Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce mutation in the Salmonella typhimurium strains both in the presence and absence of S9 metabolic activation system and hence is not likely to be mutagenic under the conditions of this study.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
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 report.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
To evaluate the mutagenic potential of test chemical in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test.
GLP compliance:
not specified
Type of assay:
bacterial forward mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 100, TA 1537, TA 98
Details on mammalian cell type (if applicable):
Not applicable.
Additional strain / cell type characteristics:
other: All strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
Cytokinesis block (if used):
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 mix was prepared from Sprague-Dawley rats livers induced with Aroclor 1254.
Test concentrations with justification for top dose:
1st Experiment
Strains: TA 1535. TA 100. TA 1537, TA 98
Dose.: 0, 20, 100, 500. 2500 and 5000 µg/plate

2nd Experiment
Strains: TA 100, TA 98
Domes: 0, 20. 100, 500 and 1000 µg/plate

3rd Experiment
Strain: TA 100
Doses: 0, 50, 100, 500, 1000 and 2500 µg/plate

4th Experiment
Strains: TA 1535, JA 1537
Doses: 0, 20, 100, 500, 1000 and 1500 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Complete solubility of the test substance in DMSO.
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: + S-9 mix 10 µg 2-aminoanthracene for the strains TA 100, TA 98, TA 1537 and TA 1535 - S-9 mix 5µg N-methyl-N-nitro-Nnitroso;TA 100 and TA 1535 10 µg 4 -nitro-o-phenylenediamine ;strain TA 98 100 µg 9-aminoacridine chloride monohydrate ; TA 1537.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Standard Plate method
- Cell density at seeding (if applicable): >108 bacteria/ml

DURATION
- Preincubation period: 16 hours
- Exposure duration:2days

DETERMINATION OF CYTOTOXICITY
- Method: A bacteriotoxic effect OTHER EXAMINATIONS:
Evaluation criteria:
A substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate
- dose-response relationship
- reproducibility of the results.
Statistics:
Yes ,,Mean ±standard deviation was observed.
Key result
Species / strain:
S. typhimurium, other: TA 1535, TA 100, TA 1537 and TA 98.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Positive controls validity:
other: As though the test substace was used as a positive control
Additional information on results:
No mutagenic effect were observed in the salmonella typhimurium strain TA 100 used in expriment number three , when exposed to test substance.
Remarks on result:
other: No mutagenic effect were observed.

TABLE 1

STRAIN: TA 1535

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

 

DOSE

MCG/PL

REVERTANTS/PLATES

TITER

DIL

QUOTIENT

-S9

M

SD

+S9*

M

SD

EXP-6

-S9

+S9*

NEGATIVE CONTROL

DMSO

15

18

21

18

3

21

17

19

19

 

2

45

41

3

1.0

1.0

20

18

16

14

16

2

21

21

26

23

3

 

0.9

1.2

100

18

20

14

17

3

22

22

24

23

1

 

1.0

1.2

500

20

24

21

22

2

28

28

24

27

2

 

1.2

1.4

2500

B

B

B

-

-

13B

12B

8B

11

3

0

0

0

-

0.6

5000

B

B

B

-

-

B

B

B

-

-

0

0

0

-

-

POSITIVE CONTROL

2-AA

10

 

 

 

320

265

242

276

40

 

 

14.5

POSITIVE CONTROL

MNNG

5

1960

2350

2300

2203

212

 

 

 

 

122.4

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND           

           

 

 

 

 

 

 

 

TABLE 2

STRAIN: TA 100

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

DOSE

MCG/PL

                         REVERTANTS/PLATES         

TITER

DIL

QUOTIENT

-S9

M

SD

+S9*

M

SD

EXP-6

-S9

+S9*

NEGATIVE CONTROL

DMSO

94

109

117

107

12

110

104

107

107

3

15

28

11

1.0

1.0

20

113

119

111

114

4

102

118

125

115

12

 

1.1

1.1

100

116

117

111

112

7

119

125

113

119

6

 

1.1

1.1

500

113

124

146

128

17

137

139

138

138

1

 

1.2

1.3

2500

B

B

B

-

-

74B

73B

80B

76

4

0

0

0

-

0.7

5000

B

B

B

-

-

B

B

B

-

-

0

0

0

-

-

POSITIVE CONTROL

2-AA

10

 

 

 

1780

1860

1910

1850

66

 

 

17.3

POSITIVE CONTROL

MNNG

5

1740

1930

1850

1840

95

 

 

 

 

17.3

 

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND                       

 

 

 

 

 

 

TABLE 3

STRAIN: TA 1537

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

DOSE

MCG/PL

                         REVERTANTS/PLATES         

TITER

DIL

QUOTIENT

-S9

M

SD

+S9*

M

SD

EXP-6

-S9

+S9*

NEGATIVE CONTROL

DMSO

13

13

10

12

2

8

10

10

9

1

16

13

11

1.0

1.0

20

9

13

8

10

3

15

11

8

11

4

 

0.8

1.2

100

11

10

11

11

1

8

10

8

9

1

 

0.9

0.9

500

16

9

11

12

4

9

9

16

11

4

 

1.0

1.2

2500

B

B

B

-

-

5B

8B

9B

7

2

0

0

0

-

0.8

5000

B

B

B

-

-

B

B

B

-

-

0

0

0

-

-

POSITIVE CONTROL

2-AA

10

 

 

 

199

194

195

196

3

 

 

21.0

POSITIVE CONTROL

MNNG

5

744

661

833

746

86

 

 

 

 

62.2

 

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND                       

 

 

 

 

 

 

TABLE 4

STRAIN: TA 98

AMES TEST WITH: 86/221

METHOD: STANDARD PLATE TEST

DOSE

MCG/PL

                         REVERTANTS/PLATES         

TITER

DIL

QUOTIENT

-S9

M

SD

+S9*

M

SD

EXP-6

-S9

+S9*

NEGATIVE CONTROL

DMSO

22

21

21

21

1

39

32

31

34

4

62

53

57

1.0

1.0

20

21

29

22

24

4

31

34

32

32

2

 

1.1

1.0

100

21

21

-

21

0

40

32

29

34

6

 

1.0

1.0

500

20

24

23

22

2

35

30

28

31

4

 

1.0

0.9

2500

B

B

B

-

-

17B

23B

17B

19

3

0

0

0

-

0.6

5000

B

B

B

-

-

B

B

B

-

-

0

0

0

-

-

POSITIVE CONTROL

2-AA

10

 

 

 

1992

1800

2000

1907

101

 

 

56.1

POSITIVE CONTROL

MNNG

5

781

839

899

840

59

 

 

 

 

39.4

 

 

* : S-9 FRACTION/COFACTORS = 3: 7         EXP :EXP. TO 10

B: REDUCED his BACKGROUND                       

 

Table – 5

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE

MCG/ PL

REVERTANTS / PLATE

QUOT IENT

-S9

M

SD

+S9*

M

SD

-S9

+S9*

NEGATIVE

CONTROL

DMSO

116

114

10

105

103

8

1.0

1.0

103

 

 

94

 

 

 

 

123

 

 

109

 

 

 

 

20

99

102

13

115

110

8

0.9

1.1

91

 

 

101

 

 

 

 

117

 

 

113

 

 

 

 

100

93

103

10

97

104

8

0.9

1.0

112

 

 

104

 

 

 

 

103

 

 

112

 

 

 

 

500

109

111

10

112

106

6

1.0

1.0

102

 

 

103

 

 

 

 

121

 

 

102

 

 

 

 

1000

93

98

9

141

129

10

0.9

1.3

92

 

 

123

 

 

 

 

108

 

 

124

 

 

 

 

POSITIVE

CONTROL

2-AA

 

 

 

2100

2033

76

 

19.8

 

 

 

2050

 

 

 

 

 

 

 

1950

 

 

 

 

10

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

1670

1703

42

 

 

 

14.9

 

1750

 

 

 

 

 

 

 

1690

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

* : S9 FRACTION/COFACTORS = 3. 7

 

Table – 6

AMES TEST WITH 86/33

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE

MCG/ PL

REVERTANTS / PLATE

QUOT IENT

-S9

M

SD

+S9*

M

SD

-S9

+S9*

NEGATIVE

CONTROL

DMSO

116

114

10

105

103

8

1.0

1.0

103

 

 

94

 

 

 

 

123

 

 

109

 

 

 

 

20

122

116

6

117

113

6

1.0

1.1

113

 

 

-

 

 

 

 

112

 

 

108

 

 

 

 

100

96

110

21

125

132

8

1.0

1.3

100

 

 

140

 

 

 

 

134

 

 

132

 

 

 

 

500

127

128

17

137

152

17

1.1

1.5

112

 

 

148

 

 

 

 

145

 

 

171

 

 

 

 

1000

149

145

7

207

206

2

1.3

2.0

150

 

 

206

 

 

 

 

137

 

 

204

 

 

 

 

POSITIVE

CONTROL

2-AA

 

 

 

2100

2033

76

 

19.8

 

 

 

2050

 

 

 

 

 

 

 

1950

 

 

 

 

10

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

1670

1703

42

 

 

 

14.9

 

1750

 

 

 

 

 

 

 

1690

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

* : S9 FRACTION/COFACTORS = 3. 7 - : CONTAMINATION

Table – 7

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA 98

DOSE

MCG/ PL

REVERTANTS / PLATE

QUOT IENT

-S9

M

SD

+S9*

M

SD

-S9

+S9*

NEGATIVE

CONTROL

DMSO

25

23

2

35

34

4

1.0

1.0

22

 

 

38

 

 

 

 

21

 

 

30

 

 

 

 

20

30

25

6

34

32

2

1.1

0.9

25

 

 

32

 

 

 

 

19

 

 

31

 

 

 

 

100

24

20

4

40

38

3

0.7

1.1

19

 

 

36

 

 

 

 

17

 

 

-

 

 

 

 

500

17

18

2

29

29

2

0.8

0.8

20

 

 

30

 

 

 

 

17

 

 

27

 

 

 

 

1000

12

10

3

33

30

3

0.5

0.9

7

 

 

30

 

 

 

 

12

 

 

27

 

 

 

 

POSITIVE

CONTROL

2-AA

 

 

 

1433

1420

26

 

41.4

 

 

 

1440

 

 

 

 

 

 

 

1390

 

 

 

 

10

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

1060

1150

95

 

 

 

50.7

 

1140

 

 

 

 

 

 

 

1250

 

 

 

 

 

 

 

10

 

 

 

 

 

 

 

 

* : S9 FRACTION/COFACTORS = 3. 7 - : CONTAMINATION

Table – 7

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE

MCG/ PL

REVERTANTS / PLATE

TITER DIL

QUOT IENT

-S9

M

SD

+S9*

M

SD

EXP-6

-S9

+S9*

NEGATIVE

CONTROL

DMSO

117

119

8

110

108

3

14

1.0

1.0

128

 

 

104

 

 

15

 

 

112

 

 

109

 

 

18

 

 

50

91

105

16

121

113

7

 

0.9

1.0

122

 

 

109

 

 

 

 

 

101

 

 

108

 

 

 

 

 

100

123

131

7

103

124

26

 

1.1

1.1

132

 

 

115

 

 

 

 

 

137

 

 

153

 

 

 

 

 

500

113

126

14

108

129

18

 

1.1

1.2

124

 

 

140

 

 

 

 

 

140

 

 

138

 

 

 

 

 

1000

127

122

8

139

154

13

22

1.0

1.4

113

 

 

162

 

 

31

 

 

127

 

 

162

 

 

17

 

 

5000

B

-

-

77

74

9

21

-

0.7

B

 

 

81

 

 

16

 

 

B

 

 

63

 

 

11

 

 

POSITIVE

CONTROL

2-AA

 

 

 

1660

1630

79

 

 

15.1

 

 

 

1690

 

 

 

 

 

 

 

 

1540

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

2150

2015

191

 

 

 

 

16.9

 

1880

 

 

 

 

 

 

 

 

-

 

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

 

 

*: S59 FRACTION/COFACTORS = 3.7 EXP : EXP. T0 10

-: CONTAMINATION B: REDUCED his- BACKOROUND

Table – 9

AMES TEST WITH 87/33

METHOD: STANDARD PLATE TEST STRAIN: TA 100

DOSE

MCG/ PL

 

REVERTANTS / PLATE

QUOT IENT

-S9

M

SD

+S9*

M

SD

TITER DIL Exp-6

-S9

+S9*

NEGATIVE

CONTROL

DMSO

117

119

8

110

108

3

14

1.0

1.0

128

 

 

104

 

 

15

 

 

112

 

 

109

 

 

18

 

 

50

127

119

7

120

119

8

 

1.0

 

115

 

 

126

 

 

 

 

 

114

 

 

110

 

 

 

 

 

100

116

113

3

136

132

5

 

0.9

1.2

110

 

 

127

 

 

 

 

 

113

 

 

133

 

 

 

 

 

500

135

141

9

132

156

26

 

1.2

1.4

152

 

 

152

 

 

 

 

 

137

 

 

183

 

 

 

 

 

1000

182

173

18

157

170

19

33

1.5

1.6

153

 

 

192

 

 

31

 

 

137

 

 

161

 

 

34

 

 

2500

211

210

16

235

250

13

26

1.8

2.3

225

 

 

253

 

 

32

 

 

193

 

 

261

 

 

31

 

 

POSITIVE

CONTROL

2-AA

 

 

 

1660

1630

79

 

 

15.1

 

 

 

1690

 

 

 

 

 

 

 

 

1540

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

2150

2015

191

 

 

 

 

16.9

 

1880

 

 

 

 

 

 

 

 

-

 

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table – 10

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA1535

 

DOSE

MCG/ PL

 

REVERTANTS / PLATE

QUOT IENT

-S9

M

SD

+S9*

M

SD

TITER DIL Exp-6

-S9

+S9*

NEGATIVE

CONTROL

DMSO

20

19

3

23

20

3

49

1.0

1.0

16

 

 

17

 

 

44

 

 

21

 

 

19

 

 

51

 

 

20

21

23

2

17

17

2

 

1.2

0.8

24

 

 

15

 

 

 

 

 

23

 

 

18

 

 

 

 

 

100

20

20

2

19

17

2

 

1.1

0.8

22

 

 

16

 

 

 

 

 

18

 

 

15

 

 

 

 

 

500

18

19

3

19

20

3

 

1.0

1.0

17

 

 

18

 

 

 

 

 

23

 

 

24

 

 

 

 

 

1000

22

20

3

18

18

1

55

1.1

0.9

17

 

 

17

 

 

64

 

 

21

 

 

19

 

 

48

1.2

1.0

1500

24

23

1

20

20

4

56

 

 

22

 

 

24

 

 

57

 

 

22

 

 

17

 

 

48

 

 

POSITIVE

CONTROL

2-AA

 

 

 

577

481

140

 

 

24.4

 

 

 

545

 

 

 

 

 

 

 

 

320

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

2050

2233

176

 

 

 

 

117.5

 

2250

 

 

 

 

 

 

 

 

2400

 

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table – 11

AMES TEST WITH 86/221

METHOD: STANDARD PLATE TEST STRAIN: TA1535

 

DOSE

MCG/ PL

 

REVERTANTS / PLATE

QUOT IENT

-S9

M

SD

+S9*

M

SD

TITER DIL Exp-6

-S9

+S9*

NEGATIVE

CONTROL

DMSO

14

14

2

14

15

1

33

1.0

1.0

12

 

 

15

 

 

27

 

 

16

 

 

15

 

 

27

 

 

20

15

15

1

14

14

1

 

1.1

1.0

14

 

 

13

 

 

 

 

 

16

 

 

15

 

 

 

 

 

100

14

13

1

13

13

3

 

1.0

0.9

13

 

 

16

 

 

 

 

 

13

 

 

10

 

 

 

 

 

500

15

13

2

10

13

3

 

0.9

0.9

13

 

 

15

 

 

 

 

 

11

 

 

13

 

 

 

 

 

1000

10

11

2

12

13

2

32

0.8

0.9

11

 

 

12

 

 

33

 

 

13

 

 

16

 

 

44

 

 

1500

12

14

2

14

15

2

24

1.0

1.0

14

 

 

18

 

 

32

 

 

15

 

 

14

 

 

30

 

 

POSITIVE

CONTROL

2-AA

 

 

 

203

221

16

 

 

15.1

 

 

 

234

 

 

 

 

 

 

 

 

226

 

 

 

 

 

10

 

 

 

 

 

 

 

 

 

POSITIVE

CONTROL

MNNG

661

702

109

 

 

 

 

50.1

 

825

 

 

 

 

 

 

 

 

619

 

 

 

 

 

 

 

 

5

 

 

 

 

 

 

 

 

 

 

 

Conclusions:
Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. Test chemical, which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test result was considered to be negative in the presence and absence of metabolic activation.
Executive summary:

Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. The test chemical, which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test result was considered to be negative in the presence and absence of metabolic activation.Genetic toxicity study was assessed for its possible mutagenic potential. For this purpose AMES test was performed according to guideline OECD 471. The test material was exposed at different concentration in 4 individual experiments. These concentrations are mentioned below :

1st Experiment

Strains: TA 1535. TA 100. TA 1537, TA 98

Doses: 0, 20, 100, 500. 2500 and 5000 µg/plate

 

2nd Experiment

Strains: TA 100, TA 98

Doses: 0, 20. 100, 500 and 1000 µg/plate

 

3rd Experiment

Strain: TA 100

Doses: 0, 50, 100, 500, 1000 and 2500 µg/plate

 

4th Experiment

Strains: TA 1535, JA 1537

Doses: 0, 20, 100, 500, 1000 and 1500 µg/plate

The test material which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test material was exposed in the presence and absence of metabolic activation. No mutagenic effects were observed in any of the strain. Thereforetest chemical was considered to be non mutagenic in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. Hence the substance cannot be classified as gene mutant in vitro.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Data available for the test chemicals were reviewed to determine the toxic nature of the test chemical. The studies are as mentioned below:

1)Test chemical was evaluated for its mutagenic potential in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. The test chemical, which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test result was considered to be negative in the presence and absence of metabolic activation.Genetic toxicity study was assessed for its possible mutagenic potential. For this purpose AMES test was performed according to guideline OECD 471. The test material was exposed at different concentration in 4 individual experiments. These concentrations are mentioned below :

1st Experiment

Strains: TA 1535. TA 100. TA 1537, TA 98

Doses: 0, 20, 100, 500. 2500 and 5000 µg/plate

 2nd Experiment

Strains: TA 100, TA 98

Doses: 0, 20. 100, 500 and 1000 µg/plate

 3rd Experiment

Strain: TA 100

Doses: 0, 50, 100, 500, 1000 and 2500 µg/plate

 4th Experiment

Strains: TA 1535, JA 1537

Doses: 0, 20, 100, 500, 1000 and 1500 µg/plate

The test material which is known to be positive in the Ames test, was investigated in parallel as a positive control substance (chemical class control) using the strain TA 100. The test material was exposed in the presence and absence of metabolic activation. No mutagenic effects were observed in any of the strain. Thereforetest chemicalwas considered to be non mutagenic in Salmonella typhimurium strain TA 1535, TA 100, TA 1537, TA 98 by Ames test. Hence the substance cannot be classified as gene mutant in vitro.

2)The spot test was performed by on Salmonella Typhimurum with strain TA1530 (hisG46, Δgal, ΔuvrB), TA1535 (hisG46, rfa, ΔuvrB) and T100 (hisG46, rfa, ΔuvrB, pKM101) for the test compound . Spot tests were performed by spreading of a stationary cell suspension (ca. 109/ml) of the indicator strains TA1530, TA1535 or TA100 on mutation plates (containing traces of L-histidine), followed by the addition of the desired amount of the test chemical in the middle of the plate. The plates were then incubated for 48 h at 37°C, and the number of his + revertant colonies were determined. An increase in the number of revertants was noted with the test compound and hence is likely to classify for gene mutation in vitro.

3) Salmonella typhimurium mutation test was performed to determine the mutagenic nature of the test compound. The study was performed using Salmonella typhimurium strain TA1535 both in the presence and absence of S9 metabolic activation system.The test compound induced base pair substitution in Salmonella typhimurium strain TA1535 and hence is likely to be mutagenic in nature.

4)Bacterial gene mutation assay was performed to determine the mutagenic nature of the test compound. Preincubation assay was performed in the Salmonella typhimurium strain TA98, TA100, TA1535 and TA1537 and E. col Wp2 uvrA in the presence and absence of S9 metabolic activation system at doses of 0.0763-5000 µg/plate.The test compound induced mutation in the Salmonella typhimurium strains TA1535, E. col Wp2 uvrA (±S9; at all doses) and TA100 (+ S9; 9.77- 1250µg/plate) and failed to induce mutation in the Salmonella typhimurium strains TA98, TA100, and TA1537.

5)The aim of the study was to determine the mutagenic activity of test chemical in Salmonella typhimurium NM5004 strain by introducing a plasmid containing both rat GSH glutathione S-transferase (GST) 5-5 cDNA and the umuC lacZ operon into the host strain Salmonella typhimurium TA1535 and used to examine whether or not GST modified the genotoxic activities of test chemical. Liver microsomes was obtained from Male Sprague-Dawley rats. The bacterial suspension was incubated with 0.1mM test chemical (dissolved in DMSO) at 37 °C for 2 h and the expressed β-galactosidase activity was determined by the method of Miller and expressed in those units or absorbance at 420 nm using o-nitrophenyl-β-D-galactopyranoside as a substrate. The genotoxicities of chemical carcinogens and mutagens were determined by measuring induction of an umu gene expression and Ames test in the two Salmonella tester strains. Cytotoxicity was observed to be 65 -80% in GST activated gene and 50 -60% in GST inactivated gene in Salmonella typhimurium NM5004 tester strain. 0 -40 revertants were observed in Salmonella typhimurium TA1535/pSK1002 strain which is considered to be negligible. The test chemical gave different responses in the two tester strains, but it could not be defined whether these chemicals are activated or inactivated by GST 5-5. Thus, the test chemical can be considered negative as satisfactory results to understand the classification were not obtained.

6) In Gene mutation study, a plasmid vector containing a cDNA for the rat f3 class GSH S-transferase (GST)5-5 was constructed and expressed in the normal [GST 5-5(+)] or in the reverse orientation [GST 5-5(-)] in SalmonellatyphimuriumTA1535 strain to observe the mutagenic nature of the test chemical. The experiment was carried out according to preincubation assay. Dibromomethane was used as a reference point for mutation system because it expressed the GST 5-5 (+) gene. Very small number of revertants were observed after the experiment. Therefore, The test chemical was a very weak mutagen in both the GST 5-5(+) and GST 5-5(-) S. typhimurium TA 1535 strains.

Based on the data available, the test chemical did not induce gene mutation in sallmonela strain in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosomal Abberation:

In vitro chromosomal aberration assay was performed to determine the mutagenic nature of the test compound both in the presence and absence of S9 metabolic activation system. The test chemical was used at dose levels of 0.05-0.2 mg/mL using CHL cells in 6 hr treatment, 18 hr recovery period (With metabolic activation) and 0.025-0.2 mg/mL using CHL cells in 24 hr and 48 hr continuous treatment period.The test compound induced structural changes in 6 hr treatment, 18 hr recovery period and structural changes in 24 hr continuous treatment period and structural and ploidy changes in the CHL cells in 48 hrs continuous treatment period and hence is positive for gene mutation in vitro.

Based on the data available, the test chemical induced chromosome aberrations in Chinese hamster cell line in the presence and absence of S9 metabolic activation system and hence it is likely to classify as a gene mutant in vitro.

Justification for classification or non-classification

Based on the experimental data available from the latest study, the target substance cannot be classified as mutagenic in nature. As though other supporting studies reviewed for the target has the positive result for gene mutation .These supporting studies have used low concentration in experiment and also detailed information are not available as per guideline. Considering all these factors for classification the target substance cannot be classified as mutant in nature. Thus the test chemical is not likely to classify as a gene mutant in vitro.