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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 06th, 1995 to September 14th, 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The Prival modification was done to substitute the study from 1981
Cross-reference
Reason / purpose:
reference to other study
Remarks:
Standard plate incorporation test with Salmonella and e.coli tester strains with and without metabolic activation from induced rat liver

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
OECD Guideline for testing of chemicals 471 Genetic Toxicology : Salmonella typhimurium, Reverse Mutation Assay, Adopted : May 26th, 1983
Deviations:
no
Qualifier:
according to
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
Version / remarks:
U.S. EPA: 798.5265 The Salmonella typhimurium reverse mutation assay Fed. Reg. 50, Subpart F, September 1985
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
EEC Directive 92/69, L 383 A, Annex B 14
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

Method

Target gene:
Histidine
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9-mix fom uninduced hamster liver
Test concentrations with justification for top dose:
The first experiment was performed with all tester strains using three plates per dose to obtain information on mutagenicity and toxicity for calculating an appropriate dose range.
The test compound was tested at doses of 4 to 5000 microgram/plate and proved to be not toxic to the bacterial strains.
Therefore 5000 microgram/plate was chosen as the highest dose in the second experiment.
Test concentrations:
0, 4, 20, 100, 500, 2500 and 5000 μg/plate
Vehicle / solvent:
On the day of the experiment the test compound was dissolved in aqua bidest. at appropriate concentrations.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
congo red
other: 2-Aminoanthracene (2-AA)
Details on test system and experimental conditions:
Preparation and storage of a liver homogenate fraction (S9)
Liver preparations were performed from the liver of non pre-treated Syrian hamsters.
The livers were removed from at least 5-6 male Syrian hamsters (7 -8 weeks old) at approx. 0 to 4 °C using cold sterile solutions and glassware, and were then pooled and washed in approx. 150 mM KCI (approximately 1 ml/g wet liver). The washed livers were cut into small pieces and homogenized in three volumes of KCI. The homogenate was centrifuged at approx. 9000 g for 10 minutes. The supernatant is the S9 fraction. This was divided into small portions, rapidly frozen and stored at approx. - 80 °C for not longer than six months.

Preparation of S9-mix
Sufficient S9 fraction was thawed immediately before each test at room temperature.
Three volumes of S9 fraction was mixed with 7 volumes of the S9 cofactor solution, which was kept on ice until used. This preparation is termed S9-mix. The concentrations of the different compounds in the S9-mix of the rat liver were:
8mM MgCI2
33mM KCI
20mM glucose-6-phosphate
2.8 units/ml glucose-6-phosphate dehydrogenase
4mM NADP+
2mM NADH
2mM FMN (Riboflavine-5'-phosphate-sodium-salt)
100mM phosphate buffer pH 7.4
According to the modification proposed by Prival using 30 minutes preincubation in the presence of 30 % (v/v) Syrian golden hamster S9-mix.

Bacteria
Bacteria were grown overnight in nutrient broth (25 g Oxoid Nutrient Broth No. 2 /liter) at approx. 37 °C. The amount of bacteria in the cell suspension was checked by nephelometry. Inoculation was performed with stock cultures which had been stored at approx. -80 °C. The compound was tested with the strains Salmonella typhimurium TA 98, TA 100, TA 1535 and TA 1537. Identification of the different bacterial strains is performed periodically and all criteria for a valid assay were fulfilled as described.

Toxicity experiments and dose range finding
The first experiment was performed with all tester strains using three plates per dose to obtain information on mutagenicity and toxicity for calculating an appropriate dose range. A reduced rate of spontaneously occurring colonies and visible thinning of the bacterial lawn were used as toxicity indicators. Thinning of the bacterial lawn was evaluated microscopically.
In combination with the second experiment, toxicity testing was performed as follows:
0.1 ml of the different dilutions of the test compound were thoroughly mixed with 0.1 ml of 106 dilution of the overnight culture of TA 100 and plated with histidine and biotin rich top agar (3 plates per dose). The solvent control is compared with the number of colonies per plate in the presence of the test compound. Results are given as a ratio of these values (= surviving fraction).

Mutagenicity test
Two independent experiments were performed for each of the two protocols (Ames, Prival).
a) - with buffer and the strains TA 98, TA 100, TA 1535 and TA 1537 in a plate incorporation test without metabolic activation
Top agar was prepared for the Salmonella strains by mixing 100 ml agar (0.6% (w/v) agar, 0.5% (w/v) NaCI) with 10 ml of a 0.5 mM histidine-biotin solution.
The following ingredients were added (in the following order) to 2 ml of molten top agar at approx. 45 °C:
0.1 ml of an overnight nutrient broth culture of the bacterial tester strain
0.1 ml test compound solution
0.5 ml buffer
After mixing, the liquid was poured into a petri dish with minimal agar (1.5 % (w/v) agar, Vogel-Bonner E medium with 2 % (w/v) glucose). After incubation for approximately 48 hours at approx. 37 °C in the dark, colonies (his+ revertants) were counted.

b)- with 30% (v/v) Syrian golden hamster S9-mix and preincubation
0.1 ml test solution, 0.1 ml bacterial suspension and 0.5 ml S9-mix were incubated at approx. 30 °C for approx. 30 minutes. Subsequently, 2 ml of soft agar containing of 100 ml agar (0.6% (w/v) agar+ 0.5% (w/v) NaCI) and 10 ml amino-acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) was added. After mixing, the samples were poured on to the VogelBonner agar plates (minimal glucose agar plates) within approximately 30 seconds.
After incubation for 48 hours at 37 °C in the dark, colonies (his+ revertants) were counted.
Rationale for test conditions:
In accordance with test guidelines.
Evaluation criteria:
A test compound is classified as mutagenic if it has either of the following effects:
a) a test compound produces at least a 2-fold increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control at complete bacterial background lawn
b) a test compound induces a dose-related increase in the mean number of revertants per plate of at least one of the tester strains over the mean number of revertants per plate of the appropriate vehicle control in at least two to three concentrations of the test compound at complete bacterial background lawn.
The test results must be reproducible.

Results and discussion

Test results
Key result
Species / strain:
other: all strains used
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:
Sterility checks and control plates
Sterility of S-9-mix and the test compound were indicated by the absence of contamination on the test material and S9-mix sterility check plates. Control plates (background control and positive controls) gave the expected number of colonies.

Solubility
The test compound did not precipitate on the plates up to the highest investigated dose of 5000 microgram/plate.

Toxicity
The test compound was tested at doses of 4 to 5000 microgram/plate and proved to be not toxic to the bacterial strains.
Therefore 5000 microgram/plate was chosen as the highest dose in the second experiment.

Mutagenicity
Experimental design
Remazol Brillantviolett 5R neu GT was tested for mutagenicity with Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 in the absence and presence of a metabolic activation system. S9-mix from Syrian golden hamsters (30 % (v/v)) was used.

Mutation results
Ames-Test:
The test compound did not cause a significant increase in the number of revertant colonies with any of the tester strains in the absence of a metabolic activation system.
No dose dependent effect was obtained.
Prival-Test:
In the presence of hamster liver S9-mix (30% (v/v)) using the preincubation method according to Prival the test compound did not show any relevant increases in the number of revertant colonies under the experimental conditions described.

Two independent experiments for each of the two protocols (Ames, Prival) were performed.

Any other information on results incl. tables

 CONTROLS – TEST 01

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 100

+S9

SOLVENT CONTROL

 

 

 

 

 

 

158.7

30.4

 

 

181

124

171

NEGATIVE CONTROLS

 

 

 

 

 

 

152.3

26.3

1.0

 

132

182

143

POSITIVE CONTROLS

 

2-AMINOANTHRACENE

 

0.5

2303.0

111.6

14.5

 

2194

2417

2298

TA 100

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

126.0

15.6

 

 

117

144

117

NEGATIVE CONTROLS

 

 

 

 

 

 

135.0

24.6

1.1

 

125

163

117

POSITIVE CONTROLS

 

SODIUM-AZIDE

 

1.0

697.3

41.2

5.5

 

711

730

651

CONTROLS – TEST 01

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 98

+S9

SOLVENT CONTROL

 

 

 

 

 

 

32.3

3.5

 

 

29

36

32

NEGATIVE CONTROLS

 

 

 

 

 

 

34.3

10.0

1.1

 

44

24

35

POSITIVE CONTROLS

 

CONGORED

 

500.0

258.3

9.5

8.0

 

249

258

268

TA 98

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

24.7

1.5

 

 

25

23

26

NEGATIVE CONTROLS

 

 

 

 

 

 

24.3

4.0

4.0

 

25

20

28

POSITIVE CONTROLS

 

2-NITROFLUORENE

 

2.5

831.7

71.8

33.7

 

910

816

769

 

TEST 01

THE EXPERIMENT WITH METABOLIC ACTIVATION WAS PERFORMED WITH 30% SYRIAN GOLDEN HAMSTER LIVER S9-MIX AND PREINCUBATION

ALL STERILITY CONTROL PLATES WERE STERILE

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVTERANTS /PLATE

PLATE 1

PLATE 2

PLATE 3

TA 100

+S9

0.

4.

20.

100.

500.

2500.

5000.

158.7

157.7

171.0

158.0

175.7

173.7

194.7

30.4

23.4

18.3

6.1

23.4

32.6

5.0

 

1.0

1.1

1.0

1.1

1.1

1.2

 

181

153

151

161

149

136

200

124

183

175

162

193

193

190

171

137

187

151

185

192

194

TA 100

-S9

0.

4.

20.

100.

500.

2500.

5000.

126.0

126.7

120.7

122.7

131.0

130.3

135.0

15.6

18.8

12.7

19.4

19.9

6.7

15.1

 

1.0

1.0

1.0

1.0

1.0

1.1

 

117

105

107

113

142

132

118

144

136

132

145

143

123

147

117

139

123

110

108

136

140

TA 98

+S9

0.

4.

20.

100.

500.

2500.

5000.

32.3

36.0

37.3

30.0

33.3

24.0

24.7

3.5

7.0

9.1

4.6

10.4

4.0

3.1

 

1.1

1.2

0.9

1.0

0.7

0.8

 

29

39

44

34

45

28

24

36

41

41

31

25

24

28

32

28

27

25

30

20

22

TA 98

-S9

0.

4.

20.

100.

500.

2500.

5000.

24.7

25.3

29.3

23.0

25.7

19.3

24.7

1.5

3.8

9.3

4.0

3.2

1.5

5.1

 

1.0

1.2

0.9

1.0

0.8

1.0

 

25

27

23

19

27

21

26

23

28

25

23

28

19

29

26

21

40

27

22

18

19

 

CONTROLS – TEST 01

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 1535

+S9

SOLVENT CONTROL

 

 

 

 

 

 

14.7

4.5

 

 

19

15

10

NEGATIVE CONTROLS

 

 

 

 

 

 

10.0

2.0

0.7

 

8

12

10

POSITIVE CONTROLS

 

2-AMINOANTHRACENE

 

0.5

260.3

3.8

17.7

 

263

262

256

TA 1535

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

11.3

2.5

 

 

14

11

9

NEGATIVE CONTROLS

 

 

 

 

 

 

12.0

3.5

1.1

 

8

14

14

POSITIVE CONTROLS

 

SODIUM-AZIDE

 

1.0

380.3

27.8

33.7

 

360

412

369

CONTROLS – TEST 01

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 1537

+S9

SOLVENT CONTROL

 

 

 

 

 

 

9.0

2.6

 

 

8

7

12

NEGATIVE CONTROLS

 

 

 

 

 

 

8.7

1.2

1.0

 

8

8

10

POSITIVE CONTROLS

 

2-AMINOANTHRACENE

 

2.5

230.0

6.6

25.6

 

223

236

231

TA 1537

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

7.7

1.5

 

 

9

6

8

NEGATIVE CONTROLS

 

 

 

 

 

 

6.3

0.6

0.8

 

6

6

7

POSITIVE CONTROLS

 

9-AMINOACRIDINE

 

50.

130.0

5.6

16.9

 

124

135

131

 

TEST 01

THE EXPERIMENT WITH METABOLIC ACTIVATION WAS PERFORMED WITH 30% SYRIAN GOLDEN HAMSTER LIVER S9-MIX AND PREINCUBATION

ALL STERILITY CONTROL PLATES WERE STERILE

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVTERANTS /PLATE

PLATE 1

PLATE 2

PLATE 3

TA 1535

+S9

0.

4.

20.

100.

500.

2500.

5000.

14.7

11.3

12.0

8.3

10.3

11.0

10.0

4.5

3.5

2.0

0.6

1.5

2.6

2.6

 

0.8

0.8

0.6

0.7

0.7

0.7

 

19

15

10

9

12

10

13

15

8

12

8

10

9

9

10

11

14

8

9

14

8

TA 1535

-S9

0.

4.

20.

100.

500.

2500.

5000.

11.3

10.0

11.0

11.0

10.0

9.7

8.7

2.5

2.0

2.6

2.6

2.0

2.1

1.2

 

0.9

1.0

1.0

0.9

0.9

0.8

 

14

12

12

9

8

9

8

11

10

13

10

12

12

10

9

8

8

14

10

8

8

TA 1537

+S9

0.

4.

20.

100.

500.

2500.

5000.

9.0

9.0

9.0

7.3

8.7

9.3

6.7

2.6

1.0

1.0

2.3

0.6

1.2

1.2

 

1.0

1.0

0.8

1.0

1.0

0.7

 

8

9

9

6

9

10

8

7

10

8

6

8

10

6

12

8

10

10

9

8

6

TA 1537

-S9

0.

4.

20.

100.

500.

2500.

5000.

7.7

8.7

8.7

8.7

7.3

9.7

9.7

1.5

1.2

1.2

2.3

2.3

9.7

9.7

 

1.1

1.1

1.1

0.9

1.3

1.3

 

9

8

10

10

6

10

9

6

8

8

10

6

10

10

8

10

8

6

10

9

10

 

CONTROLS – TEST 02

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 100

+S9

SOLVENT CONTROL

 

 

 

 

 

 

149.0

11.3

 

 

136

156

155

NEGATIVE CONTROLS

 

 

 

 

 

 

156.7

6.0

1.1

 

151

156

163

POSITIVE CONTROLS

 

2-AMINOANTHRACENE

 

0.5

2286.3

88.3

15.3

 

2359

2188

2312

TA 100

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

110.3

6.7

 

 

107

118

106

NEGATIVE CONTROLS

 

 

 

 

 

 

114.0

12.1

1.0

 

128

107

107

POSITIVE CONTROLS

 

SODIUM-AZIDE

 

1.0

703.0

25.9

6.4

 

731

680

698

 

CONTROLS – TEST 02

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 1535

+S9

SOLVENT CONTROL

 

 

 

 

 

 

10.3

0.6

 

 

10

11

10

NEGATIVE CONTROLS

 

 

 

 

 

 

11.7

2.1

1.1

 

14

10

11

POSITIVE CONTROLS

 

2-AMINOANTHRACENE

 

0.5

254.7

5.5

24.7

 

249

255

260

TA 1535

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

10.3

1.2

 

 

9

11

11

NEGATIVE CONTROLS

 

 

 

 

 

 

10.3

0.6

1.0

 

11

10

10

POSITIVE CONTROLS

 

SODIUM-AZIDE

 

1.0

344.7

30.2

33.5

 

322

379

333

 

CONTROLS – TEST 02

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 1537

+S9

SOLVENT CONTROL

 

 

 

 

 

 

9.3

1.5

 

 

8

9

11

NEGATIVE CONTROLS

 

 

 

 

 

 

8.0

2.0

0.9

 

8

6

10

POSITIVE CONTROLS

 

2-AMINOANTHRACENE

 

2.5

227.3

7.5

24.4

 

236

223

223

TA15 37

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

7.7

1.5

 

 

6

8

9

NEGATIVE CONTROLS

 

 

 

 

 

 

8.0

0.0

1.0

 

8

8

8

POSITIVE CONTROLS

 

9-AMINOACRIDINE

 

50.

127.7

9.0

16.6

 

123

122

138

 

CONTROLS – TEST 02

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 98

+S9

SOLVENT CONTROL

 

 

 

 

 

 

35.0

6.9

 

 

39

27

39

NEGATIVE CONTROLS

 

 

 

 

 

 

34.0

11.1

1.0

 

44

22

36

POSITIVE CONTROLS

 

COGORED

 

500.0

259.3

38.7

7.4

 

215

277

286

TA 98

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

23.7

3.8

 

 

28

22

21

NEGATIVE CONTROLS

 

 

 

 

 

 

27.3

2.1

1.2

 

29

28

25

POSITIVE CONTROLS

 

2-NITROFLUORENE

 

2.5

1098.0

135.0

46.3

 

961

1102

1231

 

TEST 02

THE EXPERIMENT WITH METABOLIC ACTIVATION WAS PERFORMED WITH 30% SYRIAN GOLDEN HAMSTER LIVER S9-MIX AND PREINCUBATION

ALL STERILITY CONTROL PLATES WERE STERILE

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVTERANTS /PLATE

PLATE 1

PLATE 2

PLATE 3

TA 100

+S9

0.

4.

20.

100.

500.

2500.

5000.

149.0

154.7

157.0

155.7

168.7

188.3

179.7

11.3

26.1

7.8

11.1

4.6

8.5

36.0

 

1.0

1.1

1.0

1.1

1.3

1.2

 

136

161

148

144

174

185

143

156

177

161

66

166

182

181

155

126

162

157

166

198

215

TA 100

-S9

0.

4.

20.

100.

500.

2500.

5000.

110.3

116.7

116.7

126.3

117.7

136.3

139.7

6.7

15.1

11.8

16.3

9.5

23.0

6.7

 

1.1

1.1

1.1

1.1

1.2

1.3

 

107

106

103

123

118

135

143

118

110

123

144

127

160

144

106

134

124

112

108

114

132

TA 1535

+S9

0.

4.

20.

100.

500.

2500.

5000.

10.3

11.0

12.0

12.0

12.3

11.7

11.3

0.6

2.6

0.0

2.0

2.9

0.6

2.5

 

1.1

1.2

1.2

1.2

1.1

1.1

 

10

10

12

12

9

12

14

11

14

12

10

14

11

9

10

9

12

14

14

12

11

TA 1535

-S9

0.

4.

20.

100.

500.

2500.

5000.

10.3

8.3

12.7

13.0

10.0

12.3

10.7

1.2

0.6

2.3

1.7

1.7

4.5

2.9

 

0.8

1.2

1.3

1.0

1.2

1.0

 

9

9

10

14

9

12

9

11

8

14

11

12

8

14

11

8

14

14

9

17

9

 

TEST 02

THE EXPERIMENT WITH METABOLIC ACTIVATION WAS PERFORMED WITH 30% SYRIAN GOLDEN HAMSTER LIVER S9-MIX AND PREINCUBATION

ALL STERILITY CONTROL PLATES WERE STERILE

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVTERANTS /PLATE

PLATE 1

PLATE 2

PLATE 3

TA 1537

+S9

0.

4.

20.

100.

500.

2500.

5000.

9.3

9.3

8.7

8.3

7.7

8.7

7.0

1.5

2.3

1.2

1.5

2.1

1.2

1.0

 

1.0

0.9

0.9

0.8

0.9

0.8

 

8

12

8

10

7

8

6

9

8

10

7

10

10

8

11

8

8

8

6

8

7

TA 1537

-S9

0.

4.

20.

100.

500.

2500.

5000.

7.7

8.3

10.0

8.3

9.3

9.0

7.3

1.5

1.2

0.0

0.6

0.6

1.0

1.5

 

1.1

1.3

1.1

1.2

1.2

0.9

 

6

9

10

8

9

9

6

8

7

10

8

9

10

7

9

9

10

9

10

8

9

TA 98

+S9

0.

4.

20.

100.

500.

2500.

5000.

35.0

30.7

29.0

29.7

29.7

28.0

23.0

6.9

2.5

2.6

6.5

8.6

6.2

4.4

 

0.9

0.8

0.8

0.8

0.8

0.7

 

39

28

30

30

39

23

21

27

33

26

23

22

35

20

39

31

31

36

28

26

28

TA 98

-S9

0.

4.

20.

100.

500.

2500.

5000.

23.7

25.0

24.3

25.0

21.0

22.7

23.3

3.8

4.4

2.9

1.7

1.0

2.1

3.2

 

1.1

1.0

1.1

0.9

1.0

1.0

 

28

27

26

27

22

25

27

22

20

21

24

20

21

22

21

28

26

24

21

22

21

 

CONTROLS – TEST 03

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVERTANTS/ PLATE

PLATE

1

PLATE

2

PLATE

3

TA 100 D

+S9

SOLVENT CONTROL

 

 

 

 

 

 

201.3

27.5

 

 

201

229

174

NEGATIVE CONTROLS

 

 

 

 

 

 

176.3

24.1

0.9

 

156

170

203

POSITIVE CONTROLS

 

NOT TESTED

 

 

 

*** NO RESULTS FOR THIS STRAIN ***

 

TA 100 D

-S9

SOLVENT CONTROLS

 

 

 

 

 

 

162.3

5.5

 

 

162

168

157

NEGATIVE CONTROLS

 

 

 

 

 

 

148.0

26.0

0.9

 

132

134

178

POSITIVE CONTROLS

 

NOT TESTED

 

 

 

*** NO RESULTS FOR THIS STRAIN ***

 

 

TEST 03

THE EXPERIMENT WITH METABOLIC ACTIVATION WAS PERFORMED WITH 30% SYRIAN GOLDEN HAMSTER LIVER S9-MIX AND PREINCUBATION

STRAIN

DOSE LEVELS

(μg/plate)

MEAN

STANDARD DEVIATION

RATIO: TEST/ CONTROL

BACTERIAL LAWN

NO REVTERANTS /PLATE

PLATE 1

PLATE 2

PLATE 3

TA 100 D

+S9

0.

4.

20.

100.

500.

2500.

5000.

201.3

179.3

185.3

154.7

178.3

197.3

195.0

27.5

6.0

23.2

19.7

14.6

21.2

4.4

 

0.9

0.9

0.8

0.9

1.0

1.0

 

201

180

210

167

163

178

198

229

173

182

132

192

194

197

174

185

164

165

180

220

190

TA 100 D

-S9

0.

4.

20.

100.

500.

2500.

5000.

162.3

135.3

127.7

137.3

132.3

135.0

126.7

5.5

30.8

23.3

18.6

22.7

30.6

19.5

 

0.8

0.8

0.8

0.8

0.8

0.8

 

162

125

101

122

108

122

118

168

111

138

132

136

113

113

157

170

144

158

153

170

149

 

Applicant's summary and conclusion

Conclusions:
The results led to the conclusion that the test substance is not mutagenic in the absence of a metabolic activation system using the standard Ames Test procedure. Also in the presence of hamster liver S9-mix (30 % (v/v)) and preincubation the test compound did not induce a significant increase in the number of revertant colonies. The substance did not cause any cytotoxic effects.
Executive summary:

The test substance was tested for mutagenicity with the strains TA 100, TA 1535, TA 1537 and TA 98 of Salmonella typhimurium.

 

The mutagenicity studies were conducted in the standard plate test (Ames Test) and in a modified preincubation test (Prival Test). The studies were performed in the absence and in the presence of a metabolising system derived from hamster liver homogenate. The test compound was dissolved in aqua bidest. and a dose range of 6 different doses from 4 microgram/plate to 5000 microgram/plate was used.

 

Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies.

 

Toxicity: The test compound proved to be not toxic to the bacterial strains.

 

5000 microgram/plate was chosen as top dose level for the mutagenicity study.

 

a) Ames Test:

Mutagenicity: In the absence of the metabolic activation system, the test substance did not show a dose dependent increase in the number of revertants in any of the bacterial strains.

 

b) Prival Test:

In the presence of hamster liver S9-mix (30 % (v/v)) using the preincubation method according to Prival the test substance did not induce a significant increase in the number of revertant colonies with any of the tester strains.

 

Summarising, it can be stated that the test substance is not mutagenic in the standard plate test (Ames Test) and in the preincubation method according to Prival.

The test substance is not classified according to CLP criteria.