Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames assay:

The test chemical tested negative for mutagenicity in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102 in the presence and absence of S9 metabolic activation system.

 

In vitro mammalian chromosome aberration study:

The test chemical is not expected to produce mutachromosomal effects in Chinese hamster lung-derived fibroblasts or Chinese hamster ovary cells.

 

In vitro gene mutation study in mammalian cells

The test chemical tested negative for mutagenicity in CHO cells in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity of the chemical in CHO in the presence of metabolic activation due to invalid positive control data.

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 the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
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:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
True negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: No precipitation was noted at a dose upto 5 mg/plate in the pre-experiment
- Definition of acceptable cells for analysis: No data
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: To evaluate the toxicity of the test item, a pre-experiment was performed with strains TA 98 and TA 100. Eight concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate) were tested for toxicity and mutation induction with 3 plates each (triplicates). The experimental conditions in this pre-experiment were the same as described below for the Trial-I (Plate incorporation test). Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.

In the pre-experiment, the concentration range of the test item was 0.002 – 5.0 mg/plate based on the solubility and precipitation test. In TA 98 and TA 100, cyto-toxicity was observed in the treated concentrations 1.582 and 5 mg/plate (T7 to T8), moderate inhibition was observed in the treated concentrations 0.501 mg/plate (T6) and there was no reduction in colony count as well as background lawn in any of the following concentrations tested; 0.002, 0.005, 0.016, 0.050 and 0.158 ( T1 to T5) mg/plate both in absence and in the presence of metabolic activation, when compared to that of the negative control group. Based on the results of pre-experiment following doses were selected for the main study trials: 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate, both in the absence (-S9) as well as in the presence of metabolic activation (+S9).

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

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

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

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

TABLE1- REVERTANT COUNT FOR PRE-EXPERIMENT

Dose (mg/plate)

R

Without metabolic activation (-S9)

With metabolic activation (+S9)

TA100

TA 98

TA100

TA 98

NC

(0.00)

R1

122

24

126

25

R2

118

20

119

20

R3

124

19

121

22

T1

(0.002)

R1

106

17

108

19

R2

110

19

112

17

R3

111

17

106

17

T2

(0.005)

R1

108

19

112

21

R2

102

20

108

23

R3

104

18

110

20

T3

(0.016)

R1

114

20

108

18

R2

106

22

110

16

R3

102

19

114

18

T4

(0.050)

R1

116

21

106

21

R2

112

17

114

23

R3

118

18

108

19

T5

(0.158)

R1

102

18

100

17

R2

98

17

96

18

R3

100

15

104

17

T6

(0.501)

R1

36 (+ + +)

2 (+ + +)

42 ( + + + )

4 (+ + +)

R2

26 (+ + +)

5 (+ + +)

34 (+ + +)

3 (+ + +)

R3

24 (+ + +)

3 (+ + +)

30 ( + + +)

3 (+ + +)

T7

(1.582)

R1

0 (+)

0 (+)

0 (+)

0 (+)

R2

0 (+)

0 (+)

0 (+)

0 (+)

R3

0 (+)

0 (+)

0 (+)

0 (+)

T8

(5)

R1

0 (+)

0 (+)

0 (+)

0 (+)

R2

0 (+)

0 (+)

0 (+)

0 (+)

R3

0 (+)

0 (+)

0 (+)

0 (+)

PC

R1

1088

960

1584

1242

R2

1136

992

1616

1180

R3

1168

1008

1600

1208

NC           =     Negative control

PC            =     Positive control             

R              =     Replicate

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

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

Sodium azide [10μg/plate]: TA 100,

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

 

 

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

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

16

25

126

280

R2

6

15

20

119

272

R3

8

14

22

121

260

T1

(0.002)

R1

4

13

19

108

242

R2

5

11

17

112

238

R3

5

10

17

106

232

T2

(0.005)

R1

5

12

21

112

240

R2

4

13

23

108

248

R3

4

13

20

110

252

T3

(0.016)

R1

6

14

18

108

256

R2

5

12

16

110

248

R3

4

13

18

114

264

T4

((0.050)

R1

6

15

21

106

260

R2

5

12

23

114

254

R3

5

14

19

108

268

T5

(0.158)

R1

6

14

17

100

270

R2

6

15

18

96

266

R3

5

14

17

104

258

PC

R1

168

480

1242

1584

1384

R2

186

452

1180

1616

1336

R3

170

492

1208

1600

1312

 

Dose (mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

16

24

122

276

R2

6

14

20

118

264

R3

7

13

19

124

258

T1

(0.002)

R1

5

12

17

106

232

R2

4

14

19

110

240

R3

5

11

17

111

236

T2

(0.005)

R1

6

13

19

108

242

R2

4

15

20

102

238

R3

5

13

18

104

246

T3

(0.016)

R1

5

14

20

114

240

R2

5

14

22

106

252

R3

6

15

19

102

236

T4

((0.050)

R1

6

14

21

116

254

R2

5

12

17

112

250

R3

6

13

18

118

262

T5

(0.158)

R1

6

15

18

102

256

R2

6

15

17

98

260

R3

6

14

15

100

252

PC

R1

180

1320

960

1088

1824

R2

174

1272

992

1136

1840

R3

170

1344

1008

1168

1888

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

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

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

 

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

Dose (mg/plate)

R

In the Presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

8

16

28

128

272

R2

6

14

25

123

258

R3

7

13

23

124

260

T1

(0.002)

R1

5

11

20

120

230

R2

4

10

20

118

238

R3

5

13

19

121

244

T2

(0.005)

R1

5

13

21

123

250

R2

4

12

23

120

246

R3

4

12

24

124

240

T3

(0.016)

R1

6

10

19

122

238

R2

5

14

25

119

246

R3

5

15

26

125

254

T4

((0.050)

R1

6

13

25

123

248

R2

6

12

24

124

256

R3

5

15

23

124

250

T5

(0.158)

R1

7

15

26

125

258

R2

6

14

22

123

264

R3

6

14

25

123

260

PC

R1

162

380

1344

1440

1680

R2

174

440

1360

1472

1704

R3

180

420

1384

1504

1712

 

 

Dose

(mg/plate)

R

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

NC

(0.00)

R1

7

15

26

126

260

R2

6

13

23

123

252

R3

7

13

22

120

248

T1

(0.002)

R1

4

10

21

108

232

R2

5

13

23

102

228

R3

5

11

19

104

236

T2

(0.005)

R1

4

14

18

106

234

R2

4

11

20

112

230

R3

4

12

23

110

242

T3

(0.016)

R1

5

13

24

114

248

R2

6

14

22

108

240

R3

4

12

19

111

252

T4

((0.050)

R1

6

13

23

114

250

R2

5

13

24

116

254

R3

5

14

20

120

246

T5

(0.158)

R1

6

14

24

123

258

R2

6

14

23

120

248

R3

5

13

25

121

250

PC

R1

180

1168

890

1248

1552

R2

178

1184

924

1280

1520

R3

182

1216

916

1304

1568

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

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

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

 

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

Dose (mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

7.00

1.00

15.00

1.00

22.33

2.52

122.00

3.61

270.67

10.07

T1

(0.002)

4.67

0.58

11.33

1.53

17.67

1.15

108.67

3.06

237.33

5.03

T2

(0.005)

4.33

0.58

12.67

0.58

21.33

1.53

110.00

2.00

246.67

6.11

T3

(0.016)

5.00

1.00

13.00

1.00

17.33

1.15

110.67

3.06

256.00

7.02

T4

(0.050)

5.33

0.58

13.67

1.53

21.00

2.00

109.33

4.16

260.67

8.00

T5

(0.158)

5.67

0.58

14.33

0.58

17.33

0.58

100.00

4.00

264.67

6.11

PC

174.67

9.87

474.67

20.53

1210.00

31.05

1600.00

16.00

1344.00

36.66

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

6.67

0.58

14.33

1.53

21.00

2.65

121.33

3.06

266.00

9.17

T1

(0.002)

4.67

0.58

12.33

1.53

17.67

1.15

109.00

2.65

236.00

4.00

T2

(0.005)

5.00

1.00

13.67

1.15

19.00

1.00

104.67

3.06

242.00

4.00

T3

(0.016)

5.33

0.58

14.33

0.58

20.33

1.53

107.33

6.11

242.67

8.33

T4

(0.050)

5.67

0.58

13.00

1.00

18.67

2.08

115.33

3.06

255.33

6.11

T5

(0.158)

6.00

0.00

14.67

0.58

16.67

1.53

100.00

2.00

256.00

4.00

PC

174.67

5.03

1312.00

36.66

986.67

24.44

1130.67

40.27

1850.67

33.31

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

PC= Positive control

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

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

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

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

 

TABLE 5 - MEAN REVERTANT COUNT IN PRE-INCUBATION METHOD (TRIAL II)

Dose

(mg/plate)

In the presence of Metabolic Activation (+S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

7.00

1.00

14.33

1.53

25.33

2.52

125.00

2.65

263.33

7.57

T1

(0.002)

4.67

0.58

11.33

1.53

19.67

0.58

119.67

1.53

237.33

7.02

T2

(0.005)

4.33

0.58

12.33

0.58

22.67

1.53

122.33

2.08

245.33

5.03

T3

(0.016)

5.33

0.58

13.00

2.65

23.33

3.79

122.00

3.00

246.00

8.00

T4

(0.050)

5.67

0.58

13.33

1.53

24.00

1.00

123.67

0.58

251.33

4.16

T5

(0.158)

6.33

0.58

14.33

0.58

24.33

2.08

123.67

1.15

260.67

3.06

PC

172.00

9.17

413.33

30.55

1362.67

20.13

1472.00

32.00

1698.67

16.65

 

Dose

(mg/plate)

In the Absence of Metabolic Activation (-S9)

TA 1537

TA 1535

TA 98

TA 100

TA 102

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

MEAN

SD

NC

(0.00)

6.67

0.58

13.67

1.15

23.67

2.08

123.00

3.00

253.33

6.11

T1

(0.002)

4.67

0.58

11.33

1.53

21.00

2.00

104.67

3.06

232.00

4.00

T2

(0.005)

4.00

0.00

12.33

1.53

20.33

2.52

109.33

3.06

235.33

6.11

T3

(0.016)

5.00

1.00

13.00

1.00

21.67

2.52

111.00

3.00

246.67

6.11

T4

(0.050)

5.33

0.58

13.33

0.58

22.33

2.08

116.67

3.06

250.00

4.00

T5

(0.158)

5.67

0.58

13.67

0.58

24.00

1.00

121.33

1.53

252.00

5.29

PC

180.00

2.00

1189.33

24.44

910.00

17.78

1277.33

28.10

1546.67

24.44

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

PC= Positive control

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

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

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

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

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

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

Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the negative, positive controls was tested in triplicate. Based on the solubility and precipitation test results eight different concentrations viz., 0.0 (NC), 0.002, 0.005, 0.016, 0.050, 0.158, 0.501, 1.582 and 5 mg/plate were selected for pre-experiment. Based on the pre-experiment results, the test item was tested with the following concentrations 0.0 (NC), 0.002, 0.005, 0.016, 0.050 and 0.158 mg/plate for main study, both in the presence of metabolic activation (+S9) and in the absence of metabolic activation (-S9). No substantial increase in revertant colony numbers in any of the tester strains were observed following treatment with the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

 

Endpoint:
in vitro gene mutation study in mammalian cells
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:
Data is from experimental study report.
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 test chemical 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:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
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:
other: No data
Cytotoxicity / choice of top concentrations:
other: No data
Vehicle 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
Positive controls validity:
valid
Additional information on results:
No conclusions could be drawn regarding genotoxicity or cytotoxicity of the test chemical in CHO cells in the presence of metabolic activation due to invalid positive control data.
Remarks on result:
other: No mutagenic potential was observed in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity in the presence of metabolic activation due to invalid positive control data.

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:
The test chemical tested negative for mutagenicity in CHO cells in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity of the chemical in CHO in the presence of metabolic activation due to invalid positive control data.
Executive summary:

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, chemical 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. N-ethyl-N-nitrosourea (ENU)and7,12-dimethylbenz(a) anthraceneserved as positive.The positive control used in absence of metabolic activation (i.e.N-ethyl-N-nitrosourea) produced a significant increase in the number of revertant colonies whereas the positive control used in presence of metabolic activation (i.e. 7,12 -dimethylbenz(a)anthracene) did not. Without metabolic activation, the test chemical tested negative for mutagenicity in CHO cells. No conclusions could be reached regarding the mutagenicity of the test chemical in CHO cells in the presence of the metabolic activation system.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Remarks:
Experimental data from various test chemicals
Justification for type of information:
Data for the target chemical is summarized based on the various test chemicals.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: As mentioned below
Principles of method if other than guideline:
WoE for the target CAS is summarized based on data from various test chemicals.
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
No data
Species / strain / cell type:
other: Chinese hamster lung-derived fibroblasts (CHL)
Remarks:
7
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970),

For cell lines:
- Absence of Mycoplasma contamination:
No data
- Number of passages if applicable:
4-day passages
- Methods for maintenance in cell culture:
The cell line was maintained in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum.
- Cell cycle length, doubling time or proliferation index :
The doubling time was approximately 15 hr.
- Modal number of chromosomes:
The modal chromosome number is 25
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
8
Details on mammalian cell type (if applicable):
CHO cells were grown in MEM supplemented with 10% fetal calf serum, antibiotics and sodium bicarbonate. Stock cultures were maintained in culture flasks at 37 degree Celsius in a water-saturated CO2 incubator.
Additional strain / cell type characteristics:
not specified
Cytokinesis block (if used):
7: 0.2 ug/ml colcemid was added to the culture 2 hr before cell harvesting
8: 0.1 ml of colchicine (0.01% in 2.5% Eagle's Minimal Essential Medium) was added at 16 hours post-exposure and left for 4 hours.
Metabolic activation:
with and without
Metabolic activation system:
7. no metabolic activation systems were applied
8. Aroclor-induced rat liver microsomal S9 cell fraction
Test concentrations with justification for top dose:
7. 0, 0.25 mg/ml. The top dose was expected to produce a 50% inhibition on cell growth, based on data from a pre-experiment.
8. 0 and 25 mg/ml. The top dose was half the dose that induced mitotic inhibition which was defined as one metaphase or less in 6000 CHO cells.
Vehicle / solvent:
7. DMSO
8. 2.5% Eagle's Minimal Essentil Medium (MEM supplemented with 2.5% fetal calf serum).
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
not specified
Remarks:
7
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
2.5% Eagle's Minimal Essentil Medium (MEM supplemented with 2.5% fetal calf serum).
True negative controls:
not specified
Positive controls:
other:
Remarks:
No positive controls were used, but several agents were tested for clastogenic effects in the study.
Remarks:
8
Details on test system and experimental conditions:
7. DURATION
- Exposure duration: 24 & 48 hours
SPINDLE INHIBITOR (cytogenetic assays): Colcemid
NUMBER OF CELLS EVALUATED: 100
DETERMINATION OF CYTOTOXICITY
- Method: 50% cell growth inhibition
OTHER EXAMINATIONS:
- Determination of polyploidy: yes

8. About 140,000 CHO cells were seeded on plastic dishes and kept in MEM with 10% fetal calf serum at 37°C for two to three days. Experiments were initiated when cells were confluent by 40-60%. The medium was removed from the dishes and replaced with test solutions. After 3 hours of exposure, the medium was removed, and the samples were washed with MEM, and fresh MEM was added to the dishes. Next, 0.1 ml of colchicine (0.01% in 2.5% Eagle's Minimal Essential Medium) was added at 16 hours post-exposure and left for 4 hours. Cells were then treated with 1% sodium citrate solution for 20 minutes and then immediately fixe in ethanol/acetic acid for 20 minutes. Sliders were next air-dried and stained in 2% orcein in 50% acetic acid/water, dehydrated, and mounted. 200 metaphases per sample were scored for chromosomal aberrations. The results were presented as the frequency of metaphases with chromosomal aberrations, the number of chromatid breaks per cell, and the number of chromatid exchanges per cell.


Evaluation criteria:
7. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%
8. Not specified
Statistics:
7. No data
8. Not specified
Species / strain:
other: Chinese hamster lung-derived fibroblasts (CHL)
Remarks:
7
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other:
Remarks:
Top dose was expected to produced a 50% inhibition on cell growth based on data from a pre-experiment.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
not specified
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
8
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other:
Remarks:
Top dose was half the dose that produced 100% cytotoxicity.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
other:
Remarks:
A number of chemicals tested positive without and/or with metabolic activation
Remarks on result:
other: No mutagenic potential

Study 8:

Table 1.Tabular data for solvent control, the test chemical, and for a selection of other agents that were tested for clastogenic effects in CHO cells.

Compound (conc. In mg/ml)

Activation

% metaphases with chromosome aberrations

Chromatid breaks per cell

Chromatid exchanges per cell

Solvent control (0)

With and without S9

0.7

0.01

0.00

Test chemical (10)

-S9

1.5

0.02

0.00

 

+S9

1.0

0.01

0.00

4-Methylcatechol (0.01)

-S9

20.0

0.10

0.73

 

+S9

0.0

0.00

0.00

Pyrogallol (0.1)

-S9

22.6

0.14

1.08

 

+S9

18.0

0.06

0.84

Eugenol (0.05)

-S9

0.5

0.01

0.00

 

+S9

13.9

0.17

0.24

Note: Solvent control data was presented only as mean values from the two experiments combined (with and without metabolic activation).

Conclusions:
The test chemical is not expected to produce mutachromosomal effects in Chinese hamster lung-derived fibroblasts or Chinese hamster ovary cells.
Executive summary:

The test chemical is not expected to produce mutachromosomal effects in Chinese hamster lung-derived fibroblasts or Chinese hamster ovary cells based on the data below.

 

Study 1

In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.25 mg/ml for 24 and 48 hr. DMSO was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%. The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

 

Study 2

The test chemical was tested for clastogenic effects in Chinese hamster ovary (CHO) cells at 0 (solvent control) and 25 mg/ml, with and without metabolic activation (S9). The top dose was half the dose that induced mitotic inhibition which was defined as one metaphase or less in 6000 CHO cells. 200 metaphases per sample were scored for chromosomal aberrations. The results were presented as the frequency of metaphases with chromosomal aberrations, the number of chromatid breaks per cell, and the number of chromatid exchanges per cell. No significant effect was observed following treatment with the test chemical compared to solvent control data. No positive controls were used, however, several other agents that were evaluated in the study tested clearly positive for clastogenic effects without and/or with metabolic activation. This is taken as an indication that the assay was valid for the detection of clastogenic effects in CHO cells.  

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

Genetic toxicity in vivo

Description of key information

Study 1

The test chemical was tested for clastogenic effects in mice. In the sister chromatid exchange (SCE) assay, the test chemical was given to mice in two separate trials. In trial 1, the chemical was given by oral gavage at 0 and 350 mg/kg bw (single dose). In trial 2, the test chemical was given by intraperitoneal injection at 0, 25, 50 and 100 mg/kg bw (single dose). In the chromosomal aberration assay, the test chemical was given to mice in two separate trials. In trial 1, the chemical was given to mice by oral gavage at 0 and 350 mg/kg bw (single dose). In trial 2, the test chemical was given by intraperitoneal injection at 0, 50, 100 and 200 mg/kg bw (single dose). Dose levels were selected based on the oral LD50dose for acetyl salicylic acid in mice, i.e. the oral dose was 1/3 of the oral LD50dose and the highest intraperitoneal injection dose was either 1/10 (SCE assay) or 1/5 (aberration assay) of the oral LD50dose. For all trials, the bone marrows were expelled at 24 hours after treatment to detect either SCE or chromosome aberrations (gaps excluded). The test chemical failed to produce any significant increase in the number of SCE per cell under the experimental conditions. In the chromosomal aberration assay, a weak but statistically significant positive response was observed following intraperitoneal injection at 200 mg/kg (mean, 3.5% aberrant cells at 200 mg/kg vs. mean, 1.75% at 0 mg/kg). Likewise, a weak but statistically significant positive response was observed following oral gavage treatment at 350 mg/kg (mean, 4.20% at 350 mg/kg vs. mean, 2.20% at 0 mg/kg). Treatment with the positive control cyclophosphamide at 25 mg/kg (i.p. injection) resulted in a chromosomal aberration frequency of 12.20%. The weak positive responses in the chromosome aberration assays were attributed to the ablitiy of the test chemical to biotransform to salicylic acid which per se showed no evidence of genototoxicity in the assay.

Study 2

The chemical was given via diet at 0 and 10 mM for 3 days to 1-2 day old Berlin K males. A single dose of 10 mM corresponded to the LD50 value and was regarded to be the maximally tolerated dose in the insects. Treated males were mated individually with 3 Basc virgin females. A sequence of three brood periods (each lasting 3 days) was then initiated. At the end of each 3-day breeding period, treated males were transferred to new vials and mated individually with 3 virgin females. Typically, at least 1000 F1 females were handled in each brood. Sex-linked recessive lethal mutations were scored in the F2 and F3 generations. Treatment with the test chemical resulted in no significant increase in the frequency of sex-linked recessive lethal mutation in any of the broods or in all broods combined when compared to concurrent negative control data. Treatment with 1,2 dichloroethane, which is a probable human carcinogen, at 50 mM resulted in a significant increase in the frequency of sex-linked recessive lethal mutations in brood 2 and in all broods combined when compared to control data, indicating that the assay was valid for detecting mutagenic effects.

Link to relevant study records

Referenceopen allclose all

Endpoint:
genetic toxicity in vivo, other
Remarks:
In vivo sister-chromatid exchange assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from 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 the given test chemical.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Species:
mouse
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:
Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the oral dose was 1/3 of the oral LD50 dose.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
The test chemicals were administered as a single i.p. injection 1 h after tablet implantation. Colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled
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
Control animals:
yes, concurrent vehicle
Positive control(s):
Not used.
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
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, the test chemical 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. Level of statistical significance was set at p < 0.05.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Remarks on result:
other: No mutagenic potential
Additional information on results:
No data

Table: In vivo sister chromatid exchanges induced by test chemical 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

Test chemical (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). No significant effect was observed using Student's t-test.

Conclusions:
The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice treated at 350 mg/kg bw (single dose) when compared to concurrent negative control data.
Executive summary:

In vivo sister chromatid exchange was performed to determine the mutagenic nature of the given test chemical. The study was performed using Swiss albino male mice. The test chemical was dissolved in 2% gum acacia in distilled 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, test chemical 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. The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice treated at 350 mg/kg bw (single dose) when compared to concurrent negative control data.

Endpoint:
genetic toxicity in vivo, other
Remarks:
In vivo sister chromatid exchange assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from 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 intraperitoneal route was performed to determine the mutagenic nature of the given test chemical.
GLP compliance:
not specified
Type of assay:
sister chromatid exchange assay
Species:
mouse
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:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
- Concentration of test material in vehicle: 0, 25, 50 or 100 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:
Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the highest intraperitoneal injection dose was 1/10 of the oral LD50 dose.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
The test chemicals were administered as a single i.p. injection 1 h after tablet implantation. Colchicine (4 mg/kg) was injected (i.p.) 22 h after Brdu-tablet implantation. Two hours later, the bone marrow was expelled
Remarks:
0, 25, 50 or 100 mg/Kg
No. of animals per sex per dose:
Total: 25 male mice
0 mg/Kg: 5 male mice
25 mg/Kg: 5 male mice
50 mg/Kg: 5 male mice
100 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: Intraperitoneal
- Doses / concentrations: 1.5 mg/Kg
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
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. 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 the test chemical were injected i.p. in DMSO (75 µl/mouse) to different groups of 5 animals each. Negative control mice were injected with 75 µl DMSO while mitomycin C was used as a positive control at a dose of 1.5 mg/kg of body weight

DETAILS OF SLIDE PREPARATION: 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.

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 i.p. study conducted. Level of statistical significance was set at p < 0.05.
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
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 test chemical in mice after intraperitoneal administration

Treatment

SCE/cell of 5 animals

SCE/cell

(mean ± SD)a

Replicative indices mean ± SD)a

Solvent control(DMSO)

4.5, 4.5, 4.0, 4.8, 5.5

4.66±0.55

1.86±0.09

Test chemical

 

 

 

25

5.5, 4.4, 4.5, 5.9, 4.6

4.98±0.67

1.78±0.06

50

4.5, 4.2, 5.9, 5.5, 5.4

5.10±0.71

1.88±0.09

100

4.0, 4.5, 4.9, 5.5, 5.0

4.78±0.56

1.82± 0.10

Mitomycin C (1.5 mg/Kg )

18.5, 19.5, 23.2, 18.1, 16.6

19.18±2.47

1.77±0.18

a) Mean ± SD of 5 animals (30 cells/animal).

Conclusions:
The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice i.p. treated at up to 100 mg/kg bw (single dose) when compared to concurrent negative control data. Treatment with the positive control produced expected increases in the number of SCE per cells, thus confirming the validity of the assay.
Executive summary:

In vivo sister chromatid exchange was performed to determine the mutagenic nature of the given test chemical. 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 chemical was administered as a single i.p. injection 1 h after tablet implantation. Three doses (25, 50 and 100 mg/kg) of test chemical 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. The test chemical failed to produce a significant increase in the average number of SCE per cell in bone marrow samples obtained from mice i.p. treated at up to 100 mg/kg bw (single dose) when compared to concurrent negative control data. Treatment with the positive control produced expected increases in the number of SCE per cells, thus confirming the validity of the assay.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro chromosome aberration study by the intraperitoneal route was performed to determine the mutagenic nature of the given test chemical.
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
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:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test chemical was soluble in DMSO
- Concentration of test material in vehicle: 0, 50, 100 or 200 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:
Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the highest intraperitoneal injection dose was 1/5 of the oral LD50 dose.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
After 22 hours of treatment, the animals were injected with colchicine (2 mg/kg) and 2 hours later they were sacrificed by cervical dislocation.
Remarks:
0, 50, 100 or 200 mg/Kg
No. of animals per sex per dose:
Total: 21 male mice
0 mg/Kg: 4 male mice
50 mg/Kg: 4 male mice
100 mg/Kg: 4 male mice
200 mg/Kg: 4 male mice
Positive control: 5 male mice
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide at 25 mg/kg bw
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
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.
Evaluation criteria:
Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. Chromosomal aberrations were scored following the method of WHO and previously published work. 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.
Statistics:
Statistical calculations were carried out from the percentages of aberrant cells. Student's t-test was used to compare the results of the treated series with the respective controls for chromosomal aberrations, mitotic indices and replicative indices.
Sex:
male
Genotoxicity:
positive
Remarks:
At top dose, a weak positive response was observed
Toxicity:
yes
Remarks:
At top dose, 52% degree of cytotoxicity was observed
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
No data

Table: In vivo chromosome aberration study induced by test chemical in mice after intaperitoneal administration

Treatment (mg/kg bw)

Gapsa

Aberrations/cellb

Aberrant cells % (mean±SD)c

Mitotic indices (mean±SD)c

Chromatid type

Chromosome type

Solvent control

8

 

0.015

 

0.002

 

1.75±0.50

 

2.79±0.65

Test chemical

 

 

 

 

 

50

8

0.020

0.000

2.00±0.82

3.54±0.75

100

7

0.027

0.000

2.75±1.26

3.95±0.91

250

8

0.027

0.0075

3.50±0.58**

1.69±0.33

Cyclophosphamide at 25 mg/kg bw

34

0.130

0.020

12.20±2.16

3.50±0.78

Salicylic acid

 

 

 

 

 

50

9

0.0150

0.000

1.50 ± 0.58

4.11 ± 0.69

100

7

0.0175

0.000

1.75 ± 0.50

3.46 ± 0.96

200

11

0.0150

0.000

1.75 ± 0.96

3.20 ± 0.98

a Total chromatid and chromosome gaps at each dose were recorded but not included as aberration/cell.

b Total number of aberrations (chromatid or chromosome type)/total number of cells scored per group. Results are of 4 animals (100 cells/animal).

c Results at each dose wee compared to those of the control using Student's t-test, *p < 0.02, * * p < 0.01

Conclusions:
Treatment with the test chemical at 200 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.
Executive summary:

In vivo chromosome aberration study was performed to determine the mutagenic nature of the given test chemical. The study was performed using male Swiss albino mice. Three doses (50, 100 and 200 mg/kg) were dissolved in DMSO and injected i.p. (75 µl/mouse). 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. Chromosomal aberrations were scored following previously published work. 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. Treatment with the test chemical at 200 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
Data is from peer reviewed publication
Qualifier:
according to
Guideline:
other: Refer below principle
Principles of method if other than guideline:
In vitro chromosome aberration study by the oral route was performed to determine the mutagenic nature of the test chemical
GLP compliance:
not specified
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
mouse
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: 2% gum acacia in distilled water
- 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:
Dose levels were selected based on the oral LD50 dose for acetyl salicylic acid in mice, i.e. the oral dose was 1/3 of the oral LD50 dose.
Duration of treatment / exposure:
Single dose
Frequency of treatment:
Single dose
Post exposure period:
After 22 hours of treatment, the animals were injected with colchicine (2 mg/kg) and 2 hours later they were sacrificed by cervical dislocation.
Remarks:
0 or 350 mg/Kg
No. of animals per sex per dose:
0 mg/Kg: 5 male mice
350 mg/Kg: 5 male mice
Control animals:
yes, concurrent vehicle
Positive control(s):
Not used.
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
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.
Evaluation criteria:
Mitotic indices (MI) were calculated from 1000 cells/animal and expressed as percentage. Chromosomal aberrations were scored following the method of WHO and previously published work. 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.
Statistics:
Statistical calculations were carried out from the percentages of aberrant cells. Student's t-test was used to compare the results of the treated series with the respective controls for chromosomal aberrations, mitotic indices and replicative indices.
Sex:
male
Genotoxicity:
positive
Remarks:
Weak positive response
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
No data

Table: In vivo chromosome aberration study induced by test chemical in mice after oral administration

Treatment

Gapsa

Aberrations/cellb

Aberrant cells % (mean±SD)c

Mitotic indices (mean±SD)c

Chromatid type

Chromosome type

Solvent control

9

0.022

0.00

2.20 ± 0.84

2.49 ± 0.55

Test chemical (350 mg/kg bw)

18

0.034

0.008

4.20 ± 1.09**

2.60 ± 0.37

Salicylic acid (350 mg/kg)

12

0.018

0.008

2.60 ± 0.89

3.27 ± 0.48*

a Total chromatid and chromosome gaps at each dose were recorded but not included as aberration/cell.

b Total number of aberrations (chromatid or chromosome type)/total number of cells scored per group. Results are of 5 animals (100 cells/animal).

c Results at each dose wee compared to those of the control using Student's t-test, *p < 0.05, * * p < 0.02, * * * p < 0.01.

Conclusions:
Treatment with the test chemical at 350 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.
Executive summary:

In vivo chromosome aberration study was performed to determine the mutagenic nature of the given test chemical. 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. Chromosomal aberrations were scored following the method of WHO and previously published work. 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. Treatment with the test chemical at 350 mg/kg bw produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to concurrent negative control data. The weak effect may be attributed to the ability of the test chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the study.

Endpoint:
in vivo insect germ cell study: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.5275 (Sex-linked Recessive Lethal Test in Drosophila melanogaster)
GLP compliance:
not specified
Type of assay:
Drosophila SLRL assay
Species:
Drosophila melanogaster
Strain:
other: Berlin K
Sex:
male/female
Route of administration:
oral: feed
Vehicle:
The test chemical was dissolved in 5% sucrose solution containing 2% DMSO if necessary
Details on exposure:
The test chemical was given by diet to 1-2 day old Berlin K males for 3 consecutive days, normally at the single maximally tolerated dose level (up to the LD50).
Duration of treatment / exposure:
3 days
Frequency of treatment:
daily by diet
Post exposure period:
After treatment, a sequence of three brood periods (each lasting 3 days) was then initiated. At the end of each 3-day breeding period, treated males were transferred to new vials and mated individually with 3 virgin females. Typically, at least 1000 F1 females were handled in each brood.
Remarks:
0 (vehicle and 10 mM
No. of animals per sex per dose:
Number of males not specified but at least 1000 F1 females were handled in each brood.
Control animals:
yes, plain diet
Positive control(s):
1,2 dichloroethane, which is a probable carcinogen in humans, was evaluated in addition to the test chemical for mutagenic effects in Drosophila.
Tissues and cell types examined:
X-chromosomes
Details of tissue and slide preparation:
No details given.
Evaluation criteria:
Recessive lethal mutations frequencies in brood I, II, III, and I to III, in F2 and F3 generations, following treatment with the test chemical in males, were evaluated against solven control data using statistical analysis.
Statistics:
Kastenbaum-Bowman test
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid

Table 1. Data for solvent control and for the positive control (1,2-dichloroethane at 50 mM).

Conc.

Brood

Days after treatment

Number of X-chromosomes tested

Number of lethals

Recessive lethal mutation frequency (%)

P values

0

I

0-3

9565

24

0.25

 NS

0

II

4-6

6500

4

0.06

 NS

0

III

7-9

5983

19

0.32

 NS

0

I-III

0-9

22048

47

0.21

 NS

50

I

0-3

1185

6

0.51

 NS

50

II

4-6

1179

41

3.48

<0.01

50

III

7-9

156

2

1.28

 NS

50

I-III

0-9

2520

49

1.94

<0.01

NS = not statistically significant.

Note 1: The relatively low mutation frequency in brood II at 0 mM (of 0.06%) was consistent with historical control data.

Note 2: No tabular data was presented for the test chemical at 10 mM but it was concluded by the authors to be non-mutagenic in the text.

Conclusions:
Three days treatment with the test chemical Drosophila at LD50 resulted in no significant increase in the frequency of sex-linked recessive lethal mutations in any of the broods or in all broods combined when compared to concurrent negative control data.
Executive summary:

The chemical was given via diet at 0 and 10 mM for 3 days to 1-2 day old Berlin K males. A single dose of 10 mM corresponded to the LD50 value and was regarded to be the maximally tolerated dose in the insects. Treated males were mated individually with 3 Basc virgin females. A sequence of three brood periods (each lasting 3 days) was then initiated. At the end of each 3-day breeding period, treated males were transferred to new vials and mated individually with 3 virgin females. Typically, at least 1000 F1 females were handled in each brood. Sex-linked recessive lethal mutations were scored in the F2 and F3 generations. Treatment with the test chemical resulted in no significant increase in the frequency of sex-linked recessive lethal mutation in any of the broods or in all broods combined when compared to concurrent negative control data. Treatment with 1,2 dichloroethane, which is a probable human carcinogen, at 50 mM resulted in a significant increase in the frequency of sex-linked recessive lethal mutations in brood 2 and in all broods combined when compared to control data, indicating that the assay was valid for detecting mutagenic effects.

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

Additional information

Data available from various sources was reviewed to determine the mutagenic nature of the given test chemical. The studies are as mentioned below:

Ames assay:

 Ames assay was performed to investigate the potential of the given test chemical to induce gene mutations in comparison to negative control according to the plate incorporation test (Trial I) and the pre-incubation test (Trial II) using 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 the given test chemical at any dose level in both the confirmatory trials, neither in the presence nor in the absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. The spontaneous reversion rates in the negative, positive controls are within the range of our historical data. The positive controls used for various strains showed a distinct in­crease in induced revertant colonies in both the methods i.e. Plate incorporation method and Pre-incubation method. In conclusion, it is stated that during the described mutagenicity test and under the experimental conditions reported, the test chemical did not induce gene mutations by base pair changes or frame shifts in the genome of the strains used.

In vitro mammalian chromosome aberration study:

In vitro mammalian chromosome aberration study was conducted for the given test chemical in Chinese hamster lung-derived fibroblasts (CHL) in the absence of metabolic activation system. The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo (Koyama, Utakoji & Ono, 1970), and was maintained by 4-day passages in Minimum Essential Medium (MEM; GIBCO) supplemented by 10% calf serum. The modal chromosome number is 25 and the doubling time was approximately 15 hr. The cells were exposed to chemical at three concentrations up to 0.25 mg/ml for 24 and 48 hr. DMSO was used as solvent. The top dose was expected to produce a 50% inhibition on cell growth based on data from a pre-experiment. Colcemid (final conc 0.2 microgm/ml) was added to the culture 2 hr before cell harvesting. The cells were then trypsinized and suspended in a hypotonic KCI solution (0.075 M) for 13 min at room temperature. After centrifugation the cells were fixed with acetic acid-methanol (1:3, v/v) and spread on clean glass slides. After air-drying, the slides were stained with Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 min. A hundred well-spread metaphases were observed under the microscope (x 600 with a nocover objective lens). The incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. Results were considered negative if incidence of aberrations was less than 4.9%, equivoval if it was between 5 and 9.9% and positive if it was more than 10%.The test chemical tested negative for mutachromosomal effects in Chinese hamster lung-derived fibroblasts (CHL).

 

The test chemical was tested for clastogenic effects in Chinese hamster ovary (CHO) cells at 0 (solvent control) and 25 mg/ml, with and without metabolic activation (S9). The top dose was half the dose that induced mitotic inhibition which was defined as one metaphase or less in 6000 CHO cells. 200 metaphases per sample were scored for chromosomal aberrations. The results were presented as the frequency of metaphases with chromosomal aberrations, the number of chromatid breaks per cell, and the number of chromatid exchanges per cell. No significant effect was observed following treatment with the test chemical compared to solvent control data. No positive controls were used, however, several other agents that were evaluated in the study tested clearly positive for clastogenic effects without and/or with metabolic activation. This is taken as an indication that the assay was valid for the detection of clastogenic effects in CHO cells.  

In vitro gene mutation study in mammalian cells

An in vitro mammalian cell gene mutation study was designed and conducted to determine the genotoxicity profile of the given test chemical when administered to Chinese Hamster Ovary (CHO) cells. In the genotoxicity test, chemical 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. N-ethyl-N-nitrosourea (ENU)and7,12-dimethylbenz(a) anthraceneserved as positive.The positive control used in absence of metabolic activation (i.e.N-ethyl-N-nitrosourea) produced a significant increase in the number of revertant colonies whereas the positive control used in presence of metabolic activation (i.e. 7,12 -dimethylbenz(a)anthracene) did not. Without metabolic activation, the test chemical tested negative for mutagenicity in CHO cells. No conclusions could be reached regarding the mutagenicity of the test chemical in CHO cells in the presence of the metabolic activation system.

Justification for classification or non-classification

Sodium salicylate has tested negative for mutagenicity in bacteria in a study according to OECD Test Guideline 471. Read-across chemical No.: 1 has tested negative for clastogenic effects in CHO cells with and without metabolic activation. In mice, treatment with sodium salicylate produced a weak increase in the frequency of chromosomal aberrations in bone marrow samples when compared to solvent control data. This weak response was attributed to the ability of the chemical to biotransform to salicylic acid which per se showed no evidence of genotoxicity in the same study. In an in vitro mammalian gene mutation assay, treatment with sodium salicylate tested negative for mutagenicity in CHO cells in the absence of metabolic activation. No conclusions could be drawn regarding the mutagenicity of the chemical in the presence of metabolic activation due to invalid positive control data. However, in a sex-linked recessive lethal test in Drosophila melanogaster, three days of dosing with read-across chemical No.: 2 at LD50 failed to produce any significant increase in the frequency of sex-linked recessive lethal mutations. Therefore, by weight of evidence, sodium salicylate is regarded to be classified as Not Classified for Germ cell mutagenicity.