OF ANALYSIS: Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases

OTHER: No data
Evaluation criteria:
The bone marrow cells were observed for sister chromatid exchanges
Statistics:
Student's t-test was used to compare the results of the treated series with the respective controls for SCE, MI and RI in the oral study conducted.
Sex:
male
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: No mutagenic potential
Additional information on results:
No data

Table: In vivo sister chromatid exchanges induced by sodium salicylate in mice after oral administration

Treatment

SCE/cell of 5 animals

SCE/cell

(mean ± SD)a

Replicative indices mean ± SD)a

Solvent control(Gum acacia)

4.4, 3.9, 5.1, 4.6, 4.7

4.54±0.43

1.83±0.10

SS (350 mg/kg)

5.6, 5.9, 4.3, 5.1, 4.7

5.12±0.64

1.82±0.07

a Mean ± SD of 5 animals (30 cells/animal). Results at each dose were compared with the control using Students, t-test (* p < 0.001).

Conclusions:
Sodium salicylate did not induce sister chromatid exchange in the bone marrow cells of mice treated orally and hence it is not likely to classify as a gene mutant in vivo.
Executive summary:

In vivo sister chromatid exchange was performed to determine the mutagenic nature of Sodium salicylate. The study was performed using Swiss albino male mice. The test chemical was dissolved in 2% gum acacia in ditilled water at dose level of 0 or 350 mg/Kg. Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. In the single-dose oral study, SS was gavaged with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half an hour after tablet implantation to different groups of 5 animals each. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases. No significant increase in SCE or RI was observed for SS for all the doses tested when compared with solvent control. Sodium salicylate did not induce sister chromatid exchange in the bone marrow cells of mice treated orally and hence it is not likely to classify as a gene mutant in vivo.

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

Additional information

Gene mutation in vitro:

Data available for the target chemical was reviewed to determine the mutagenic nature of Soduim salicylate (CAS no 54 -21 -7). The studies are as mentioned below:

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 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, 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.

 

An another in vitro mammalian cell gene mutation study was designed and conducted by Sustaiinability Support Services (Europe) AB to determine the genotoxicity profile of Sodium salicylate (CAS No. 54-21-7) when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, sodium salicylate was administered to CHO cells for 3 hrs at the dose levels of 0.0625, 0.125, 0.25 or 0.5 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 asN-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. Only the positive control ENU gave a clear indication of gene mutations occurring while no other treatment gave rise to gene toxicity. Two very diffuse colonies were seen in one well out of four at the concentration 0.0625 mM and in the presence with 4% S9 liver microsomal fraction. These diffuse colonies are not regarded to be relevant since the two spots were only mildly colored by crystal violet, thus indicating that it was a small cluster of apoptotic cells taking their last breath instead of cells surviving the TG-selection. This is further supported by the results of the higher tested concentrations of sodium salicylate, i.e. these concentrations did not show any evidence of diffuse or clear colonies present. When the mutation frequency was determined, a frequency of 3.08 x 10-4 was shown after a 3 hour exposure of ENU as the positive control and in the absence of S9 liver microsomal fraction. Since no other tested concentration of sodium salicylate in the absence or presence of S9 liver microsomal fraction resulted in colonies, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposed in vitro to the test chemical at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs. Based on the results of the current study, we conclude that sodium salicylate does not give rise to gene mutations when CHO cells are exposed to the test chemical in vitro at 0, 0.0625, 0.125, 0.25 or 0.5 mM for 3 hrs, in the presence or absence of metabolic activation.

Gene mutation toxicity study was performed by Demerc et al (The American Naturalist, 1951) to determine the mutagenic nature of Sodium salicylate (CAS no 54 -21 -7). The study was performed using E. coli Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4. The test chemical was dissolved in distilled water and used at dose levels of 0, 1.0, 2.0, 2.0 or 3.0%. For every experiment, bacteria were grown for 24 hours at 37˚C in an aerated broth culture containing 10 micrograms of streptomycin per milliliter. They reached a saturation titer of approximately 2 to 3 x 109cells per milliliter. Each culture was started from an inoculum, usually large, taken from streptomycin-agar slants kept in a refrigerator. Before treatment the bacteria were washed in saline and resuspended in distilled water. A sample of the new suspension was added to the desired solution of chemical in distilled water, and incubated at 37˚C for a certain period of time; no growth occurs under these conditions. Another sample of the same suspension was added to an equal amount of distilled water and incubated for the same period of time, as a control. At the end of the treatment period, both treated and control suspensions were assayed by plating suitable dilutions on streptomycin-agar plates. At the same time they were plated (0.1 ml per Petri dish), either undiluted or diluted not more than 1:10 in plain broth, onto a number of streptomycin-free plates, using a glass spreader and a turntable. The assay plates were incubated for 48 hours, after which it was possible to count the colonies and calculate the titers of the two suspensions at the end of treatment, and the percentage of survivors. The streptomycin-free plates (mutant plates) were incubated for at least six days. After this time the colonies were scored, and the frequency of mutants calculated by dividing the number of colonies by the number of (viable) bacteria plated. Sodum salicylate did not increase the frequency of revertant mutants in the strains of E. coli used.Sodium salicylate did not induce gene mutation inE. coli Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4 and hence it is not likely to classify as a gene mutant in vitro.

Based on the prediction done using the OECD QSAR toolbox version 3.3 with log kow as the primary descriptor and considering the five closest read across substances, chromosomal aberration was predicted for sodium salicylate. The study assumed the use of Chinese hamster ovary (CHO) cell line with and without S9 metabolic activation system. Soduim salicylate was predicted to not induce chromosomal aberrations in Chinese hamster ovary (CHO) cell line in the presence and absence of S9 metabolic activation system and hence, according to the prediction made, it is not likely to classify as a gene mutant in vitro.

In another study by Kuboyama and Fuji et al (The Journal Of Nihon University School Of Dentistry, 1992) Salmonella/micosome mutagenicity assay was performed to determine the mutagenic nature of sodium salicylate (CAS no 54 -21 -7). The study was performed by the preincubatoin method using Salmonella typhimurium strains TA98 and TA100 with and without addition of S9 liver fractions isolated from polychlorobiphenyl induced rats, mice, hamsters and guinea pigs. The test chemical was dissolved in DMSO and used at dose level of 0.1 mg/plate. A test tube containing a suspension of one strain of Salmonella typhimurium (or E. coli) plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37º C with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen*. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine-manufacturing gene. The number of colonies is usually counted after 2 days. In an Ames test ,Sodium salicylate , in dimethyl sulphoxide at dose 0.1 mg/plate was not mutagenic in Salmonella typhimurium strains TA98 and TA100 with and without addition of S9 liver fractions isolated from polychlorobiphenyl induced rats, mice, hamsters and guinea pigs.

In the same study by Kuboyama and Fuji et al (1992), Rec assay by the cold incubation method was performed to determine the mutagenic nature of Sodium salicylate (CAS no 54 -21 -7). The study was performed using Bacillus subtilis strains H17 and M45. Two strains of B. subtilis H17 and M45 were grown at 37˚C in B-2 broth for 14 h. Each culture was streaked on the dry surface of a B-2 agar plate in a standard Petri dish, taking care not to let them touch one another. Test compounds, 0.05 ml (0.25-5 mg/disc), were dropped on a paper disc (diameter 8 mm), which had been placed at the starting point of the streaks. All the plates were kept at 4˚C for 24 h, and then incubated at 37˚C for 24 h. The length of the inhibition zone was then measured. When the inhibition zone of cellular growth was more than 2 mm of zonedifference between growth inhibition zones for Rec+ and Rec- strains, it was considered that the test compound had a damaging effect on cellular DNA. Sodium salicylate did not induce DNA damage in the Bacillus subtilis strains H17 and M45 and hence it is not likely to classify as a gene mutant in vitro.

Gene mutation in vivo:

Data available for the target chemical was reviewed to determine the mutagenic nature of Sodium salicylate in vivo:

In vivo sister chromatid exchange was performed by Giri et al (Mutation research, 1996) to determine the mutagenic nature of Sodium salicylate (CAS no 54 -21 -7). The study was performed using Swiss albino male mice. The test chemical was dissolved in 2% gum acacia in ditilled water at dose level of 0 or 350 mg/Kg. Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. In the single-dose oral study, SS was gavaged with distilled water in 2% gum acacia (0.3 ml/mouse) at the dose of 350 mg/kg half an hour after tablet implantation to different groups of 5 animals each. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first (M 1), second (M 2) and third (M 3) division metaphases. No significant increase in SCE or RI was observed for SS for all the doses tested when compared with solvent control. Sodium salicylate did not induce sister chromatid exchange in the bone marrow cells of mice treated orally and hence it is not likely to classify as a gene mutant in vivo.

In the same study by Giri et al (1996), In vivo sister chromatid exchange was performed to determine the mutagenic nature of Sodium salicylate (CAS no 54 -21 -7). The study was performed using Swiss albino male mice. The test chemical was dissolved in DMSO at dose level of 0, 25, 50 or 100 mg/Kg. Paraffin-coated (approx. 80% of the surface) BrdU tablets (50 mg each) were implanted subcutaneously in the flank of the mice under ether anaesthesia. The test chemicals were administered as a single i.p. injection 1 h after tablet implantation. Three doses (25, 50 and 100 mg/kg) of SS were injected i.p. in DMSO (75µl/mouse) to different groups of 5 animals each. Negative control mice were injected with 75µl DMSO while mitomycin C was used as a positive control at a dose of 1.5 mg/kg of body weight. For SCE analysis, colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled with 0.075 M KCl. After hypotonic treatment (0.075 M KCl at 37°C) for 20 min, the cells were fixed 3 times with methanol/acetic acid (3 : 1). The slides were prepared, and the chromosomes were differentially stained with fluorescence-plus- Giemsa technique. All the slides were coded and 30 second division metaphase cells (40 + 2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored per dose tested. No significant increase in SCE or RI was observed for SS for all the doses tested when compared with solvent control. Based on the observations made, Sodium salicylate did not induce sister chromatid exchange in the bone marrow cells of mice treated intraperitoneally and hence it is not likely to classify as a gene mutant in vivo.

In vitro chromosome aberration study was also performed by Giri et al (Mutatoin Research, 1996) to determine the mutagenic nature of Sodium salicylate (CAS no 54 -21 -7). The study was performed using male Swiss albino mice. Three doses (50, 100 and 200 mg/kg) SS was dissolved in DMSO and injected i.p. (75µl/mouse) following the protocol of Preston et al.Bone marrow chromosomes were prepared and slides were stained with Giemsa. All the slides were coded and 100 well spread metaphase cells were scored per animal. Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. CA were scored following the method of WHO and Preston et al. The aberrations frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell. Sodium salicylate did not induce chromosome aberrations in the bone marrow of male mice at 50 and 100 mg/Kg but it induced chromosome aberration at the highest dose of 200 mg/Kg.

Giri et al (1996) also performed an in vitro chromosome aberration study to determine the mutagenic nature of Sodium salicylate (CAS no 54 -21 -7). The study was performed using male Swiss albino mice. The test chemical was dissolved in 2% gum acacia in distilled water at a single oral dose of 0 or 350 mg/Kg. Negative control mice were gavaged only 2% gum acacia in distilled water. After 22 h of chemical treatment the animals were injected with colchicine (2 mg/kg) and 2 h later they were killed by cervical dislocation.Bone marrow chromosomes were prepared and slides were stained with Giemsa. All the slides were coded and 100 well spread metaphase cells were scored per animal. Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. CA were scored following the method of WHO and Preston et al. The aberrations frequencies of chromatid and chromosome types per cell were calculated. Gaps were recorded but not included in the frequency of aberrations per cell. A significant increase in CA was also observed for SS when compared with solvent control. Based on the observations made, Sodium salicylate induced chromosome aberration in the bone marrow cells of mice treated orally.

Based on the data available for the target chemical, Sodium salicylate (CAS no 54 -21 -7) does not exhibit gene mutation in vitro and in vivo. The in vivo chromosme aberration study though indicates some mutagenic nature, but according to the in vivo data summarized the test chemical is not mutagenic upto a dose of 100 mg/kg. Considering this, the test chemical is not likely to classify as a gene mutant in vitro and in vivo.

Justification for classification or non-classification

Based on the data available for the target chemical, Sodium salicylate (CAS no 54 -21 -7) does not exhibit gene mutation in vitro and in vivo. The in vivo chromosme aberration study though indicates some mutagenic nature, but according to the in vivo data summarized the test chemical is not mutagenic upto a dose of 100 mg/kg. Considering this, the test chemical is not likely to classify as a gene mutant in vitro and in vivo.