Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 9, 1992 to October 19, 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:
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
Deviations:
no
Qualifier:
according to
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:
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
Qualifier:
according to
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 Salmonella typhimurium in the presence of flavine mononucleotide and hamster liver S-9. Mut. Res., 103- 106 (1982).
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Reactive Red 158

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 applicable
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 or 5 μl per plate were used as the highest dose.
The results of the first plate incorporation experiment was considered a pre-test for toxicity.
Doses of repeats for the plate incorporation test were chosen on the basis of the results obtained in the first experiment.
The doses of the preincubation test trial were determined on the basis of the results of the plate incorporation assay.

0, 8, 40, 200, 1000, 5000 μg/plate - Plate incorporation test
0, 8, 40, 200, 1000, 5000 μg/tube - Preincubation test

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. The following doses were used for the first trial (plate incorporation test): 0, 8, 40, 200, 1000, 5000 μg/plate.
The doses of the preincubation test were determined on the basis of the results of the plate incorporation test. In the plate incorporation test, there was no indication of a bacteriotoxic effect of C.I. Reactive Red 158 at doses of up to and including 200 µg per plate. Higher doses had a weak, strain-specific bacteriotoxic effect, but could nevertheless be used for assessment up to and including 5000 µg per plate. Therefore, the following doses were used for the second trial (preincubation test): 0, 8, 40, 200, 1000, 5000 μg/tube.
Vehicle / solvent:
C.I. Reactive Red 158 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-NOPD); 2-aminoanthracene (2-AA); Benzadine
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, 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 or 5 μl per plate were 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 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 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 pre-incubation 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. 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 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 fivefold 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, 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 effect 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 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 not met, an assay was accepted if it showed mutagenic activity of the test compound.

The following doses per plate were evaluated:
μg per plate
1. Negative control 0
2. C.I. Reactive Red 158 5000
3. C.I. Reactive Red 158 1000
4. C.I. Reactive Red 158 200
5. C.I. Reactive Red 158 40
6. C. I. Reactive Red 158 8
7. Positive control, sodium azide 10 (only TA 1535)
8. Positive control, nitrofurantoin 0.2 (only TA 100)
9. Positive control, 4-nitro-1,2-phenylene diamine 10 (only TA 1537)
10. Positive control, 4-nitro-1,2-phenylene diamine 0.5 (only TA 98)
11. Positive control, 2-aminoanthracene 3
12. Positive control, benzidine 4 (only TA 98)
13. Positive control, Congo red 50 (only TA 98)

The solvent employed for Congo red was deionized water, and for the other positive controls DMSO. C.I. Reactive Red 158 was dissolved in deionized water.
No "untreated" negative control was set up for deionized water, 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 bacterial strains used.
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:
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 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.
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 ≥ 1000 µg per plate and, in the second test, doses ≥ 200 μg/tube, had a 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 ≥ 1000 µg per plate and, in the second test, doses ≥ 200 μg/tube, had a 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 ≥ 1000 µg per plate and, in the second test, doses ≥ 200 μg/tube, had a 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 ≥ 1000 µg per plate and, in the second test, doses ≥ 200 μg/tube, had a 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
There was no indication of a bacteriotoxic effect at doses of up to and including 200 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. Higher doses had a weak, strain-specific bacteriotoxic effect, but could nevertheless be used for assessment up to and including 5000 μg per plate.
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.
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.

Prival Assay
There was no indication of a bacteriotoxic effect at doses of to and including 40 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 strain-specific bacteriotoxic effect, and could only partly be used for assessment up to and including 5000 μg per tube.
None of the four strains concerned showed a dose related and biologically relevant increase in mutant count 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 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 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

 

14

13

12

15

12

11

695

 

52

72

76

73

63

42

 

260

 

10

8

9

11

7

3

 

 

56

 

21

27

24

19

23

9

 

 

143

μg/tube

0

8

40

200

1000

5000

Na-azide

NF

4-NPDA

 

12

13

14

13

11

15

784

 

71

79

62

49

67

55

 

223

 

10

10

12

11

11

8

 

 

49

 

12

16

19

18

20

17

 

 

54

 

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

 

21

18

20

21

16

12

143

 

100

112

115

117

138

56

386

 

23

22

18

17

13

14

77

 

34

37

43

45

42

24

402

μg/tube

0

8

40

200

1000

5000

Benzidine

Congo red

2-AA

 

19

13

16

19

14

12

 

 

101

 

133

151

144

140

135

84

 

 

892

 

20

23

16

18

16

10

 

 

187

 

39

32

29

26

24

22

103

71

 

Summary of historical negative and positive controls of experiments performed from January to June 1991 using mean value 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

14

754

369

2) Ethylene glycol dimethylether

 

Summary of historical negative and positive controls of experiments performed from July to December 1991 using mean value 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

107

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

 

 

79

 

 

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

 

Summary of historical negative and positive controls of experiments performed from January to June 1992 using mean value 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

-

-

-

-

-

-

16

15

13

15

13

17

2

4

3

4

2

-

93

81

68

91

59

69

11

9

4

15

12

-

9

9

7

7

7

7

2

1

1

2

1

-

23

23

19

25

19

14

4

4

3

2

1

-

Na-azide

NF

4-NPDA

-

-

-

660

147

 

285

 

65

 

 

52

 

 

7

 

 

74

 

 

13

30%

Water

DMSO

DMF

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

 

23

22

18

25

17

19

 

4

5

2

-

3

-

 

124

120

89

145

79

119

 

18

20

5

-

9

-

 

12

12

11

9

8

8

 

1

2

1

-

2

-

 

31

31

27

38

24

18

 

6

5

1

-

4

-

2-AA

+

151

17

669

208

62

13

382

111

10%

Water

DMSO

DMF

Ethanol

Acetone

EGDE2

 

+

+

+

+

+

+

 

20

17

17

24

15

11

 

4

3

-

4

3

-

 

118

111

87

98

69

48

 

17

15

-

5

7

-

 

12

10

9

9

12

11

 

3

2

-

2

6

-

 

33

32

34

37

29

22

 

5

4

-

5

5

-

2-AA

+

159

35

1148

332

246

21

1126

292

2) Ethylene glycol dimethylether

Applicant's summary and conclusion

Conclusions:
The test item was considered to be non-mutagenic without and with an exogenous metabolic activation system in the plate incorporation as well as in the Prival modification of the Salmonella/microsome test. The substance is not to be classified in accordance with CLP criteria.
Executive summary:

Reactive Red 158 was investigated for mutagenic effects using the Salmonella/ microsome plate incorporation test 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 153 and TA 98.

In the plate incorporation assay doses of up to and including 200 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 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 Reactive Red 158 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-nitro1,2-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.

In addition, Reactive Red 158 was investigated in 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 40 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. A higher doses, the substance had a strain-specific bacterotoxic effect, so that this range could only be used to a limited extent up to 5000 μg per tube for assessment purposes.

Evidence of mutagenic activity of Reactive Red 158 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 seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, Reactive Red 158 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.

The substance is not classified in accordance with CLP criteria.