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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:
The experimental phase of the study was performed between 11 February 2010 and 25 March 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:
Bentonite, acid-leached
EC Number:
274-324-8
EC Name:
Bentonite, acid-leached
Cas Number:
70131-50-9
Molecular formula:
unspecified for this UVCB substance
IUPAC Name:
Bentonite, acid-leached
Details on test material:
- Name of test material (as cited in study report):Bentonite acid-leached
- Physical state: Solid
- Purity.: 100%
- Expiration date of the lot/batch: 31 December 2010
- Storage condition of test material: 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.

- Justification for choice of vehicle: The test material was insoluble and could not be sufficiently suspended 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 the best doseable suspension in polyethylene glycol 400 and was therefore 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 were 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 were considered first, statistical methods, as recommended by the UKEMS were also used as an aid to evaluation, however, statistical significance was not 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 vehicle (polyethylene glycol 400) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

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 particulate precipitate was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

No toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method. A small, statistically significant increase in TA100 revertant colony frequency was observed in the presence of S9 at 50 μg/plate in Experiment 2. This increase was considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at 50 μg/plate were within the in-house historical untreated/vehicle control range for the tester strain and the fold increase was only 1.19 times the concurrent vehicle control.













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

97

 

20

 

22

 

18

 

7

 

91

(96)

24

(21)

29

(24)

23

(19)

11

(11)

99

 

19

 

20

 

15

 

14

 

EXPERIMENT 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

114

 

21

 

25

 

15

 

14

 

105

(102)

24

(22)

13

(19)

12

(17)

12

(12)

86

 

20

 

19

 

25

 

11

 

Table2              Test Results: Experiment 1 – Without Metabolic Activation

Test Period

From: 15 March 2010

To: 18 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

129

130

115

(125)

8.4#

20

25

22

(22)

2.5

23

33

29

(28)

5.0

26

21

35

(27)

7.1

10

11

14

(12)

2.1

-

50

124

109

113

(115)

7.8

24

25

19

(23)

3.2

26

14

20

(20)

6.0

21

20

22

(21)

1.0

11

10

11

(11)

0.6

-

150

115

111

119

(115)

4.0

27

23

30

(27)

3.5

23

25

20

(23)

2.5

24

23

33

(27)

5.5

14

10

10

(11)

2.3

-

500

135

101

119

(118)

17.0

19

29

30

(26)

6.1

24

33

31

(29)

4.7

21

26

23

(23)

2.5

16

11

15

(14)

2.6

-

1500

111 P

137 P

119 P

(122)

13.3

36 P

23 P

13 P

(24)

11.5

25 P

16 P

22 P

(21)

4.6

30 P

26 P

24 P

(27)

3.1

11 P

8 P

12 P

(10)

2.1

-

5000

108 P

126 P

111 P

(115)

9.6

20 P

23 P

25 P

(23)

2.5

25 P

25 P

28 P

(26)

1.7

23 P

21 P

24 P

(23)

1.5

7 P

12 P

13 P

(11)

3.2

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

586

624

731

(647)

75.2

154

111

162

(142)

27.4

242

227

242

(237)

8.7

102

95

118

(105)

11.8

1270

1027

935

(1077)

173.1

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation   Table 3: Test results: Experiment 1 - With metabolic activation

Test Period

From: 15 March 2010

To: 18 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

106

130

119

(118)

12.0#

24

15

12

(17)

6.2

23

21

30

(25)

4.7

29

26

27

(27)

1.5

14

12

16

(14)

2.0

+

50

117

108

112

(112)

4.5

14

25

15

(18)

6.1

20

23

22

(22)

1.5

26

33

23

(27)

5.1

18

12

14

(15)

3.1

+

150

103

114

101

(106)

7.0

13

12

19

(15)

3.8

22

16

22

(20)

3.5

35

20

24

(26)

7.8

8

8

11

(9)

1.7

+

500

98

104

113

(105)

7.5

18

19

21

(19)

1.5

21

33

24

(26)

6.2

30

31

27

(29)

2.1

15

11

9

(12)

3.1

+

1500

128 P

97 P

135 P

(120)

20.2

18 P

16 P

23 P

(19)

3.6

26 P

33 P

24 P

(28)

4.7

31 P

27 P

29 P

(29)

2.0

13 P

15 P

11 P

(13)

2.0

+

5000

117 P

121 P

123 P

(120)

3.1

19 P

16 P

15 P

(17)

2.1

22 P

28 P

26 P

(25)

3.1

25 P

21 P

23 P

(23)

2.0

12 P

11 P

9 P

(11)

1.5

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

305

310

280

(298)

16.1

145

200

156

(167)

29.1

181

146

184

(170)

21.1

168

148

119

(145)

24.6

131

124

155

(137)

16.3

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

#        Standard deviation

Table 4: Test results: Experiment 2 - Without metabolic activation

Test Period

From: 22 March 2010

To: 25 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

142

126

128

(132)

8.7#

13

21

13

(16)

4.6

20

12

26

(19)

7.0

24

16

22

(21)

4.2

5

10

8

(8)

2.5

-

50

130

124

106

(120)

12.5

24

23

24

(24)

0.6

14

33

15

(21)

10.7

29

19

24

(24)

5.0

12

5

4

(7)

4.4

-

150

135

129

110

(125)

13.1

13

22

14

(16)

4.9

18

12

19

(16)

3.8

20

34

25

(26)

7.1

14

12

4

(10)

5.3

-

500

156

128

154

(146)

15.6

23

12

15

(17)

5.7

27

22

26

(25)

2.6

27

14

25

(22)

7.0

3

1

7

(4)

3.1

-

1500

148 P

139 P

135 P

(141)

6.7

21 P

15 P

14 P

(17)

3.8

22 P

22 P

27 P

(24)

2.9

32 P

29 P

32 P

(31)

1.7

7 P

15 P

8 P

(10)

4.4

-

5000

135 P

124 P

150 P

(136)

13.1

18 P

12 P

18 P

(16)

3.5

18 P

29 P

23 P

(23)

5.5

20 P

24 P

29 P

(24)

4.5

3 P

12 P

9 P

(8)

4.6

Positive

controls

 

S9-Mix

 

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

438

422

470

(443)

24.4

366

385

443

(398)

40.1

605

584

623

(604)

19.5

178

196

184

(186)

9.2

2484

3236

2411

(2710)

456.7

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

4NQO 4-Nitroquinoline-1-oxide

9AA    9-Aminoacridine

P        Precipitate

#        Standard deviation

Table 5: Test results: Experiment 2 - With metabolic activation

Test Period

From: 22 March 2010

To: 25 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

153

168

139

(153)

14.5#

18

11

11

(13)

4.0

27

24

25

(25)

1.5

35

32

30

(32)

2.5

12

16

9

(12)

3.5

+

50

168

200

178

*

(182)

16.4

21

14

13

(16)

4.4

22

19

16

(19)

3.0

30

27

20

(26)

5.1

14

16

9

(13)

3.6

+

150

141

136

132

(136)

4.5

10

13

20

(14)

5.1

19

23

20

(21)

2.1

25

42

26

(31)

9.5

9

22

4

(12)

9.3

+

500

144

153

162

(153)

9.0

16

22

21

(20)

3.2

32

34

30

(32)

2.0

30

33

36

(33)

3.0

9

15

8

(11)

3.8

+

1500

139 P

113 P

135 P

(129)

14.0

24 P

11 P

20 P

(18)

6.7

32 P

37 P

16 P

(28)

11.0

43 P

23 P

48 P

(38)

13.2

12 P

7 P

10 P

(10)

2.5

+

5000

139 P

140 P

159 P

(146)

11.3

12 P

14 P

14 P

(13)

1.2

19 P

18 P

16 P

(18)

1.5

38 P

21 P

23 P

(27)

9.3

11 P

3 P

8 P

(7)

4.0

Positive

controls

 

S9-Mix

 

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1399

1269

1343

(1337)

65.2

249

202

265

(239)

32.8

297

283

284

(288)

7.8

379

321

323

(341)

32.9

319

227

180

(242)

70.7

2AA    2-Aminoanthracene

BP      Benzo(a)pyrene

P        Precipitate

*         p£0.05

#        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.

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