Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 11, 1992 to March 30, 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guidelines for Testing of Chemicals "Genetic Toxicology: Salmonella typhimurium, Reverse Mutation Assay" Adopted: 26 May 83, No. 471
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 84/449/EEC B.14. Other Effects – Mutagenicity Salmonella typhimurium Reverse Mutation Test
Qualifier:
according to guideline
Guideline:
other: M. J. Prival, V. D; Mitchell
Version / remarks:
M. J. Prival, V. D; Mitchell: Analysis of a method for testing azo dyes for mutagenicity in Sallmonella typhimurium in the presence of flavin mononucleo­ tide and hamster liver S-9. Mut. Res.,103- 116 (1982).
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt
EC Number:
286-839-5
EC Name:
7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt
Cas Number:
85391-83-9
Molecular formula:
C21H14ClF2N5O8S2.xNa
IUPAC Name:
7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt
Test material form:
solid: particulate/powder
Details on test material:
Reactive Red 123

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 μg /plate was used as the highest dose.
The following doses per pate were evaluated in the first test:
0, 8, 40, 200, 1000, 5000 μg/plate
Due to the substance's weak toxicity, doses ranging from 8 μg to 5000 μg per tube were chosen for the repeat tests.
The following doses per plate were evaluated in repeat tests:
0, 8, 40, 200, 1000, 5000 μg/plate
Vehicle / solvent:
Levafix Scharlach E-2GA was dissolved in deionized water.
The solvent used was chosen out of the following solvents, in the order given: water, methanol, ethanol, acetone &, DMSO, DMF, and ethylene glycol dimethylether according to information given by the internal sponsor.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
congo red
other: Nitrofurantoin (NF); 4-nitro-1,2-phenylene diamine (4-NPDA); 2-Aminoanthracene (2-AA); Benzidine
Details on test system and experimental conditions:
Test Design
For the mutant count, four plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained four plates per strain. The amount of solvent for the test substance and for the controls was 0.1 ml/plate.

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 μg /plate was used as the highest dose. At least four additional doses were routinely used as progressive dilutions of the top dose. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtain in the first experiment.

Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound. Preincubation was performed in a water bath at 30 °C for 30 minutes. At the end of the preincubation period 2 ml of molten soft agar were added to the tubes, the content mixed and plated.

For the mutant count, four plates were used for each strain and dose. An equal-number of plates, filled with the solvent minus the test substance, comprised the negative control.
Each positive control also contained four plates per strain. In experiments without S9 mix buffer was used as replacement.

The doses of this trial were determined on the basis of the results of the plate incorporation assay.

The toxicity of the substance was assessed in three ways.
The first method was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables by the letter "b" after the mutant count. Where only a single "b", without any other values, is noted for a concentration, this "b" represents four plates with reduced background growth. (The same applies to the signs "c", "v", "p", "n" or "%", which may also be used in the tables.) Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls. Thirdly, the titer, was determined. Total bacterial counts were taken on two plates for each concentration studied with S9 mix. However, if an evaluation was performed only without S9 mix, the bacterial count was taken without S9 mix.

The bacterial suspensions were obtained from 17-hour cultures in nutrient broth, which had been incubated at 37 °C and 90 rpm. These suspensions were used for the determination of mutant counts. No standardized procedure was employed to set S the bacterial suspensions at a defined density of viable cells per millilitre, since the chosen method of incubation normally produces the desired density. However, the numbers of viable cells were established in a parallel procedure by determining the titers.

The dilution of bacterial suspensions used for the determination of titers was 1:1,000,000. Titers were determined under the same conditions as were the mutations, except that the histidine concentration in the soft agar was increased from 0.5 mM to 2.5 mM to permit the complete growth of bacteria.

The tests were performed both with and without S9 mix.

The count was made after the plates had been incubated for 48 hours at 37 °C. If no immediate count was possible, plate were temporarily stored in a refrigerator.
Rationale for test conditions:
The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound.
Evaluation criteria:
The following criteria determined the acceptance of an assay:
a) The negative controls had to be within the expected range, as defined by published data (e.g. Maron and Ames, 1983) and/or the laboratories' own historical data.
b) The positive controls had to show sufficient effects, as defined by the laboratories' experience.
c) Titer determinations had to demonstrate sufficient bacterial density in the suspension.

An assay which did not comply with at least one of the above criteria was not used for assessment. Furthermore, the data generated in this assay needed to be confirmed by two additional independent experiments. Even if the criteria for points (a), (b) and (c) were it met, an assay was accepted if it showed mutagenic activity of the test compound.

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result.
For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.

In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
bacteriotoxic effects at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
bacteriotoxic effects at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
bacteriotoxic effects at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
bacteriotoxic effects at 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Plate Incorporation Method
There was no indication of a bacteriotoxic effect of Levafix Scharlach E-2GA at doses of up to and including 1000 μg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 μg per plate had a weak, strain-specific bacteriotoxic effect, but could nevertheless be used for assessment.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix.

Prival Assay
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.
There was no indication of a bacteriotoxic effect of Levafix Scharlach E-2GA at doses of up to and including 1000 μg per tube. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. 5000 μg per plate, a weak, strain-specific bacteriotoxic effect, but could nevertheless be used for assessment.
None of the four strains concerned showed a dose-related and biologically relevant increase in mutant counts over those of the negative controls and thus confirmed the results of the plate incorporation method.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, benzidine, Congo red and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Any other information on results incl. tables

Summary of the Results with Levafix Scharlach E-2GA in the Salmonella/Microsome Test

Plate Incorporation Method

S9 mix

TA 1535

TA 100

TA 1537

TA 98

Without

-ve

-ve

-ve

-ve

With

-ve

-ve

-ve

-ve

-ve = negative

 

Summary of the Results with Levafix Scharlach E-2GA in the Salmonella/Microsome Test

Prival Assay

S9 mix

TA 1535

TA 100

TA 1537

TA 98

Without

-ve

-ve

-ve

-ve

With

-ve

-ve

-ve

-ve

-ve = negative

 

Summary of Mean Values Without S9 Mix

Group

Strain

TA 1535

TA 100

TA 1537

TA 98

μg/plate

0

8

40

200

1000

5000

Na-azide

NF

4-NPDA

 

19

23

18

21

17

17

875

 

107

124

124

105

106

82

 

388

 

5

7

6

6

4

4

 

 

62

 

19

17

23

17

20

10

 

 

82

μg/tube

0

8

40

200

1000

5000

Na-azide

NF

4-NPDA

 

11

12

10

11

10

10

883

 

82

103

109

115

94

86

 

316

 

13

13

13

14

13

11

 

 

44

 

37

32

30

30

27

28

 

 

75

 

Summary of Mean Values With S9 Mix

Group

Strain

TA 1535

TA 100

TA 1537

TA 98

μg/plate

0

8

40

200

1000

5000

2-AA

 

26

20

28

27

24

17

140

 

150

140

156

155

140

102

865

 

6

5

6

9

7

5

47

 

28

27

27

33

22

15

245

μg/tube

0

8

40

200

1000

5000

Benzidine

Congo red

2-AA

 

17

22

21

23

20

15

 

 

166

 

150

168

176

180

164

137

 

 

1392

 

24

21

22

19

23

17

 

 

251

 

55

56

47

53

53

42

119

85

 

Historical Controls of Plate Incorporation Method

 

Summary of historical negative and positive controls of experiments performed from January to June 1988 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

DMSO

DMF

Ethanol

Acetone

EGDME2

-

-

-

-

-

-

14

13

12

15

10

18

2

2

2

3

2

97

94

87

69

85

117

9

15

11

7

10

8

8

8

7

7

10

1

1

1

1

1

17

17

19

22

18

21

2

2

3

3

2

Na-azide

NF

4-NPDA

-

-

-

839

115

 

382

 

46

 

 

90

 

 

13

 

 

109

 

 

20

30%

Water

DMSO

DMF

Ethanol

Acetone

EGDME2

 

+

+

+

+

+

+

 

14

15

14

20

14

18

 

3

3

3

2

1

 

134

124

113

105

134

159

 

10

14

9

6

25

 

8

9

9

6

11

9

 

2

2

2

1

2

 

29

29

31

30

34

35

 

3

3

5

3

3

2-AA

+

282

63

601

164

66

17

532

160

10%

Water

DMSO

DMF

Ethanol

Acetone

 

+

+

+

+

+

 

147

14

--

23

13

 

4

2

 

123

111

72

87

85

 

4

13

 

6

 

9

8

9

8

7

 

 

1

 

33

33

27

38

29

 

 

5

2-AA

+

357

67

1422

428

298

65

1323

323

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1988 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

DMSO

DMF

Ethanol

Acetone

-

-

-

-

-

14

14

12

10

15

3

2

2

2

2

97

93

70

71

138

9

25

4

2

10

8

8

7

7

8

2

1

1

1

3

20

19

13

21

39

5

10

1

3

6

Na-azide

NF

4-NPDA

-

-

-

822

137

 

412

 

42

 

 

88

 

 

19

 

 

124

 

 

25

30%

Water

DMSO

DMF

Ethanol

Acetone

 

+

+

+

+

+

 

12

16

14

17

13

 

2

2

3

3

4

 

144

124

117

90

177

 

15

15

14

4

35

 

10

10

9

8

9

 

2

2

2

1

2

 

35

32

31

39

43

 

6

5

6

2

8

2-AA

+

261

69

755

196

93

21

583

171

10%

DMSO

DMF

 

+

+

 

7

11

 

1

 

110

121

 

12

 

9

6

 

1

 

32

26

 

5

2-AA

+

348

70

1544

572

416

75

1499

423

 

Summary of historical negative and positive controls of experiments performed from January to June 1989 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

DMSO

DMF

Ethanol

Acetone

EGDE2

-

-

-

-

-

-

10

9

7

10

9

8

3

3

1

2

-

2

91

84

60

73

100

69

11

16

4

12

--

16

7

7

6

7

7

6

1

2

1

2

-

2

18

16

14

18

18

17

3

2

2

4

-

4

Na-azide

NF

4-NPDA

-

-

-

721

110

 

359

 

61

 

 

75

 

 

13

 

 

119

 

 

35

30%

Water

DMSO

DMF

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

 

14

14

14

17

15

14

 

2

3

2

3

-

2

 

133

114

100

118

138

115

 

12

18

9

12

--

25

 

9

9

8

10

13

11

 

2

1

2

2

-

2

 

32

28

25

37

32

27

 

8

4

4

8

-

8

2-AA

+

195

33

633

127

63

28

392

133

10%

DMSO

DMF

 

+

+

 

12

--

 

2

-

 

105

--

 

28

--

 

7

7

 

2

-

 

25

31

 

4

-

2-AA

+

267

27

1455

348

283

64

1547

289

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1989 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

DMSO

DMF

Ethanol

Acetone

EGDE2

-

-

-

-

-

10

9

8

15

8

4

4

3

-

-

72

57

57

96

63

6

15

12

--

--

7

8

6

6

6

4

2

1

-

-

16

17

14

13

21

5

5

6

-

-

Na-azide

NF

4-NPDA

-

-

-

853

147

 

326

 

47

 

 

91

 

 

26

 

 

87

 

 

25

30%

DMSO

DMF

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

 

14

15

11

21

13

 

2

3

6

-

-

 

89

87

79

96

87

 

7

6

13

--

--

 

11

11

 

2

4

2

-

-

 

23

26

23

20

26

 

2

4

5

-

-

2-AA

+

157

42

500

83

73

22

498

101

10%

DMSO

Ethanol

 

+

+

 

14

11

 

5

-

 

91

53

 

7

-

 

10

4

 

1

-

 

24

18

 

4

-

2-AA

+

158

54

1464

152

289

117

1294

113

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to June 1990 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

-

-

-

-

-

-

-

15

12

10

17

13

10

14

3

2

4

 

3

1

4

74

72

65

87

77

69

95

10

13

10

 

11

4

14

7

8

7

7

8

6

8

1

2

2

 

2

1

1

22

17

10

19

19

11

18

5

3

6

 

2

2

5

Na-azide

NF

4-NPDA

-

-

-

799

108

 

268

 

48

 

 

52

 

 

12

 

 

81

 

 

14

30%

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

+

 

18

18

13

22

19

13

15

 

2

3

3

 

3

1

2

 

108

86

97

121

98

104

97

 

17

11

17

 

15

8

9

 

9

9

7

11

8

7

9

 

2

2

3

 

2

3

3

 

27

27

20

28

29

22

28

 

5

3

5

 

4

3

8

2-AA

+

161

39

509

130

48

15

379

54

10%

DMSO

Ethanol

Acetone

 

+

+

+

 

18

16

 

2

 

89

85

107

 

20

 

11

8

 

4

 

30

29

17

 

6

2-AA

+

214

49

1196

181

235

38

1140

284

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1990 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

-

-

-

-

-

-

-

13

14

13

13

12

12

13

2

2

2

1

3

2

2

105

105

82

105

93

116

112

16

7

16

16

14

2

15

9

8

6

8

9

6

8

1

1

2

1

1

1

2

21

21

12

21

22

23

18

4

3

4

4

3

1

3

Na-azide

NF

4-NPDA

-

-

-

882

114

 

380

 

60

 

 

48

 

 

9

 

 

71

 

 

15

30%

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

+

 

18

17

15

22

19

13

18

 

3

2

3

2

3

1

3

 

143

137

109

144

118

131

135

 

15

5

14

16

18

4

14

 

11

10

10

11

10

9

11

 

2

2

1

2

1

1

2

 

29

28

23

33

39

26

32

 

3

4

3

3

7

1

5

2-AA

+

175

41

800

243

84

17

485

93

10%

DMSO

Acetone

EGDE2

 

+

+

+

 

16

12

 

2

 

127

124

140

 

19

 

9

10

 

3

 

32

26

 

5

2-AA

+

179

69

1321

148

298

39

1206

168

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to June 1990 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

-

-

-

-

-

-

-

12

13

9

11

12

10

11

3

2

-

2

1

-

3

111

113

80

105

96

55

108

10

14

--

14

15

--

5

9

10

7

8

9

5

8

2

2

-

2

2

-

1

28

30

23

29

31

21

23

5

3

-

5

5

-

8

Na-azide

NF

4-NPDA

-

-

-

623

102

 

398

 

56

 

 

49

 

 

10

 

 

89

 

 

20

30%

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

+

 

16

18

11

23

19

14

15

 

3

3

-

5

3

-

4

 

152

154

84

152

127

84

132

 

15

11

--

7

17

--

6

 

12

12

9

10

10

14

8

 

2

2

-

3

3

-

1

 

38

40

29

48

43

18

40

 

7

7

-

10

6

-

9

2-AA

+

182

33

800

163

86

24

472

105

10%

Water

DMSO

Methanol

 

+

+

+

 

15

16

--

 

-

3

-

 

102

132

150

 

-

5

-

 

5

10

--

 

-

1

-

 

46

39

--

 

-

4

-

2-AA

+

208

48

1408

216

314

147

754

369

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1991 using mean values presented as medians (Z) and semi-Q range (QR)

Compound and S9 Mix

Strain

TA 1535

TA 100

TA 1537

TA 98

Z

QR

Z

QR

Z

QR

Z

QR

Water

Buffer

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

-

-

-

-

-

-

-

-

12

13

12

7

10

12

12

14

3

2

3

 

1

4

2

3

89

97

92

75

84

80

87

108

10

10

15

 

11

8

6

22

9

8

9

7

8

8

8

8

3

1

1

 

1

3

1

1

27

25

24

17

25

23

26

26

4

2

4

 

3

4

4

5

Na-azide

NF

4-NPDA

-

-

-

605

122

 

339

 

52

 

 

53

 

 

9

 

 

71

 

 

17

30%

Water

Buffer

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

+

+

 

19

17

19

11

25

18

18

22

 

4

 

3

 

 

5

2

4

 

138

159

130

142

134

119

111

144

 

21

 

11

 

 

19

9

11

 

13

13

10

9

12

11

13

13

 

2

 

2

 

 

2

 

3

 

33

38

33

32

37

37

28

32

 

4

 

4

 

 

2

11

3

2-AA

+

164

38

727

139

91

32

520

161

10%

Water

Buffer

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

+

+

 

16

14

16

15

16

19

17

20

 

4

 

2

 

 

3

 

2

 

113

94

118

114

111

94

112

153

 

18

 

14

6

 

6

 

11

 

10

10

10

11

9

12

11

11

 

3

 

3

 

 

2

 

1

 

33

34

31

21

29

32

32

34

 

5

 

3

 

 

2

 

5

2-AA

+

197

50

1431

260

304

116

1097

207

2) Ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:
Due to the results, the test item has to be regarded as non-mutagenic. The substance is not classifiable according to CLP criteria.
Executive summary:

Levafix Scharlach E-2GA was initially investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to 5000 μg per plate on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

 

In the plate incorporation assay doses of up to and including 1000 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At 5000 μg per plate, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes.

 

In the plate incorporation assay evidence of mutagenic activity of Levafix Scharlach E-2GA was not seen. No biologically relevant increase in the mutant count, in comparison with the negative, controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

Levafix Scharlach E-2GA was investigated in an independent repeat using the Salmonella/microsome test, modified with S9 mix according to Prival and Mitchell, for point mutagenic effects in doses of up to 5000 μg per tube on the same strains. Without S9 mix preincubation was used.

 

Doses of up to and including 1000 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At 5000 μg per plate, the substance had a weak, strain-specific bacteriotoxic effect, so that this range could nevertheless be used for assessment purposes.

 

Evidence of mutagenic activity of Levafix Scharlach E-2CA was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

 

The positive controls sodium azide, nitrofurantoin, 4-nitro--1,2-phenylene diamine, benzidine, Congo red and 2-aminoanthracene had a marked mutagenic effect, as was see by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

 

Therefore, Levafix Scharlach E-2GA was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the Prival modification of the Salmonella/microsome test.

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