Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 July 2018 and 23 July 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
This deviation is considered to have no impact on either the result or integrity of the study.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
This deviation is considered to have no impact on either the result or integrity of the study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
This deviation is considered to have no impact on either the result or integrity of the study.
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
This deviation is considered to have no impact on either the result or integrity of the study.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Chemical Name: Reaction mass of Magnesium dioxido(oxo)silane and Silicone dioxide (MgSiO3, Si and SiO2)
Physical state/Appearance: Black powder
Batch Number: Y180510A
Purity: > 95%
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark

Method

Target gene:
Salmonella typhimurium: histidine
Escherichia coli (WP2uvrA): tryptophan
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:
rat liver homogenate metabolizing system (10% liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
The maximum concentration was 5000 µg/plate (the OECD TG 471 maximum recommended dose level)
Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500 and 5000 µg/plate.
Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
Vehicle / solvent:
Vehicle: DMSO
The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Controlsopen allclose all
Untreated negative controls:
yes
Remarks:
Untreated plates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (> 99.9%)
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Absence of S9-mix
Untreated negative controls:
yes
Remarks:
Untreated plates
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (> 99.9%)
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
Microsomal Enzyme Fraction
The S9 Microsomal fractions (CD Sprague-Dawley) were pre-prepared using standardized in house procedures (outside the confines of this study). Lot No. PB/βNF S9 25 May 2018 was used in this study.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.
S9 5.0 mL
1.65 M KCl/0.4 M MgCl2 1.0 mL
0.1 M Glucose-6-phosphate 2.5 mL
0.1 M NADP 2.0 mL
0.2 M Sodium phosphate buffer (pH 7.4) 25.0 mL
Sterile distilled water 14.5 mL
A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 7193746 04/2022) and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd (lot numbers 48097 08/2018 and 48043 08/2018).

Test Item Preparation and Analysis
The test item was accurately weighed and, on the day of each experiment, approximate half log dilutions prepared in pre-dried dimethyl sulphoxide by mixing on a vortex mixer and sonication for 20 minutes at 40 °C. Formulated concentrations were adjusted to allow for the stated water/impurity content (95%) of the test item.
All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 – Plate Incorporation Method
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9 mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Manual counts were performed on various plates due to spreading colonies/artefact , thereby obtaining an accurate count for reporting.

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
Without Metabolic Activation
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.

With Metabolic Activation
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9 mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.

Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
The study was based on the in vitro technique described by Ames et al., (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These Salmonella typhimurium strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium, revertants may be produced after exposure to a chemical mutagen which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.
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 item and the bacteria are also incubated in the presence of a liver microsomal preparation (S9-mix) prepared from rats pre treated with a mixture known to induce an elevated level of these enzymes.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

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
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
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
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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:
Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and the S9-mix used in both experiments was shown to be sterile. The test item formulation was also shown to be sterile. These data are not given in the report.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The vehicle (dimethyl sulphoxide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

Experiment 1 (plate incorporation)
The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate.
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item precipitate (black and fibrous in appearance) was noted by eye at 1500 and 5000 µg/plate in the absence and presence of metabolic activation (S9-mix), respectively. This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Experiment 2 (pre-incubation)
The maximum dose level of the test item in the second experiment was the same as for Experiment 1 (5000 µg/plate).
There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix).
A test item precipitate (black and fibrous in appearance) was noted by eye at 5000 µg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.
There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix).

Any other information on results incl. tables

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

143

 

18

 

15

 

25

 

20

 

113

(130)

26

(24)

24

(24)

28

(26)

11

(15)

133

 

28

 

33

 

24

 

13

 

Experiment 2

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA

TA98

TA1537

161

 

19

 

29

 

23

 

12

 

146

(149)

32

(23)

37

(32)

27

(26)

17

(13)

140

 

19

 

29

 

27

 

11

 

 

Test Results: Experiment 1 – Without Metabolic Activation (Plate Incorporation)

Test Period

From: 17 July 2018

To: 20 July 2018

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

128

113

120

(120)

7.5#

25

30

25

(27)

2.9

27

29

27

(28)

1.2

13

15

26

(18)

7.0

11

17

10

(13)

3.8

1.5 µg

132

139

119

(130)

10.1

26

25

27

(26)

1.0

25

23

33

(27)

5.3

22

23

21

(22)

1.0

10

13

15

(13)

2.5

5 µg

130

129

130

(130)

0.6

31

23

23

(26)

4.6

15

31

23

(23)

8.0

10

16

17

(14)

3.8

11

18

15

(15)

3.5

15 µg

133

121

120

(125)

7.2

25

26

36

(29)

6.1

27

16

19

(21)

5.7

18

18

16

(17)

1.2

19

8

22

(16)

7.4

50 µg

131

122

138

(130)

8.0

27

24

26

(26)

1.5

29

27

27

(28)

1.2

22

14

19

(18)

4.0

20

13

13

(15)

4.0

150 µg

121

125

129

(125)

4.0

22

24

28

(25)

3.1

23

22

40

(28)

10.1

18

19

15

(17)

2.1

15

11

11

(12)

2.3

500 µg

96

121

112

(110)

12.7

25

21

30

(25)

4.5

26

24

30

(27)

3.1

26

20

28

(25)

4.2

11

7

10

(9)

2.1

1500 µg

107 P

134 P

112 P

(118)

14.4

27 P

26 P

26 P

(26)

0.6

27 P

23 P

28 P

(26)

2.6

13 P

21 P

18 P

(17)

4.0

7 P

10 P

11 P

(9)

2.1

5000 µg

119 P

113 P

121 P

(118)

4.2

31 P

21 P

24 P

(25)

5.1

29 P

27 P

18 P

(25)

5.9

24 P

15 P

24 P

(21)

5.2

9 P

14 P

18 P

(14)

4.5

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

681

670

655

(669)

13.1

710

835

1023

(856)

157.6

900

864

779

(848)

62.1

159

157

146

(154)

7.0

407

332

366

(368)

37.6

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

4NQO      4-Nitroquinoline-1-oxide

9AA        9-Aminoacridine

P            Test Item Precipitate

#            Standard deviation

Test Results: Experiment 1 – With Metabolic Activation (Plate Incorporation)

Test Period

From: 17 July 2018

To: 20 July 2018

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

152

141

145

(146)

5.6#

12

13

14

(13)

1.0

35

32

33

(33)

1.5

22

18

24

(21)

3.1

15

10

10

(12)

2.9

1.5 µg

134

120

142

(132)

11.1

18

8

15

(14)

5.1

47

42

37

(42)

5.0

28

30

28

(29)

1.2

12

9

9

(10)

1.7

5 µg

151

133

151

(145)

10.4

10

19

16

(15)

4.6

28

31

21

(27)

5.1

23

30

29

(27)

3.8

13

16

12

(14)

2.1

15 µg

126

134

132

(131)

4.2

14

11

13

(13)

1.5

35

33

33

(34)

1.2

20

25

23

(23)

2.5

15

11

14

(13)

2.1

50 µg

134

132

127

(131)

3.6

13

19

5

(12)

7.0

30

27

29

(29)

1.5

24

24

32

(27)

4.6

16

12

12

(13)

2.3

150 µg

141

134

130

(135)

5.6

15

8

17

(13)

4.7

31

32

29

(31)

1.5

20

25

31

(25)

5.5

18

10

12

(13)

4.2

500 µg

118

128

110

(119)

9.0

8

16

9

(11)

4.4

25

45

35

(35)

10.0

15

26

28

(23)

7.0

10

15

8

(11)

3.6

1500 µg

119

111

138

(123)

13.9

13

9

12

(11)

2.1

40

36

31

(36)

4.5

30

26

24

(27)

3.1

13

10

15

(13)

2.5

5000 µg

120 P

111 P

107 P

(113)

6.7

10 P

8 P

12 P

(10)

2.0

33 P

32 P

38 P

(34)

3.2

28 P

29 P

26 P

(28)

1.5

13 P

16 P

12 P

(14)

2.1

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

1559

1657

2216

(1811)

354.4

295

331

310

(312)

18.1

226

220

241

(229)

10.8

151

121

131

(134)

15.3

306

353

395

(351)

44.5

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#            Standard deviation

Test Results: Experiment 2 – Without Metabolic Activation (Pre-Incubation)

Test Period

From: 20 July 2018

To: 23 July 2018

S9-Mix

(-)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

154

123

153

(143)

17.6#

22

15

19

(19)

3.5

21

38

34

(31)

8.9

29

16

24

(23)

6.6

11

15

6

(11)

4.5

15 µg

137

158

157

(151)

11.8

23

18

20

(20)

2.5

28

27

38

(31)

6.1

27

28

13

(23)

8.4

17

16

14

(16)

1.5

50 µg

154

149

149

(151)

2.9

22

19

23

(21)

2.1

33

28

24

(28)

4.5

36

32

27

(32)

4.5

15

13

24

(17)

5.9

150 µg

141

144

152

(146)

5.7

16

18

14

(16)

2.0

30

36

22

(29)

7.0

24

19

26

(23)

3.6

12

11

14

(12)

1.5

500 µg

129

154

150

(144)

13.4

27

20

26

(24)

3.8

32

29

30

(30)

1.5

19

24

22

(22)

2.5

15

17

23

(18)

4.2

1500 µg

136

141

135

(137)

3.2

21

31

23

(25)

5.3

34

34

32

(33)

1.2

32

21

26

(26)

5.5

18

14

7

(13)

5.6

5000 µg

138 P

150 P

137 P

(142)

7.2

31 P

30 P

17 P

(26)

7.8

39 P

30 P

33 P

(34)

4.6

20 P

22 P

24 P

(22)

2.0

17 P

23 P

9 P

(16)

7.0

Positive controls

S9-Mix

(-)

Name

Dose Level

No. of Revertants

ENNG

ENNG

ENNG

4NQO

9AA

3 µg

5 µg

2 µg

0.2 µg

80 µg

987

864

946

(932)

62.6

1013

986

1034

(1011)

24.1

1104

1060

1124

(1096)

32.7

262

281

280

(274)

10.7

483

486

510

(493)

14.8

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

4NQO         4-Nitroquinoline-1-oxide

9AA           9-Aminoacridine

P               Test item precipitate

#               Standard deviation

Test Results: Experiment 2 – With Metabolic Activation (Pre-Incubation)

Test Period

From: 20 July 2018

To: 23 July 2018

S9-Mix

(+)

Dose Level

Per Plate

Number of revertants (mean) +/- SD

Base-pair substitution strains

Frameshift strains

TA100

TA1535

WP2uvrA

TA98

TA1537

Solvent Control

(DMSO)

152

129

149

(143)

12.5#

11

14

17

(14)

3.0

47

31

38

(39)

8.0

28

29

37

(31)

4.9

16

25

14

(18)

5.9

15 µg

134

149

148

(144)

8.4

8

18

20

(15)

6.4

36

39

35

(37)

2.1

35

24

34

(31)

6.1

15

11

20

(15)

4.5

50 µg

134

129

119

(127)

7.6

13

18

17

(16)

2.6

38

32

44

(38)

6.0

32

40

26

(33)

7.0

9

22

18

(16)

6.7

150 µg

132

121

128

(127)

5.6

17

14

18

(16)

2.1

35

42

37

(38)

3.6

28

29

31

(29)

1.5

16

12

13

(14)

2.1

500 µg

132

124

111

(122)

10.6

14

18

16

(16)

2.0

37

40

42

(40)

2.5

30

28

23

(27)

3.6

23

19

15

(19)

4.0

1500 µg

134

127

155

(139)

14.6

28

15

18

(20)

6.8

41

41

36

(39)

2.9

27

36

31

(31)

4.5

22

20

22

(21)

1.2

5000 µg

142 P

126 P

180 P

(149)

27.7

16 P

22 P

12 P

(17)

5.0

36 P

55 P

37 P

(43)

10.7

17 P

29 P

30 P

(25)

7.2

8 P

14 P

32 P

(18)

12.5

Positive controls

S9-Mix

(+)

Name

Dose Level

No. of Revertants

2AA

2AA

2AA

BP

2AA

1 µg

2 µg

10 µg

5 µg

2 µg

2346

2185

2031

(2187)

157.5

386

426

411

(408)

20.2

144

151

147

(147)

3.5

87

120

145

(117)

29.1

386

314

337

(346)

36.8

BP          Benzo(a)pyrene

2AA        2-Aminoanthracene

P            Test item precipitate

#            Standard deviation

Applicant's summary and conclusion

Conclusions:
In this Reverse Mutation Assay ‘Ames Test’ using strains of Salmonella typhimurium and Escherichia coli (OECD TG 471) the test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide did not induce an increase in the frequency of revertant colonies at any of the dose levels used either with or without metabolic activation (S9-mix). Under the conditions of this test Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was considered to be non-mutagenic.
Executive summary:

Introduction

The test method was designed to be compatible with the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF, the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Regulation (EC) number 440/2008 of 30 May 2008, the ICH S2(R1)guideline adopted June 2012 (ICH S2(R1) Federal Register. Adopted 2012; 77:33748-33749)and the USA, EPA OCSPP harmonized guideline - Bacterial Reverse Mutation Test.

Methods

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with suspensions of the test item using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 (plate incorporation) was based on OECD TG 471 and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and was 15 to 5000 µg/plate. Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non‑toxic dose levels and the potential toxicity of the test item following the change in test methodology.

Results

The vehicle (dimethyl sulphoxide) 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 and without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The maximum dose level of the test item in the first experiment was selected as the OECD TG 471 recommended dose level of 5000 µg/plate. There was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method). A test item precipitate (black and fibrous in appearance) was noted by eye at 1500 and 5000 mg/plate in both the presence and absence of metabolic activation (S9-mix), respectively. This observation did not prevent the scoring of revertant colonies.

Based on the results of Experiment 1, the same maximum dose level (5000 µg/plate) was employed in the second mutation test (pre-incubation method). Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix). A test item precipitate (black and fibrous in appearance) was by eye at 5000 mg/plate in both the presence and absence of metabolic activation (S9-mix). This observation did not prevent the scoring of revertant colonies.

There were no significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). 

Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre‑incubation method). 

Conclusion

Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was considered to be non-mutagenic under the conditions of this test.