Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 19, 1992 to July 27, 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
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
Principles of method if other than guideline:
Prival M.J. and V.D. Mitchell: Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9. Mutation Res. 97, 103-116, 1982
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 Orange 64
Specific details on test material used for the study:
No further details specified in the study report.

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:
0, 8, 40, 200, 1000, 5000 μg/plate

The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 ug or 5 ul per plate were used as the highest dose.
Vehicle / solvent:
The solvent employed for Congo red was deionized water, and for other positive controls DMSO. Levafix orange E-3GA 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; 4-nitro-1,2-phenylene diamine; 2-aminoanthracene; benzidine
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 59 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 plate 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 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 pre-incubation test. Pre-incubation for 30 minutes at 37 °C was used.
The doses of this trial were determined on the basis of the results of the plate incorporation assay.
Due to the negative outcome of these two trials and due to the structure of the compound an additional repeat was performed according to Prival and Mitchell (1982) due to the chemical structure of the compound. Pre-incubation 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 pre-incubation 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 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 milliliter, 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, plates 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).
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 not 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 modification until a final evaluation is possible.
Statistics:
Not specified in the study report.

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:
no cytotoxicity
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:
no cytotoxicity
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:
no cytotoxicity
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:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Plate incorporation assay
As may be seen, there was no indication of a bacteriotoxic effect of Levafix Orange E-3GA at doses of up to and including 5000 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.
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.

Pre-incubation assay
As may be seen, there was no indication of a bacteriotoxic effect of Levafix Orange E-3GA 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 had only a weak, strain-specific bacteriotoxic effect, and could nevertheless be used for assessment purposes.
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 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.

Assay according to Prival and Mitchell
As may be seen, there was no indication of a bacteriotoxic effect of Levafix Orange E-3GA at doses of up to and including 5000 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.
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 and pre-incubation 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 Orange E-3GA in the Salmonella Microsome Test Plate Incubation 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 Orange E-3GA in the Salmonella/Microsome Test Pre-incubation 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 Orange E-3GA 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

 

17

16

17

17

10

15

296

 

66

59

70

78

55

67

 

203

 

7

8

7

7

4

4

 

 

52

 

18

16

18

17

13

19

 

 

67

μg/tube

0

8

40

200

1000

5000

Na-azide

NF

4-NPDA

 

8

8

6

8

6

6

387

 

66

56

63

54

56

63

 

295

 

8

7

10

10

9

5

 

 

55

 

17

17

18

15

13

12

 

 

70

μg/tube

0

8

40

200

1000

5000

Na-azide

NF

4-NPDA

 

7

7

7

10

7

6

121

 

78

84

80

71

84

75

 

277

 

8

4

4

7

6

5

 

 

43

 

22

12

11

16

16

14

 

 

44

 

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

 

18

18

22

20

18

14

98

 

79

79

98

91

72

83

539

 

11

11

9

7

7

5

69

 

22

28

24

23

19

20

442

μg/tube

0

8

40

200

1000

5000

2-AA

 

10

10

8

8

9

7

51

 

65

75

78

84

67

62

460

 

7

6

8

9

8

6

130

 

23

20

20

20

16

11

560

μg/tube

0

8

40

200

1000

5000

Benzidine

Congo red

2-AA

 

14

14

9

10

10

9

 

 

90

 

155

159

170

170

157

157

 

 

1329

 

17

16

18

16

19

7

 

 

264

 

24

31

26

22

21

19

79

53

 

Summary of historical negative and positive controls of experiments performed from January to June 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

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

83

24

472

405

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 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

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

 

55

 

 

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

Applicant's summary and conclusion

Conclusions:
Levafix Orange E-3GA is 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.
Therefore, the substance is not classified in accordance with CLP criteria.
Executive summary:

Levafix Orange E-3GA was initially investigated using the Salmonella/ microsome plate incorporation test for point mutagenic effects in doses of up to 5000 μg per plate of 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 5000 μg per plate did not cause any bacteriotoxic effects:

Total bacteria counts remained unchanged and no inhibition of growth was observed.

 

In the plate incorporation assay evidence of mutagenic activity of Levafix Orange E-3GA 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.

 

In an independent repeat Levafix Orange E-3GA was investigated using the Salmonella/microsome test for point mutagenic effects in doses up to 5000 μg per plate after pre-incubation for 30 minutes on four Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537 and TA 98.

 

Doses 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 only a weak strain-specific baceriotoxic effect, so that this dose could nevertheless be used for assessment purposes.

 

Evidence of mutagenic activity of Levafix Orange E-3GA 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 Orange E-3GA was investigated in second 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.

 

Doses of up to and including 5000 μg per tube did not cause any bacteriotoxic effects: Total bacteria counts remained unchanged and no inhibition of growth was observed.

 

Evidence of mutagenic activity of Levafix Orange E-3GA 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, Levafix Orange E-3GA 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.