Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 21, 1995 to November 30, 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 92/69/EEC B.14. Salmonella typhimurium Reverse Mutation Test
Deviations:
no
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
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: US EPA: HG - Gene Muta - S. typhimurium, October 1984
Version / remarks:
New and Revised Health Effects Test Guidelines October 1984 (U.S.) Environmental Protection Agency Washington, DC (PB 84-233295) .
HG - Gene Muta - S. typhimurium, October 1984
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride
EC Number:
228-800-7
EC Name:
2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride
Cas Number:
6359-50-8
Molecular formula:
C22H25N2.Cl
IUPAC Name:
2-[2-(2,3-dihydro-2-methyl-1H-indol-1-yl)vinyl]-1,3,3-trimethyl-3H-indolium chloride
Test material form:
solid: particulate/powder
Details on test material:
Basic Yellow 21

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The doses of this trial were determined on the basis of the results of the plate incorporation assay. Doses are given as μg/tube for better separation of plate incorporation and preincubation trials, despite the fact that μg/plate and μg/tube could be used synonymously.

0, 16, 50, 158, 500, 1581 and 5000 μg/plate – with and without S9 mix
0, 45, 90, 180, 360, 720 and 1440 μg/tube – with and without S9 mix

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µI per plate were used as the highest dose. At least five additional doses were routinely used. The doses used in the first trial (plate incorporation test) were: 0, 16, 50, 158, 500, 1581 and 5000 μg/plate.
Doses up to and including 158 µg per plate did not cause any bacteriotoxic effects. At higher doses, the substance had a strong, strain-specific bacteriotoxic effect, so that this range could only be used to a limited extent up to and including 1581 µg per plate for assessment purposes.
Due to the substance's toxicity, doses ranging from 45 µg to 1440 µg per tube were chosen for the repeat tests. The doses used in the second trial (preincubation test) were: 0, 45, 90, 180, 360, 720 and 1440 μg/tube (equivalent to 0, 16, 50, 158, 500, 1581 and 5000 μg/plate).
Vehicle / solvent:
The test substance was formulated in deionized water and formed a light-yellow suspension (5000 μg per plate) and light yellow clear solutions (1581 μg per plate and below) respectively. The positive controls were dissolved in DMSO.
The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethylether, and DMF according to information given by the internal sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
cumene hydroperoxide
other: Nitrofurantoin (NF); 4-nitro-1,2-phenylene diamine (4-NPDA); 2-aminoanthracene (2-AA)
Details on test system and experimental conditions:
The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
For the mutant count, three 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 three 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 or 5 μl per plate were used as the highest dose. At least five additional doses were routinely used. 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 obtained in the first experiment.
Because the first assessable trial showed no mutagenic effects of the test substance, the repeat was performed as preincubation in a water bath at 37 °C for 20 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, three 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 three 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. Doses are given as μg/tube for better separation of plate incorporation and preincubation trials, despite the fact that μg/plate and μg/tube could be used synonymously.
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. 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 the bacterial suspensions at a defined density of viable cells per millilitre, since the chosen method of incubation normally produces the designed 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 fivefold to per -it 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, plates were temporarily stored in a refrigerator.

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.
Only trials which complied with all three of the above criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.
The following doses per plate were evaluated in the first test:
μg per plate
1. Negative control 0
2. ASTRAZON Gelb 7GLL 5000
3. ASTRAZON Gelb 7GLL 1581
4. ASTRAZON Gelb 7GLL 500
5. ASTRAZON Gelb 7GLL 158
6. ASTRAZON Gelb 7GLL 50
7. ASTRAZON Gelb 7GLL 16
8. Positive control, sodium azide 10 (only TA 1535)
9. Positive control, nitrofurantoin 0.2 (only TA 100)
10. Positive control, 4-nitro-1,2-phenylene diamine 10 (only TA 1537)
11. Positive control, 4-nitro-1,2-phenylene diamine 0.5 (only TA 98)
12. Positive control, Cumene hydroperoxide 50 (only TA 102)
13. Positive control, 2-aminoanthracene 3

Due to the substance's toxicity, doses ranging from 45 μg to 1440 μg per tube were chosen for the repeat tests.
ASTRAZON Gelb 7GLL was formulated in deionized water and formed a light-yellow suspension (5000 μg per plate) and light yellow clear solutions (1581 μg per plate and below) respectively. The positive controls were dissolved in DMSO.
No ''untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature (e.g. Maron and Ames, 1983) and from the laboratories own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.
Rationale for test conditions:
The initial plate incorporation test followed the directions of Ames et al. (1973a, 1975) and Maron and Ames (1983).
Evaluation criteria:
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. For TA 102 an increase of about 150 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgement.
In case questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
None

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:
In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.
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:
In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.
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:
In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.
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:
In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In first test, doses ≥ 500 µg per plate and, in the second test, doses ≥ 180 μg/tube, had a strong, strain-specific bacteriotoxic effect.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Plate Incorporation Method
As may be seen, there was no indication of a bacteriatoxic effect of ASTRAZON Gelb 7GLL at doses of up to and including 158 μg per plate. The total bacteria count's consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. Higher doses had a strong, strain-specific bacteriotoxic effect. Therefore they could only partly be used for assessment purposes up to and including 1581 μg per plate.
None of the five 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.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, cumene hydroperoxide 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.

Preincubation Method
As may be seen, there was no indication of a bacterioxic effect of ASTRAZON Gelb 7GLL at doses of up to and including 90 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. Higher doses had a strong, strain-specific bacterio-toxic effect. Therefore they could only partly be used for assessment purposes up to and including 720 μg per tube.
None of the five 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, cumene hydroperoxide 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 ASTRAZON Gelb 7GLL in the

Salmonella/Microsome Test Plate Incorporation Method

S9 mix

TA 1535

TA 100

TA 1537

TA 98

TA 102

Without

-ve

-ve

-ve

-ve

-ve

With

-ve

-ve

-ve

-ve

-ve

-ve = Negative

 

Summary of the Results with ASTRAZON Gelb 7GLL in the

Salmonella/Microsome Test Preincubation Method

S9 mix

TA 1535

TA 100

TA 1537

TA 98

TA 102

Without

-ve

-ve

-ve

-ve

-ve

With

-ve

-ve

-ve

-ve

-ve

-ve = Negative

 

Summary of Mean Values Without S9 Mix

group

Strain

TA 1535

TA 100

TA 1537

TA 98

TA 102

μg/plate

0

16

50

158

500

1581

5000

Na-azide

NF

4-NPDA

Cumene

 

8

7

9

10

6

3

--

822

 

100

94

92

89

81

19

0

 

288

 

11

10

8

12

12

10

--

 

 

182

 

22

27

34

29

33

17

--

 

 

210

 

278

309

351

330

305

241

---

 

 

 

685

μg/tube

0

45

90

180

360

720

1440

Na-azide

NF

4-NPDA

Cumene

 

10

9

9

9

7

6

--

711

 

91

91

81

84

77

88

40

 

493

 

13

11

2

11

9

6

5

 

 

168

 

31

28

28

33

28

22

20

 

 

201

 

330

446

458

421

298

248

209

 

 

 

708

 

Summary of Mean Values With S9 Mix

Group

Strain

TA 1535

TA 100

TA 1537

TA 98

TA 102

μg/plate

0

16

50

158

500

1581

5000

2-AA

 

11

10

9

13

8

3

--

224

 

103

112

100

113

94

49

--

1583

 

12

9

11

12

8

6

--

472

 

37

29

37

37

38

37

--

1509

 

361

370

380

407

349

324

---

1179

μg/tube

0

45

90

180

360

720

1440

2-AA

 

14

12

13

15

10

7

--

264

 

122

131

22

118

116

80

18

1602

 

13

14

13

13

14

12

7

320

 

38

44

41

47

41

33

30

1261

 

403

440

479

463

432

403

241

749

 

Historical Controls

Plate Incorporation Method

 

Summary of historical negative and positive controls of experiments performed from January to June 1993 using mean values present 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

-

-

-

-

-

-

13

11

10

10

14

14

2

2

-

1

1

2

96

80

70

77

88

95

13

9

-

-

11

14

10

8

11

10

9

3

1

-

-

1

25

22

16

26

25

4

3

-

-

3

Na-azid

NF

4-NPDA

-

-

-

683

86

 

337

 

52

 

 

57

 

 

9

 

 

82

 

 

15

30%

Water

DMSO

Ethanol

EGDE2

 

+

+

+

+

 

17

15

28

36

 

-

2

-

-

 

116

111

135

137

 

-

7

-

-

 

11

9

10

14

 

-

2

-

-

 

36

30

38

45

 

-

5

-

-

2-AA

+

157

35

571

190

82

258

494

76

10%

Water

DMSO

DMF

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

 

18

14

13

29

16

16

 

4

2

-

-

3

-

 

118

101

87

113

114

118

 

9

13

-

-

5

9

 

10

9

10

13

11

10

 

3

1

-

-

1

-

 

39

31

23

45

43

36

 

5

3

-

-

7

-

2-AA

+

154

34

1177

246

225

100

1236

195

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1993 using mean values present 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

Ethanol

Acetone

EGDE2

-

-

-

-

-

11

11

17

16

17

2

2

-

-

3

82

86

121

92

95

21

15

-

-

20

7

8

8

9

9

3

2

-

-

2

21

19

27

20

18

2

2

-

-

5

Na-azid

NF

4-NPDA

-

-

-

643

145

 

284

 

74

 

 

64

 

 

11

 

 

58

 

 

9

30%

Water

DMSO

EGDE2

 

+

+

+

 

16

18

15

 

-

4

-

 

127

105

140

 

-

16

-

 

6

10

10

 

-

3

-

 

26

37

27

 

-

9

-

2-AA

+

220

23

448

203

88

21

478

86

10%

Water

DMSO

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

 

12

14

25

15

18

 

4

3

-

-

-

 

100

98

125

113

118

 

19

21

-

-

11

 

8

10

13

10

12

 

2

2

-

-

1

 

27

29

47

32

29

 

7

6

-

-

3

2-AA

+

145

39

812

342

164

40

1127

223

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to December 1994 using mean values present 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

-

-

-

-

-

-

-

10

9

12

12

7

7

11

2

2

-

-

2

3

3

89

82

94

95

56

79

79

14

9

-

-

10

12

10

8

9

9

11

8

6

8

2

2

-

-

2

2

2

22

21

25

15

24

18

16

3

4

-

-

11

3

6

Na-azid

NF

4-NPDA

-

-

-

706

98

 

263

 

33

 

 

105

 

 

35

 

 

138

 

 

23

30%

Water

DMSO

EGDE2

 

+

+

+

 

13

11

10

 

-

-

-

 

98

96

115

 

-

-

-

 

7

5

7

 

-

-

-

 

29

22

22

 

-

-

-

2-AA

+

152

24

594

140

72

29

489

93

10%

Water

DMSO

DMF

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

+

 

13

11

13

20

10

10

14

 

2

2

-

-

3

-

3

 

112

105

106

119

84

106

114

 

15

15

-

-

11

11

18

 

8

8

9

11

4

6

8

 

2

2

-

-

2

3

2

 

30

27

36

32

28

28

29

 

7

5

-

-

4

2

10

2-AA

+

171

49

1111

330

103

73

1301

208

2) Ethylene glycol dimethylether

 

Preincubation Method

 

Summary of historical negative and positive controls of experiments performed from January to June 1993 using mean values present 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

Acetone

-

-

-

15

13

15

2

3

-

129

94

123

10

8

-

13

10

13

2

1

-

34

26

27

5

4

-

Na-azid

NF

4-NPDA

-

-

-

689

106

 

475

 

67

 

 

60

 

 

10

 

 

101

 

 

22

10%

Water

DMSO

Acetone

 

+

+

+

 

20

16

16

 

4

3

-

 

140

114

141

 

10

11

-

 

12

10

14

 

2

1

-

 

45

34

33

 

4

5

-

2-AA

+

166

20

1176

149

255

114

1196

206

 

Summary of historical negative and positive controls of experiments performed from July to December 1993 using mean values present 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

Ethanol

Acetone

EGDE2

-

-

-

-

-

13

15

10

15

14

2

4

-

-

1

128

120

168

108

126

13

21

-

-

20

10

9

7

1

1

1

2

-

-

-

22

24

26

24

20

1

3

-

-

2

Na-azid

NF

4-NPDA

-

-

-

754

162

 

443

 

46

 

 

65

 

 

13

 

 

73

 

 

8

10%

Water

DMSO

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

 

15

15

19

19

16

 

2

2

-

-

2

 

153

129

131

147

152

 

25

20

-

-

23

 

9

11

10

12

12

 

1

1

-

-

3

 

26

34

28

36

30

 

3

5

-

-

8

2-AA

+

167

25

1188

138

226

44

1100

198

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from January to December 1994 using mean values present 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

Methanol

Ethanol

Acetone

EGDE2

-

-

-

-

-

-

11

10

12

9

11

13

2

2

-

2

-

2

131

99

139

103

113

105

18

10

-

10

-

22

8

8

6

10

8

10

2

2

-

4

-

2

19

22

18

20

23

20

6

5

-

4

-

5

Na-azid

NF

4-NPDA

-

-

-

787

116

 

402

 

41

 

 

98

 

 

33

 

 

137

 

 

26

10%

Water

DMSO

Methanol

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

 

13

11

20

14

14

13

 

2

3

-

2

-

2

 

163

115

139

118

175

142

 

25

13

-

9

-

19

 

9

8

7

9

7

10

 

2

2

-

3

-

2

 

29

29

24

24

31

33

 

7

6

-

6

-

7

2-AA

+

185

35

1357

166

178

55

1268

208

2) Ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:
Basic Yellow 21 was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.
The substance is not classified in accordance with CLP criteria.
Executive summary:

Basic Yellow 21 was initially investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 μg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102.

 

Doses up to and including 158 μg per plate did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriatoxic effect, so that this range couldonly be used to a limited extent up to and including 158 μg per plate for assessment purposes.

 

Evidence of mutagenic activity of the test substance 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, cumene hydroperoxide 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.

 

The test substance was investigated in an independent repeat using the Salmonella/microsome test for point mutagenic effects in doses up to 1440 μg per tube after preincubation for 20 minutes at 37 °C on five Salmonella typhimurium LT2. mutants.

These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 und TA 102.

 

Doses up to and including 90 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts, remained unchanged and no inhibition of growth was observed. At higher doses, the substance had a strong, strain-specific bacteriatoxic effect, so that this range could only be used to a limited extent up to and including 720 μg per tube for assessment purposes.

 

Evidence of mutagenic activity of Basic Yellow 21 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, cumene hydroperoxide 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.

 

Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.

The substance is not classified in accordance with CLP criteria.