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Genetic toxicity: in vitro

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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:
Study was performed between 25 February 2010 and 23 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to GLP and internationally accepted guideline

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Kieselguhr, soda ash flux-calcined
EC Number:
272-489-0
EC Name:
Kieselguhr, soda ash flux-calcined
Cas Number:
68855-54-9
Molecular formula:
SiO2
IUPAC Name:
Kieselguhr, soda ash flux-calcined
Details on test material:
Sponsor's identification: Soda-ash flux calcined kieselguhr
Description : White powder
Purity : 100%
Batch number : 211184
Date received : 10 February 2010
Expiry date : 31 December 2013
Storage conditions: Room temperature in the dark

Method

Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix (rat liver homogenate metabolising system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Mutation test - Experiments 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate
Vehicle / solvent:
- Vehicle: Polyethylene glycol 400

- Justification for choice of vehicle: For compounds that are completely insoluble in the standard Ames solvents as recommended by Maron et al (1980) the testing laboratory found that Polyethylene glycol 400 is an excellent suspending agent. However, as this vehicle is not on the recommended lists for acceptability, in-house testing was performed to ensure that PEG 400 is non-toxic to either the bacteria or the microsomal S9 enzymes and that it does not inhibit the mutagenic effect of the standard positive controls. All results were within acceptable ranges and therefore PEG 400 is considered to be satisfactory for use in the reverse mutation assay.

The test material was insoluble in sterile distilled water, dimethyl sulphoxide, acetone, dimethyl formamide and acetonitrile at 50 mg/ml and tetrahydrofuran at 200 mg/ml in solubility checks performed in–house. The test material formed a good, doseable suspension in polyethylene glycol 400 at 12.5 and 25 mg/ml, therefore, this suspending agent was selected as the vehicle
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Concurrent untreated controls
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Migrated to IUCLID6: 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100, 5 μg/plate for TA1535 - without S9-mix
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Migrated to IUCLID6: 80 μg/plate for TA1537 - without S9-mix
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Migrated to IUCLID6: 0.2 μg/plate for TA98 - without S9-mix
Positive controls:
yes
Positive control substance:
other: 2-Aminoanthracene (2AA) with S9-mix: 1 μg/plate for TA100; 2 μg/plate for TA1535 and TA1537; 10 μg/plate for WP2uvrA-
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
Migrated to IUCLID6: 5 μg/plate for TA98 - with S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2:

Preliminary Toxicity Test: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1: Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counter.

Mutation Test - Experiment 2: The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 μg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and foreach concentration of test material both with and without S9-mix. Manual counts were required after employing the pre-incubation method at 5000 μg/plate (absence of S9-mix only) because of a particulate test material precipitation.

DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1)


NUMBER OF REPLICATIONS: Plates were prepared in triplicate


NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 1 to 9.9 x 10^09 bacteria per mL.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. A fine, black particulate precipitate was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the strains of bacteria, at any dose level either with or without metabolic activation or exposure method.

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table1              Spontaneous Mutation Rates (Concurrent Negative Controls)

EXPERIMENT 1

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

93

 

23

 

29

 

23

 

8

 

88

(91)

24

(23)

30

(27)

18

(20)

11

(10)

93

 

22

 

21

 

20

 

12

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

125

 

27

 

29

 

23

 

15

 

120

(114)

22

(24)

31

(26)

21

(22)

8

(12)

98

 

22

 

19

 

23

 

13

 

 

Table2:   Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 12 March 2010

To: 15 March 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

144

167

175

(162)

16.1#

22

27

18

(22)

4.5

29

20

21

(23)

4.9

32

22

34

(29)

6.4

23

16

16

(18)

4.0

-

50

187

174

181

(181)

6.5

23

18

20

(20)

2.5

21

29

20

(23)

4.9

26

29

33

(29)

3.5

20

20

12

(17)

4.6

-

150

161

181

152

(165)

14.8

21

18

23

(21)

2.5

21

21

19

(20)

1.2

22

26

32

(27)

5.0

18

16

15

(16)

1.5

-

500

191

173

141

(168)

25.3

22

21

23

(22)

1.0

18

26

16

(20)

5.3

27

22

29

(26)

3.6

15

19

20

(18)

2.6

-

1500

166

166

153

(162)

7.5

16

24

23

(21)

4.4

26

27

26

(26)

0.6

29

16

27

(24)

7.0

19

16

21

(19)

2.5

-

5000

148 P

139 P

137 P

(141)

5.9

20 P

16 P

21 P

(19)

2.6

25 P

19 P

24 P

(23)

3.2

23 P

32 P

21 P

(25)

5.9

18 P

16 P

18 P

(17)

1.2

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

528

665

583

(592)

68.9

301

305

283

(296)

11.7

197

188

187

(191)

5.5

128

118

119

(122)

5.5

618

673

649

(647)

27.6

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation Table3              Test Results: Experiment 1 – With Metabolic Activation

Test Period

From: 12 March 2010

To: 15 March 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

185

157

172

(171)

14.0#

13

13

14

(13)

0.6

27

29

26

(27)

1.5

33

26

34

(31)

4.4

15

10

11

(12)

2.6

+

50

168

168

176

(171)

4.6

18

14

12

(15)

3.1

21

21

18

(20)

1.7

27

30

27

(28)

1.7

13

11

12

(12)

1.0

+

150

174

147

185

(169)

19.6

10

13

11

(11)

1.5

18

22

24

(21)

3.1

31

32

33

(32)

1.0

18

13

12

(14)

3.2

+

500

179

156

174

(170)

12.1

14

11

15

(13)

2.1

16

26

24

(22)

5.3

30

23

31

(28)

4.4

14

13

13

(13)

0.6

+

1500

185

180

136

(167)

27.0

13

16

13

(14)

1.7

23

21

29

(24)

4.2

33

30

25

(29)

4.0

11

16

16

(14)

2.9

+

5000

158 P

177 P

167 P

(167)

9.5

13 P

18 P

10 P

(14)

4.0

23 P

21 P

21 P

(22)

1.2

31 P

24 P

33 P

(29)

4.7

12 P

13 P

15 P

(13)

1.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1062

962

874

(966)

94.1

163

172

186

(174)

11.6

377

330

318

(342)

31.2

212

172

175

(186)

22.3

343

401

385

(376)

30.0

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        Standard deviation Table4              Test Results: Experiment 2 – Without Metabolic Activation

Test Period

From: 20 April 2010

To: 23 April 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

-

0

168

156

159

(161)

6.2#

26

24

23

(24)

1.5

23

21

18

(21)

2.5

26

29

30

(28)

2.1

9

13

8

(10)

2.6

-

50

154

136

152

(147)

9.9

21

24

29

(25)

4.0

21

24

14

(20)

5.1

25

29

31

(28)

3.1

9

10

11

(10)

1.0

-

150

151

142

144

(146)

4.7

21

26

29

(25)

4.0

25

20

15

(20)

5.0

20

27

26

(24)

3.8

11

11

7

(10)

2.3

-

500

151

158

141

(150)

8.5

26

23

25

(25)

1.5

19

16

14

(16)

2.5

24

26

25

(25)

1.0

9

11

10

(10)

1.0

-

1500

155

156

151

(154)

2.6

23

27

22

(24)

2.6

20

18

21

(20)

1.5

26

20

26

(24)

3.5

12

9

10

(10)

1.5

-

5000

156 P

152 P

139 P

(149)

8.9

27 P

25 P

26 P

(26)

1.0

20 P

21 P

22 P

(21)

1.0

18 P

22 P

22 P

(21)

2.3

10 P

8 P

8 P

(9)

1.2

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

407

420

368

(398)

27.1

236

244

172

(217)

39.5

363

387

423

(391)

30.2

663

510

291

(488)

187.0

2374

2028

2084

(2162)

185.7

ENNG N-ethyl-N'-nitro-N-nitrosoguanidine

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation Table5              Test Results: Experiment 2 – With Metabolic Activation

Test Period

From: 20 April 2010

To: 23 April 2010

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

+

0

173

159

157

(163)

8.7#

16

20

16

(17)

2.3

32

33

22

(29)

6.1

31

34

30

(32)

2.1

12

16

14

(14)

2.0

+

50

154

178

143

(158)

17.9

16

12

14

(14)

2.0

31

20

18

(23)

7.0

31

27

31

(30)

2.3

8

12

7

(9)

2.6

+

150

144

164

141

(150)

12.5

14

18

13

(15)

2.6

20

23

19

(21)

2.1

28

34

29

(30)

3.2

15

10

13

(13)

2.5

+

500

162

158

163

(161)

2.6

16

11

13

(13)

2.5

30

33

26

(30)

3.5

25

31

29

(28)

3.1

12

14

8

(11)

3.1

+

1500

194

147

184

(175)

24.8

13

13

14

(13)

0.6

27

23

26

(25)

2.1

31

26

33

(30)

3.6

15

14

13

(14)

1.0

+

5000

168 P

174 P

135 P

(159)

21.0

12 P

17 P

16 P

(15)

2.6

29 P

26 P

23 P

(26)

3.0

32 P

34 P

29 P

(32)

2.5

14 P

13 P

15 P

(14)

1.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1398

1669

1671

(1579)

157.0

329

311

354

(331)

21.6

187

219

199

(202)

16.2

235

202

268

(235)

33.0

173

243

291

(236)

59.3

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        Standard deviation

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.