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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-6-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to the respective OECD guideline and under GLP conditions.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Principles of method if other than guideline:
The bacterial reverse mutation test is commonly employed as an initial screen for genotoxic activity and, in particular, for point mutation-inducing activity of chemicals. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain. Since many compounds do not exert a mutagenic effect until they have been metabolized by enzyme systems not available in the bacterial cell, the test compound and test organisms are also incubated in the presence of a liver microsomal fraction.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Charcoal (Probe 1: C-Fix = 73.3%)
IUPAC Name:
Charcoal (Probe 1: C-Fix = 73.3%)
Constituent 2
Chemical structure
Reference substance name:
Charcoal
EC Number:
240-383-3
EC Name:
Charcoal
Cas Number:
16291-96-6
Molecular formula:
C
IUPAC Name:
Charcoal
Details on test material:
Name:charcoal (Probe 1: C-Fix = 73.3%)Batch No.:19062009 1PPAppearance:charcoal typicCAS-No.:16291-96-6EINECS-No.:240-383-3Purity:C-Fix = 73.3%Date of expiry:Dec. 2030Storage conditions: room temperature 20 ± 5 °C, keep away from humidity

Method

Target gene:
Tester strains TA98 and TA 97a is reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Strain TA 102 is sensitive to oxidizing DNA damage based on AT base pairs at the site of the mutation.
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 97
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutations in hisD6610, uvrB, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 98
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutations in hisD3052, uvrB, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Mutations in hisG46, uvrB, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
other: Mutations in hisG428, pKM 101, and rfa
Species / strain / cell type:
S. typhimurium TA 1535
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
other: Mutations in hisG46, uvrB, and rfa
Metabolic activation:
with and without
Metabolic activation system:
Rat liver homogenate (S9 mix) obtained from male Sprague-Dawley rats pretreated with Aroclor 1254 (500 mg/kg body weight)
Test concentrations with justification for top dose:
plate incorporation method: 50, 150, 500, 1,500, and 5,000 µg/platepre-incubation method: 313, 625, ,1250, 2,500, and 5,000 µg/plate
Vehicle / solvent:
Ethanol
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1,2-phenylene diamine
Remarks:
concentration: 20 µg/plate; strains: TA 97a, TA 98 and TA 102 in the absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide
Remarks:
concentration: 1 µg/plate; strains: TA 100 and TA 1535 in the absence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Amino-anthracene
Remarks:
concentration per plate: 1 µg; strains: TA 97a, TA 100, TA 102 and TA 1535 in the presence of metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
concentration:20 µg; strain: TA 98; in the presence of metabolic activation
Details on test system and experimental conditions:
An extract was prepared from 250 g of the test item by extraction with 1 L acetone:n-hexane 50:50 (v/v) for 1 h at 50°C in an ultrasonic bath. The sample was washed with an additional 250 mL of the solvent. The combined extracts were reduced at 50°C in a rotary evaporator to approximately 150 mL and filled quantitatively in a 250 mL volumetric flask. At the start of the experiment, a stock solution containing 50 g/L of the test item in Ethanol was prepared and used for the dilutions.
Evaluation criteria:
The colonies were counted visually, the numbers were recorded.The increase factor f(I) of revertant induction (mean revertants divided by mean spontaneous revertants) and the absolute number of revertants (“Rev. abs.”, mean revertants minus mean spontaneous revertants) were calculated
Statistics:
The use of statistics was limited to the calculation of means and standard deviations.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
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:
valid
Positive controls validity:
valid
Additional information on results:
A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration related increase over the range tested can also be taken as a sign of mutagenic activity.
Remarks on result:
other: other: plate incorporation method and pre-incubation method
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mean revertants incorporation method

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

Mean

99

122

9

13

78

98

185

186

11

5

sd

2.2

3.1

2.2

1.9

3.4

11.6

10.8

5.9

1.7

1.0

DMSO

Mean

113

128

9

9

108

112

196

192

6

14

sd

18.2

4.4

5.0

3.4

12.4

7.0

19.5

9.3

1.7

3.1

Ethanol

Mean

123

106

9

16

113

108

211

184

6

14

sd

10.2

13.4

6.6

1.0

6.6

8.8

11.5

7.5

2.4

0.8

Solvent control

Mean

97

122

7

9

104

90

212

189

8

14

sd

1.5

14.1

1.6

2.5

5.4

12.5

14.1

10.0

3.0

3.7

Positive Controls

Mean

950

958

868

603

642

898

556

968

645

554

sd

120

147

258

73

232

149

206

151

141

107

f(I)

8.41

7.48

96.44

67.00

8.23

8.02

2.84

5.04

58.64

39.57

5000 µg/pl.

Mean

116

122

13

13

101

73

187

196

8

14

sd

14

18

1

5

9

7

4

1

2

1

f(I)

1.20

1.00

1.86

1.44

0.97

0.81

0.88

1.04

1.00

1.00

1500 µg/pl.

Mean

110

98

10

10

84

77

166

178

9

11

sd

13

2

2

3

6

14

18

4

1

3

f(I)

1.13

0.80

1.43

1.11

0.81

0.86

0.78

0.94

1.13

0.79

500 µg/pl.

Mean

116

109

6

9

103

99

183

190

7

6

sd

18

9

1

3

10

7

11

8

1

1

f(I)

1.20

0.89

0.86

1.00

0.99

1.10

0.86

1.01

0.88

0.43

150 µg/pl.

Mean

109

120

6

17

110

81

183

170

10

14

sd

16

7

2

2

7

17

10

4

2

2

f(I)

1.12

0.98

0.86

1.89

1.06

0.90

0.86

0.90

1.25

1.00

50 µg/pl.

Mean

98

113

8

17

103

103

203

199

11

7

sd

4

12

1

2

14

10

2

3

1

2

f(I)

1.01

0.93

1.14

1.89

0.99

1.14

0.96

1.05

1.38

0.50

 

Mean revertants preincubation method

Strain

TA97a

TA98

TA100

TA102

TA1535

Induction

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

-S9

+S9

H2O

Mean

104

122

12

13

104

142

145

147

13

12

sd

17.2

6.4

2.6

5.6

16.1

33.5

16.0

32.7

4.2

2.4

DMSO

Mean

114

104

14

16

120

96

128

213

10

13

sd

5.0

7.2

2.2

2.9

28.0

10.2

12.4

35.9

0.0

1.0

Ethanol

Mean

114

111

15

16

116

113

150

232

11

13

sd

6.4

5.2

3.7

2.2

13.1

27.3

16.7

20.3

4.1

2.1

Solvent control

Mean

124

129

11

10

137

143

172

212

8

11

sd

6.2

36.3

1.0

4.7

26.1

7.1

30.4

31.9

3.9

1.5

Positive Controls

Mean

486

423

677

148

734

569

729

653

111

491

sd

46

28

33

43

50

95

104

135

4

114

f(I)

4.26

4.07

48.36

9.25

7.06

5.93

5.70

3.07

8.54

37.77

5000 µg/pl.

Mean

111

127

10

12

129

129

130

201

6

9

sd

11

24

2

5

15

14

9

96

1

5

f(I)

0.90

0.98

0.91

1.20

0.94

0.90

0.76

0.95

0.75

0.82

2500 µg/pl.

Mean

105

102

12

12

109

102

132

165

14

8

sd

12

13

1

3

16

13

16

40

1

2

f(I)

0.85

0.79

1.09

1.20

0.80

0.71

0.77

0.78

1.75

0.73

1250 µg/pl.

Mean

111

114

5

13

129

119

155

216

10

10

sd

4

4

2

2

9

11

16

17

2

5

f(I)

0.90

0.88

0.45

1.30

0.94

0.83

0.90

1.02

1.25

0.91

625 µg/pl.

Mean

118

126

11

9

120

163

194

232

9

13

sd

10

9

2

4

9

5

31

58

1

3

f(I)

0.95

0.98

1.00

0.90

0.88

1.14

1.13

1.09

1.13

1.18

313 µg/pl.

Mean

111

111

9

10

115

138

179

261

8

13

sd

7

5

1

3

4

20

20

25

1

2

f(I)

0.90

0.86

0.82

1.00

0.84

0.97

1.04

1.23

1.00

1.18

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):negative with an without metabolic activationIt can be stated, that under the test conditions, the test item charcoal (Probe 1: C-Fix = 73.3%) is not mutagenic in the bacterial reverse mutation test using Salmonella typhimurium, strains TA97a, TA98, TA100, TA102, and TA1535.
Executive summary:

The test item charcoal (Probe 1: C-Fix = 73.3%) was tested in the bacterial reverse mutation assay (Ames test) using the Salmonella typhimurium tester strains TA97, TA 98, TA100, TA102, and TA1535 in the presence and absence of metabolic activation by rat liver S9 mix. The assay was performed using both the plate incorporation and the preincubation method. Under the conditions of this study, charcoal (Probe 1: C-Fix = 73.3%) was concluded to be negative in all tester strains in the presence and absence of metabolic activation.

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