Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
December 3, 1991 to December 13, 1991
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
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 Yellow 27

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 and 5000 μg/plate (or tube)
Vehicle / solvent:
The test item was dissolved in deionized water.
Controls
Untreated negative controls:
yes
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 diamain (4-NPDA); 2-Aminoanthracene (2-AA); Benzidine
Details on test system and experimental conditions:
S9 mix was used to simulate the mammalian metabolism of the test substance. It was made from the livers of at least six adult male Sprague Dawley rats, of approximately 200 to 300 g in weight. For enzyme induction, the animals received a single intraperitoneal injection of Aroclor 1254, dissolved in
corn oil, at a dose of 500 mg/kg body weight, five days prior to sacrifice. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames ( 1983). .

The rats were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4 deg C until all animals had been prepared. All the remaining steps were carried out under sterile condition at 4 deg C.

The livers were washed with cold (4 deg C), 0.15 M KCl solution (approximately 1 ml KCl per 1 g liver), and then homogenized in fresh, cold (4 deg C), 0.15 M KCl (approximately 3 ml KCl per
1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4 deg C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was stored at -80 deg C in small portions.

These portions were slowly thawed before using. The S9 mix was freshly prepared (Ames et al., 1973a) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix cold.

Seventy ml of cofactor solution are composed as follows:
MgCl2 X 6 H20: 162.6 mg
KCl: 246.0 mg
glucose-6-phosphate, disodium salt: 179.1 mg
NADP, disodium salt: 315.0 mg
phosphate buffer: 100.0 mM

S9 mix consists of this cofactor solution, S9 fraction and if needed, 0.15 M KCl. The S9 mix comprised the amount of (fraction (X%), 70% cofactor solution and (30-X%) 0,15 M KCl, The S9 fraction was derived from the preparation dated July 31 1991 (protein content 25.4 mg per ml). Prior to first use, each batch was checked for its metabolising capacity by using reference mutagen(s); appropriate activity was demonstrated. At the beginning of each experiment, 4 aliquots of the S9 mix were plated (0.5 ml/plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37 deg C. No indication of contamination of S9 mix was found.

S9 Mix According to Prival
S9 mix was used to simulate the mammalian metabolism of the test substance. It was made from the livers of at least six adult male syrian hamsters, approximately 8 weeks old. No enzyme induction was performed. The animals were prepared unfasted, following the directions of Ames et al. (1975) and Maron and Ames ( 1983) .

The hamsters were terminated. Livers were removed under sterile conditions immediately after sacrifice and kept at 4 deg C until all animals had been prepared. All the remaining steps were carried out under sterile conditions at 4 deg C.

The livers were washed with cold (4 deg C), 0.15 M KCl solution (approximately 1 ml KCl per 1 g liver), and then homogenized in fresh, cold (4 deg C), 0.15 M KCl (approximately 3 ml KCl per
1 g liver). The homogenate was then centrifuged in a cooling centrifuge at 4 deg C and 9000 g for 10 minutes. The supernatant (the S9 fraction) was not stored but used freshly.

The S9 mix was freshly prepared (Prival and Mitchell 1982) and used only on the same day. It was placed in a vessel with a double glass wall until used. The hollow wall was filled with ice to keep the S9 mix cold.

S9 mix is composed as follows:

MgCl2 X 6 H20: 8.0 mM
KCl: 33.0 mM
glucose-6-phosphate, disodium salt: 20.0 mM
NADP, disodium salt: 2.0 mM
NADPH: 2.0 mM
FMN: 2.0 mM
phosphate buffer: 100.0 mM
glucose-6-phosphate dehydrogenase: 2.8 units / ml

S9 mix consists of 70% of this cofactor solution, and 30% (v/v) S9 fraction and if needed, 0.15 M KCl. The S9 fraction was derived from the preparation dated December 11, 1991 (protein content 32.8 mg per ml). Prior to first use, each batch was checked for its metabolising capacity by using reference mutagen(s); appropriate activity was demonstrated. At the beginning of each experiment, 4 aliquots of the S9 mix were plated (0.5 ml/plate) in order to assess its sterility. This was repeated after completion of test tube plating. The sterility control plates were then incubated for 48 hours at 37 deg C. No indication of contamination of S9 mix was found.

The initial plate incorporation test followed the direction 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 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 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 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", “y" “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 usd 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 desires, 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.tmM 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:
In accordance with test guidelines.
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 (i.e. Maron and Ames, 183) and the laboratories' own historical data.
b) The positive controls had to show sufficient affects, 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. Exe n if the criteria for points (a), (b) and (c) were not an assay was accepted if it showed mutagenic activity of the test compound.
Statistics:
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.

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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Plate incorporation assay
There was no indication of a bacteriotoxic effect of Levafix Goldgelb E-G trocken FW 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 to both the test with and without S9 mix.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-pheynlene 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 and Mitchell assay
There was no indication of a bacteriotoxic effect of Levafix Goldgelb E-G trocken FW at dose 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 fours 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 Goldgelb E-G trocken FW 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 Goldgelb E-G trocken FW 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-azid

NF

4-NPDA

 

8

7

8

10

7

7

554

 

80

71

91

100

91

80

 

312

 

7

5

5

5

5

6

 

 

36

 

14

15

12

15

14

11

 

 

81

μg/tube

0

8

40

200

1000

5000

Na-azid

NF

4-NPDA

 

12

12

11

11

8

8

697

 

110

106

114

113

98

86

 

314

 

8

6

5

4

5

4

 

 

32

 

21

21

17

13

15

16

 

 

42

 

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

 

16

15

16

16

13

11

137

 

109

106

109

100

96

107

466

 

12

9

8

5

8

7

36

 

24

19

22

21

19

15

229

μg/tube

0

8

40

200

1000

5000

Benzidine

Congo red

2-AA

 

12

13

14

11

14

10

 

 

73

 

165

166

170

166

155

125

 

 

410

 

23

18

21

18

21

15

 

 

144

 

22

24

22

19

17

20

73

71

 

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

EDGE2

-

-

-

-

-

-

-

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-azid

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

EDGE2

-

-

-

-

-

-

-

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-azid

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 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

EDGE2

-

-

-

-

-

-

-

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-azid

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

+

161

39

509

130

48

15

379

54

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

 

Applicant's summary and conclusion

Conclusions:
Levafix Goldgelb E-G trocken FW 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.
Executive summary:

Levafix Goldgelb E-G trocken FW 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 5000 μg per plate did not cause any bacteriotoxic effects:

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

 

Evidence of mutagenic activity of Levafix Goldgelb E-G trocken FW in the plate incorporation assay 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 Goldgelb E-G trocken FW 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 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 Goldgelb E-G trocken FW was not seen. No biologically releyant 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 Goldgelb E-G trocken FW 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.