Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD TG 471): negative (non-mutagenic)

Chromosome Aberration test (OECD TG 473): negative (non-clastogenic)

MLA (OECD TG 490): negative (non-mutagenic)

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
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
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
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
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. QA statement)
Type of assay:
bacterial reverse mutation assay
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
Target gene:
Salmonella typhimurium: histidine
Escherichia coli (WP2uvrA): tryptophan
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.
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.
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).

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

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 14 August 2018 and 10 October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
Number of lymphocyte cells counted. It was considered that deviation did not have a major impact on the outcome or integrity of the study.
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
Number of lymphocyte cells counted. It was considered that deviation did not have a major impact on the outcome or integrity of the study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
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 Silicon and Silicon dioxide (MgSiO3, Si and SiO2)
Physical state/Appearance: Black powder
Batch: Y180510A
Purity: 95%
Expiry Date: 31 May 2019
Storage Conditions: Room temperature in the dark
Formulated concentrations were adjusted to allow for the stated water/impurity content (5%) of the test item.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non smoking volunteer (aged 18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.
The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: female, aged 32 years
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver homogenate metabolizing system (S9), at a 2% final concentration
Test concentrations with justification for top dose:
The test item was considered to be a UVCB and therefore the maximum recommended dose was initially set at 5000 µg/mL.

The dose levels of the controls and the test item are given below:
4(20)-hour without S9: 0*, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, 20, MMC 0.2*
4(20)-hour with S9 (2%): 0*, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, 20, CP 2*
24-hour without S9: 0*, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, 20, MMC 0.1*

* Dose levels selected for metaphase analysis.
Vehicle / solvent:
Vehicle: MEM
The test item was insoluble in dimethyl sulphoxide and acetone at 500 mg/mL but was suspendable in MEM at 50 mg/mL in solubility checks performed in house.
Prior to each experiment, the test item was accurately weighed, formulated in MEM and appropriate serial dilutions prepared.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Eagle's minimal essential medium with HEPES buffer (MEM)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Absence of S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Eagle's minimal essential medium with HEPES buffer (MEM)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Presence of S9-mix
Details on test system and experimental conditions:
Cell Culture:
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10 % foetal bovine serum (FBS), at approximately 37 ºC with 5 % CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Microsomal Enzyme Fraction and S9-Mix:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures (outside the confines of this study). Lot No. PB/βNF S9 29/03/18 and 31/08/18 were used in this study.
The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed at a 10% volume of S9-mix into culture media, was 2%.

Experimental Design and Study Conduct:
Test Item Preparation and Analysis
The purity of the test item was 95% and was accounted for in the test item formulations.
Prior to each experiment, the test item was accurately weighed, formulated in MEM and appropriate serial dilutions prepared.
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm (Scott et al., 1991).

The pH and osmolality readings are presented in the following table:
Dose level µg/mL 0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000
pH 7.48 7.43 7.47 7.47 7.42 7.41 7.42 7.39 7.40 7.39
Osmolality mOsm 316 322 - 316 - 325 - 323 320 316
- = Not performed for this dose concentration
The test item was formulated within two hours of it being applied to the test system; the test item formulations were assumed to be stable. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation because it is not a requirement of the guidelines. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.
It should be noted that a precipitate of the test item was observed at and above 78.13 µg/mL and, therefore, the maximum recommended dose level in the study would be reduced to 300 µg/mL.

Culture conditions:
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9):
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1mL of 20% S9¯mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and Main Experiment.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the original culture medium. The cells were then re-incubated for a further 20 hours at approximately 37 ºC in 5% CO2 in humidified air.

4-Hour Exposure Without Metabolic Activation (S9):
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The total volume for each culture was a nominal 10 mL.
After 4 hours at approximately 37 ºC, 5% CO2 in humidified air, the cultures were centrifuged and the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium. The cells were then returned to the incubator for a further 20 hours.

24-Hour Exposure Without Metabolic Activation (S9):
As the exposure was continuous the cultures were established, at a nominal volume of 9.0 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal final volume of each culture was 10 mL. The cultures were then incubated at approximately 37 ºC, 5% CO2 in humidified air for 24 hours.
The preliminary toxicity test was performed using all three of the exposure conditions as described for the Main Experiment but using single cultures only.

Preliminary Toxicity Test:
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 20-hour recovery period in treatment-free media, 4(20)-hour exposure.
iii) 24-hour continuous exposure to the test item without S9-mix.
The dose range of test item used was 1.17 to 300 µg/mL, the maximum dose was limited by the precipitate observation in the solubility check.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for mitotic index evaluation. Mitotic index data was used to estimate test item toxicity and for selection of the dose levels for the Main Experiment.

Main Experiment:
Three exposure groups were used for the Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.31 to 20 µg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by 20-hour culture in treatment-free media prior to cell harvest. The dose range of test item used was 0.31 to 20 µg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix prior to cell harvest. The dose range of test item used was 0.31 to 20 µg/mL.

Cell Harvest:
Mitosis was arrested by addition of demecolcine (Colcemid 0.1 µg/mL) two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075M hypotonic KCl. After approximately fourteen minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC to ensure complete fixation prior to slide preparation.

Preparation of Metaphase Spreads:
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labeled with the appropriate identification data.

Staining:
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation of Response:
Qualitative Slide Assessment
The slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation, if any, of the test item. These observations were used to select the dose levels for mitotic index evaluation.

Coding:
The slides were coded using a computerized random number generator.

Mitotic Index:
Prior to the start of the study the GSP was updated to state that 1000 lymphocyte cells would be counted. Due to an error by the study director, a total of 1000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded for the Preliminary Toxicity testing and 2000 lymphocyte cell nuclei for the main experiment. This study was conducted under the previous GSP which stated 2000 lymphocytes to be counted in both cases. It was considered that deviation did not have a major impact on the outcome or integrity of the study.
Both sets of results obtained were expressed as the mitotic index and as a percentage of the vehicle control value. The resulting percentages are relative to the amount of lymphocytes counted.

Scoring of Chromosome Damage:
Where possible, 300 consecutive well-spread metaphases from each concentration were counted (150 per duplicate), where there were at least 15 cells with aberrations (excluding gaps), slide evaluation was terminated. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing and the ISCN (1985). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) (including the incidence of cells with endoreduplicated chromosomes) was also reported. Many experiments with human lymphocytes have established a range of aberration frequencies acceptable for control cultures in normal volunteer donors.
Evaluation criteria:
The following criteria were used to determine a valid assay:
• The frequency of cells with structural chromosome aberrations (excluding gaps) in the vehicle control cultures was within the laboratory historical control data range.
• All the positive control chemicals induced a positive response (p≤0.01) and demonstrated the validity of the experiment and the integrity of the S9-mix
• The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline
• The required number of cells and concentrations were analyzed

Criteria for determining the Study Conclusion
Providing that all of the acceptability criteria are fulfilled, a test item can be considered to be clearly negative if, in any of the experimental conditions examined:
1) The number of cells with structural aberrations in all evaluated dose groups should be within the range of the laboratory historical control data.
2) No toxicologically or statistically significant increase of the number of cells with structural chromosome aberrations is observed following statistical analysis.
3) There is no concentration-related increase at any dose level

A test item can be classified as genotoxic if:
1) The number of cells with structural chromosome aberrations is outside the range of the laboratory historical control data.
2) At least one concentration exhibits a statistically significant increase in the number of cells with structural chromosome aberrations compared to the concurrent negative control.
3) The observed increase in the frequency of cells with structural aberrations is considered to be dose-related
When all of the above criteria are met, the test item can be considered able to induce chromosomal aberrations in human lymphocytes.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include numerical aberrations in the form of polyploidy and endoreduplicated cells.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test. (Richardson et al. 1989).
A toxicologically significant response is recorded when the p value calculated from the statistical analysis of the frequency of cells with aberrations excluding gaps is less than 0.05 when compared to its concurrent control and there is a dose-related increase in the frequency of cells with aberrations which is reproducible. Incidences where marked statistically significant increases are observed only with gap-type aberrations will be assessed on a case by case basis.
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Preliminary Toxicity Test:
The dose range for the Preliminary Toxicity Test was 1.17 to 300 µg/mL. The maximum dose level was based on the precipitate observed in the solubility test.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 75 µg/mL, in all three exposure groups. However precipitate was also observed during microscopic assessment at 9.38 µg/mL in the 4(20)-hour exposure groups and at 4.69 µg/mL in the continuous exposure group.
Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 300 µg/mL in the 4(20)-hour exposures in the presence and absence of metabolic activation (S9) and in the 24 hour continuous exposure. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was based on the lowest precipitating dose level and was 20 µg/mL for the 4(20)-hour exposure groups and the continuous exposure group.

Chromosome Aberration Test – Main Experiment:
The qualitative assessment of the slides determined that precipitate was similar to that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present up to 20 µg/mL in all three exposure groups.

Precipitate observations were made at the end of exposure in blood-free cultures and was noted at and above 5 µg/mL, in the 4(20)-hour exposure group in the absence of S9, and at and above 2.5 µg/mL, in the 4(20)-hour exposure group in the presence of S9 and at and above 10 µg/mL in the 24-hour continuous exposure group.

The mitotix index data for the main experiment confirm the qualitative observations in that a slight dose-related inhibition of mitotic index was observed. In the 4(20)-hour exposure group in the absence of S9, 17 and 23% mitotic inhibition was achieved at 5 and 10 µg/mL respectively.

In the 4(20)-hour exposure group in the presence of S9, 35% mitotic inhibition was achieved at 5 µg/mL. However, it should be noted that no mitotic inhibition was observed at 10 µg/mL.

A slight inhibition of mitotic index of 18 and 4% was noted at 5 and 10 µg/mL respectively in the 24-hour continuous exposure group.

The maximum dose level selected for metaphase analysis was the lowest precipitating dose level (10 µg/mL in the absence of S9 and 5 µg/mL in the presence of S9).

The chromsome aberration data are given in attached background material.

The assay was considered valid as it met all of the following criteria:
The frequency of cells with chromosome aberrations (excluding gaps) in the vehicle control cultures were within the current historical control data range.

All the positive control chemicals induced a demonstrable positive response (p≤0.01) and confirmed the validity and sensitivity of the assay and the integrity of the S9-mix.

The study was performed using all three exposure conditions using a top concentration which meets the requirements of the current testing guideline.

The required number of cells and concentrations were analyzed.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation.

The test item did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in all of the exposure groups.

Chromosome Aberration Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Group

Final concentration of Mg-SiO(µg/mL)

4(20)-hour without S9

0*, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, 20, MMC0.2*

4(20)-hour with S9 (2%)

0*, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, 20, CP2*

24-hour without S9

0*, 0.31, 0.63, 1.25*, 2.5*, 5*, 10*, 20, MMC0.1*

*  = Dose levels selected for metaphase analysis

MMC = Mitomycin C

CP = Cyclophosphamide

Mitotic Index - Preliminary Toxicity Test

Dose Level

(µg/mL)

4(20)-Hour Without S9

4(20)-Hour With S9

24-Hour Without S9

Mitotic Index

% of Control

Mitotic Index

% of Control

Mitotic Index

% of Control

0

7.00

100

5.90

100

8.10

100

1.17

-

-

-

-

9.40

116

2.34

5.70

81

7.50

127

14.20

175

4.69

6.30

90

7.80

132

9.70 P

120

9.38

6.30 P

90

9.20 P

156

7.90 P

98

18.75

6.80 P

97

8.10 P

137

- P

-

37.5

- P

-

- P

-

- P

-

75

- P

-

- P

-

- P

-

150

- P

-

- P

-

- P

-

300

- P

-

- P

-

- P

-

- = Not assessed for mitotic index

P = Precipitate observed at end of exposure period in blood-free cultures

Mitotic Index – Main Experiment (4(20)-hour Exposure Groups)

Dose Level (mg/mL)

4(20)-Hour Without S9

4(20)-Hour With S9

A

B

Mean

% of Control

A

B

Mean

% of Control

0

6.85

6.05

6.45

100

7.85

5.75

6.80

100

0.31

-

-

-

-

-

-

-

-

0.63

-

-

-

-

-

-

-

-

1.25

7.15

5.25

6.20

96

6.90

9.15

8.03

118

2.5

6.70

5.55

6.13

95

7.05 P

9.05 P

8.05

118

5.0

6.05 P

4.70 P

5.38

83

5.40 P

3.45 P

4.43

65

10

4.65 P

5.30 P

4.98

77

8.10 P

7.15 P

7.63

112

20

- P

- P

-

-

- P

- P

-

-

MMC0.2

4.15

3.55

3.85

60

NA

NA

NA

NA

CP2

NA

NA

NA

NA

3.90

4.95

4.43

65

MMC = Mitomycin C

CP = Cyclophosphamide

P = Precipitateat end of exposure period in blood-free cultures

NA = Not applicable

-  = Not assessed for mitotic index

Mitotic Index – Main Experiment (24-hour Exposure Group)

Dose Level

(µg/mL)

24-Hour Without S9

A

B

Mean

% of Control

0

5.50

3.80

4.65

100

0.31

-

-

-

-

0.63

-

-

-

-

1.25

3.45

4.40

3.93

84

2.5

3.75

5.30

4.53

97

5.0

2.90 P

4.70 P

3.80

82

10

3.40 P

5.55 P

4.48

96

20

- P

- P

-

-

MMC0.1

0.45

1.40

0.93

20

MMC  = Mitomycin C

P = Precipitateat end of exposure period in blood-free cultures

NA = Not applicable

-  = Not assessed for mitotic index

Mean Frequency of Polyploid Cells (%)

Main Experiment

Dose Level

(µg/mL)

Exposure Group

4(20)-Hour Without S9

4(20)-Hour With S9

24-Hour Without S9

0

0.3

0

0

0.31

-

-

-

0.63

-

-

-

1.25

0

0

0

2.5

0

0

0

5.0

0

0

0

10

0

-

0

20

-

-

-

MMC 0.4

0

NA

NA

MMC 0.2

NA

NA

0

CP 5

NA

0

NA

MMC    Mitomycin C

CP        Cyclophosphamide

NA       Not applicable

Conclusions:
Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide did not induce a statistically significant increase in the frequency of cells with chromosome aberrations, in either the absence or presence of a liver enzyme metabolizing system. The test item was, therefore, considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction

This report describes the results of anin vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991). 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated;4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Experiment were as follows:

Group

Final concentration of test itemReactionmass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide(µg/mL)

4(20)-hour without S9

0, 0.31, 0.63, 1.25, 5, 10, 20

4(20)-hour with S9 (2%)

0, 0.31, 0.63, 1.25, 5, 10, 20

24-hour without S9

0, 0.31, 0.63, 1.25, 5, 10, 20

Results

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion

The test item, Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 28 August 2018 and 18 September 2018.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
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 Silicon and Silicon dioxide (MgSiO3, Si and SiO2)
Batch Number: Y180510A
Purity: 95%
Physical State/Appearance: Black powder
Expiry Date: 31 May 2019
Storage Conditions: Room temperature, in the dark
Target gene:
thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
20% S9-mix (i.e. 2% final concentration of S9)
Test concentrations with justification for top dose:
The test item was a multi-constituent therefore the maximum proposed dose level in the solubility test was set at 5000 µg/mL, the maximum recommended dose level, and a correction for the purity of the test item of 95% was applied.

Preliminary Toxicity Test
Due to the high levels of precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 1.17 to 300 µg/mL for all three of the exposure groups.
0, 1.17, 2.34, 4.69, 9.38, 18.75, 37.5, 75, 150 and 300 µg/mL

Mutagenicity Test
0.015 to 2 µg/mL for all three of the exposure groups:
0.015, 0.03, 0.06, 0.13, 0.25, 0.5, 1 and 2 µg/mL

Formulated concentrations were adjusted to allow for the stated water/impurity content (5%) of the test item.
Vehicle / solvent:
R0 medium
The test item was found to form a suspension suitable for dosing in culture medium.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
True negative controls:
no
Positive controls:
yes
Remarks:
400 µg/mL and 150 µg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups
Positive control substance:
ethylmethanesulphonate
Remarks:
The positive controls were formulated in DMSO. In the absence of metabolic activation.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
R0 medium
True negative controls:
no
Positive controls:
yes
Remarks:
1.5 µg/mL in the 4 hr exposure group
Positive control substance:
cyclophosphamide
Remarks:
The positive controls were formulated in DMSO. In the presence of metabolic activation.
Details on test system and experimental conditions:
Cell Culture:
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction:
Lot No. PB/βNF S9 29/03/18 was used in this study, and was pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a copy of the certificate of S9 efficacy is presented in Appendix 2.
S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0.
20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the Preliminary Toxicity Test and Mutagenicity Test.

Experimental Design and Study Conduct:
Cell Cleansing:
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Test Item Preparation:
There was no significant change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated (Scott et al., 1991). The pH and osmolality readings from the solubility check are in the following table:
µg/mL 0 19.53 39.06 78.13 156.25 312.5 625 1250 2500 5000
pH 7.48 7.43 7.47 7.47 7.42 7.41 7.42 7.39 7.40 7.39
mOsm 316 322 - 316 - 325 - 323 320 316
- = not determined
No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of it being applied to the test system. It is assumed that the formulation was stable for this duration. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Preliminary Toxicity Test:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure period without S9. Due to the high levels of precipitate observed in the solubility test, the dose range used in the preliminary toxicity test was 1.17 to 300 µg/mL for all three of the exposure groups. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10^5 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 10^5 cells/mL in which case all the cells were maintained. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiment. Maximum dose levels were selected using the following criteria:

i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB*) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.
ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.
iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Mutagenicity Test:
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10^6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, and 0.3 x 10^6 cells/mL in 10 mL cultures were established in 25 cm^2 tissue culture flasks for the 24-hour exposure group in the absence of metabolic activation. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration) at eight dose levels of the test item (0.015 to 2 µg/mL for all three of the exposure groups), vehicle and positive controls. To each universal was added 2 mL of S9 mix if required, 2 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL (R10 was used for the 24 hour exposure group).

The exposure vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.
Rationale for test conditions:
Dose selection for the mutagenicity experiments was made using data from the preliminary toxicity test in an attempt to obtain the desired levels of toxicity. This optimum toxicity is approximately 20% survival (80% toxicity), but no less than 10% survival (90% toxicity). Relative Total Growth (RTG) values are the primary factor used to designate the level of toxicity achieved by the test item for any individual dose level. However, under certain circumstances, %RSG values may also be taken into account when designating the level of toxicity achieved. Dose levels that have RTG survival values less than 10% are excluded from the mutagenicity data analysis, as any response they give would be considered to have no biological or toxicological relevance.
Evaluation criteria:
An approach for defining positive and negative responses is recommended to assure that the increased MF is biologically relevant. In place of statistical analysis generally used for other tests, it relies on the use of a predefined induced mutant frequency (i.e. increase in MF above the concurrent control), designated the Global Evaluation Factor (GEF) of 126 x 10^-6, which is based on the analysis of the distribution of the vehicle control MF data from participating laboratories.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in MF above the concurrent background exceeds the GEF and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Preliminary Cytotoxicity Test:
The dose range of the test item used in the preliminary toxicity test was 1.17 to 300 μg/mL.
There was evidence of very modest dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. Precipitate of the test item was observed at and above 1.17 μg/mL in all three of the exposure groups. Therefore, following the recommendations of the OECD 490 guideline, the maximum dose levels in the Mutagenicity Test were limited by test item precipitate in all three of the exposure groups.


Mutagenicity Test:
As was seen previously, there was evidence of very modest dose-related toxicity in cells treated with the test item in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation, as indicated by the RTG and / or %RSG values. There was no evidence of any reductions in viability (%V) in any of the three exposure indicating that residual toxicity had not occurred. Acceptable levels of toxicity were seen with the positive control substances.

Unexpectedly, precipitate of the test item was observed at all of the dose levels in the 4-hour exposure group in the absence of metabolic activation, at and above 0.03 in the 4-hour exposure group in the presence of metabolic activation, and at and above 0.13 µg/mL in the 24-hour exposure group in the absence of metabolic activation, at the end of the exposure periods. However, it was considered that a maximum dose level lower than 0.015 µg/mL would be beyond the accuracy of the pipettors and this study type. There was also no evidence of any response at the non-precipitating dose levels in the 4-hour exposure group in the presence of metabolic activation and the 24-hour exposure group in the absence of metabolic activation. It should also be noted that no precipitate was observed post-washing below 0.13 µg/mL in the 4-hour exposure group in the absence of metabolic activation. Therefore, the test item was considered to have been adequately tested. The upper two dose levels of 1 and 2 µg/mL were not plated as they were considered to be surplus to requirements with precipitate present at the lower dose levels.

The vehicle controls had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion, indicating that the test system was operating satisfactorily, and that the metabolic activation system was functional.

The test item did not induce any toxicologically significant or dose related increases in the mutant frequency x 10^-6 per viable cell at any of the dose levels, or in any of the three exposure groups.

Preliminary Cytotoxicity Test

The dose range of the test item used in the preliminary toxicity test was 1.17 to 300 µg/mL. The results for the Relative Suspension Growth (%RSG) were as follows:

Dose

(mg/mL)

% RSG (-S9)

4-Hour Exposure

% RSG (+S9)

4-Hour Exposure

% RSG (-S9)

24-Hour Exposure

0

100

100

100

1.17

81

92

99

2.34

88

80

91

4.69

95

80

96

9.38

101

79

94

18.75

92

74

91

37.5

92

84

96

75

90

82

79

150

97

89

76

300

119

75

76

Summary of Results

Main Experiment

Concentration

(µg/mL)

4-Hours-S9

Concentration

(µg/mL)

4-Hours+S9

 

%RSG

RTG

MF§

 

%RSG

RTG

MF§

0

 

100

1.00

130.57

 

0

 

100

1.00

166.34

 

0.015

 

108

1.09

135.48

 

0.015

 

84

1.05

120.77

 

0.03

 

102

0.94

134.50

 

0.03

 

90

0.92

154.45

 

0.06

 

107

1.06

156.50

 

0.06

 

93

1.05

113.30

 

0.13

 

102

1.01

137.57

 

0.13

 

85

0.91

143.07

 

0.25

 

110

1.06

134.00

 

0.25

 

85

0.86

149.50

 

0.5

 

110

1.06

143.00

 

0.5

 

86

0.85

170.78

 

1

Ø

106

 

 

 

1

Ø

82

 

 

 

2

Ø

108

 

 

 

2

Ø

83

 

 

 

MF threshold for a positive response = 256.57

MF threshold for a positive response = 292.34

Positive control

 

 

Positive control

 

 

EMS

 

 

 

 

 

CP

 

 

 

 

 

400

 

89

0.62

1344.49

 

1.5

 

62

0.45

1043.98

 

               

Concentration

(µg/mL)

24-Hours-S9

 

%RSG

RTG

MF§

0

 

100

1.00

168.32

 

0.015

 

87

0.91

172.49

 

0.03

 

98

1.01

171.05

 

0.06

 

88

1.02

137.78

 

0.13

 

88

0.92

158.82

 

0.25

 

92

0.99

163.62

 

0.5

 

76

0.84

169.62

 

1

Ø

61

 

 

 

2

Ø

57

 

 

 

MF threshold for a positive response = 294.32

Positive control

 

 

EMS

 

 

 

 

 

150

 

26

0.22

1867.74

 

 

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration

(µg/mL)

Cell counts $

Viability §

after day 2

2 cells/well

Resistant mutants §

after day 2

2000 cells/well

 

 

 

0h

 24h

48h

 

 

 

 

 

 

 

 

 

0

A

B

9.22

8.54

7.44

8.69

7.03

7.23

76

70

70

72

78

76

77

70

15

16

20

14

22

15

16

15

 

0.015

A

B

8.77

8.73

7.78

8.52

7.85

7.68

72

70

78

75

 

 

 

 

19

19

17

14

 

 

 

 

 

0.03

A

B

8.47

8.29

9.08

7.48

6.88

8.18

73

69

70

73

 

 

 

 

12

16

21

15

 

 

 

 

 

0.06

A

B

8.77

8.80

7.47

8.24

8.17

7.60

69

72

78

75

 

 

 

 

17

23

19

19

 

 

 

 

 

0.13

A

B

8.49

7.27

8.22

7.75

7.57

8.93

78

72

75

68

 

 

 

 

16

15

18

20

 

 

 

 

 

0.25

A

B

8.73

8.39

7.95

7.73

8.41

8.36

73

76

69

72

 

 

 

 

18

16

15

17

 

 

 

 

 

0.5

A

B

8.76

8.30

8.76

7.92

7.17

8.68

72

78

70

70

 

 

 

 

18

22

18

12

 

 

 

 

 

1

A

B

8.30

7.79

7.96

8.39

8.32

8.19

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

 

2

A

B

9.12

8.21

7.47

8.25

7.55

8.64

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

 

Positive control EMS (µg/mL)

400

A

B

8.73

8.36

6.18

6.06

8.57

8.81

65

68

54

59

 

 

 

 

72

75

71

69

 

 

 

 

 

Summary Analysis: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

14.38

100

72.82

1.00

130.57

0.015

 

15.82

108

73.10

1.09

135.48

0.03

 

15.59

102

67.78

0.94

134.50

0.06

 

15.48

107

72.54

1.06

156.50

0.13

 

16.47

102

71.99

1.01

137.57

0.25

 

16.43

110

70.37

1.06

134.00

0.5

 

16.52

110

70.37

1.06

143.00

1

Ø

16.87

106

 

 

 

2

Ø

15.91

108

 

 

 

Positive control EMS

Concentration

(µg/mL)

SG

%RSG

%V

RTG

MF§

400

 

13.30

89

51.17

0.62

1344.49

 

GEF =126, therefore MF threshold for a positive response = 256.57

Large and Small ColoniesAnalysis: Mutagenicity Test (-S9) 4-Hour Exposure

Concentration

(µg/mL)

Viability #

after day 2

Small colonies #

after day 2

Large colonies #

after day 2

0

A

B

76

70

70

72

78

76

77

70

6

6

9

7

7

6

6

3

9

10

11

7

15

9

10

12

0.015

A

B

72

70

78

75

 

 

 

 

8

7

6

5

 

 

 

 

11

12

11

9

 

 

 

 

0.03

A

B

73

69

70

73

 

 

 

 

4

4

9

6

 

 

 

 

8

12

12

9

 

 

 

 

0.06

A

B

69

72

78

75

 

 

 

 

5

9

9

6

 

 

 

 

12

14

10

13

 

 

 

 

0.13

A

B

78

72

75

68

 

 

 

 

6

8

6

8

 

 

 

 

10

7

12

12

 

 

 

 

0.25

A

B

73

76

69

72

 

 

 

 

8

4

6

4

 

 

 

 

10

12

9

13

 

 

 

 

0.5

A

B

72

78

70

70

 

 

 

 

8

7

7

6

 

 

 

 

10

15

11

6

 

 

 

 

400 EMS

A

B

65

68

54

59

 

 

 

 

36

49

39

36

 

 

 

 

36

26

32

33

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutation frequencies

Concentration

(µg/mL)

 

Small colonies

Large colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

179

768

718

768

46.2

685

768

78.5

0.38

0.015

 

89

384

358

384

48.0

341

384

81.2

0.38

0.03

 

99

384

361

384

45.6

343

384

83.3

0.36

0.06

 

90

384

355

384

54.1

335

384

94.1

0.37

0.13

 

91

384

356

384

52.6

343

384

78.4

0.41

0.25

 

94

384

362

384

41.9

340

384

86.5

0.33

0.5

 

94

384

356

384

53.8

342

384

82.3

0.40

400 EMS

 

138

384

224

384

526.7

257

384

392.4

0.56

Cell and 96-Well Plate Counts: Mutagenicity Test (+S9) 4-Hour Exposure

Concentration

(µg/mL)

Cell counts $

Viability §

after day 2

2 cells/well

Resistant mutants §

after day 2

2000 cells/well

 

 

 

0h

 24h

 48h

 

 

 

 

 

 

 

 

 

0

A

B

8.63

8.66

8.26

7.96

8.59

8.46

72

73

74

76

79

79

76

80

23

22

24

22

23

17

22

24

 

0.015

A

B

8.82

8.15

7.78

7.98

7.51

7.53

79

85

85

81

 

 

 

 

21

20

20

20

 

 

 

 

 

0.03

A

B

7.62

8.19

8.82

8.52

7.63

8.08

78

78

75

77

 

 

 

 

21

19

20

25

 

 

 

 

 

0.06

A

B

8.70

7.70

7.88

7.93

8.58

8.66

81

76

82

80

 

 

 

 

15

20

16

19

 

 

 

 

 

0.13

A

B

8.47

7.86

8.16

7.70

7.39

8.32

83

75

80

76

 

 

 

 

21

18

21

23

 

 

 

 

 

0.25

A

B

8.51

8.45

8.49

7.83

7.28

7.32

78

79

72

78

 

 

 

 

19

22

17

24

 

 

 

 

 

0.5

A

B

8.17

7.87

8.34

8.88

8.11

6.86

75

73

77

77

 

 

 

 

24

25

15

25

 

 

 

 

 

1

A

B

7.82

7.83

8.99

9.11

7.43

6.47

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

 

2

A

B

8.00

7.69

8.65

8.84

8.06

6.46

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

 

Positive control CP (µg/mL)

1.5

A

B

8.29

7.95

6.86

7.27

6.60

6.34

63

66

63

70

 

 

 

 

63

60

68

77

 

 

 

 

 

Summary Analysis: Mutagenicity Test (+S9) 4-Hour Exposure 

Concentration

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

17.28

100

78.74

1.00

166.34

0.015

 

14.81

84

98.08

1.05

120.77

0.03

 

17.03

90

81.00

0.92

154.45

0.06

 

17.04

93

88.81

1.05

113.30

0.13

 

15.57

85

85.11

0.91

143.07

0.25

 

14.89

85

80.34

0.86

149.50

0.5

 

16.11

86

77.20

0.85

170.78

1

Ø

15.72

82

 

 

 

2

Ø

15.87

83

 

 

 

Positive control CP

Concentration

(µg/mL)

SG

%RSG

%V

RTG

MF§

1.5

 

11.43

62

57.33

0.45

1043.98

 

GEF =126, therefore MF threshold for a positive response = 292.34

Large and Small Colonies Analysis: Mutagenicity Test (+S9) 4-Hour Exposure

Concentration

(µg/mL)

Viability #

after day 2

Small colonies #

after day 2

Large colonies #

after day 2

0

A

B

72

73

74

76

79

79

76

80

7

10

7

8

9

3

8

9

16

12

17

14

14

14

14

15

0.015

A

B

79

85

85

81

 

 

 

 

9

9

5

8

 

 

 

 

12

11

15

12

 

 

 

 

0.03

A

B

78

78

75

77

 

 

 

 

6

6

8

11

 

 

 

 

15

13

12

14

 

 

 

 

0.06

A

B

81

76

82

80

 

 

 

 

6

1

4

9

 

 

 

 

9

19

12

10

 

 

 

 

0.13

A

B

83

75

80

76

 

 

 

 

7

7

6

11

 

 

 

 

14

11

15

12

 

 

 

 

0.25

A

B

78

79

72

78

 

 

 

 

9

7

4

12

 

 

 

 

10

15

13

12

 

 

 

 

0.5

A

B

75

73

77

77

 

 

 

 

9

10

6

13

 

 

 

 

15

15

9

12

 

 

 

 

1.5 CP

A

B

63

66

63

70

 

 

 

 

36

40

39

48

 

 

 

 

27

20

29

29

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutation frequencies

Concentration

(µg/mL)

 

Small colonies

Large colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

159

768

707

768

52.5

652

768

104.0

0.34

0.015

 

54

384

353

384

42.9

334

384

71.1

0.38

0.03

 

76

384

353

384

52.0

330

384

93.6

0.36

0.06

 

65

384

364

384

30.1

334

384

78.5

0.29

0.13

 

70

384

353

384

49.5

332

384

85.5

0.37

0.25

 

77

384

352

384

54.2

334

384

86.8

0.39

0.5

 

82

384

346

384

67.5

333

384

92.3

0.43

1.5 CP

 

122

384

221

384

481.8

279

384

278.6

0.61

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 24-Hour Exposure

Concentration

(µg/mL)

Cell counts $

Viability §

after day 2

2 cells/well

Resistant mutants §

after day 2

2000 cells/well

 

 

 

0h

 24h

 48h

 

 

 

 

 

 

 

 

 

0

A

B

11.83

11.63

7.47

7.50

6.79

7.22

72

73

78

81

78

77

79

76

22

22

22

24

20

25

22

25

 

0.015

A

B

11.50

12.14

7.31

7.41

5.97

6.17

75

81

78

79

 

 

 

 

22

24

26

25

 

 

 

 

 

0.03

A

B

12.29

12.02

6.44

6.88

6.99

7.43

80

76

81

77

 

 

 

 

25

23

24

25

 

 

 

 

 

0.06

A

B

12.36

10.41

6.64

8.50

6.48

6.52

82

80

79

79

 

 

 

 

24

18

20

22

 

 

 

 

 

0.13

A

B

11.42

11.50

5.90

7.01

8.28

6.62

79

80

80

72

 

 

 

 

20

23

22

24

 

 

 

 

 

0.25

A

B

10.99

11.57

6.90

6.94

7.57

7.46

73

76

82

81

 

 

 

 

22

20

24

26

 

 

 

 

 

0.5

A

B

12.23

11.29

5.12

7.28

4.63

8.13

80

80

76

79

 

 

 

 

26

24

25

22

 

 

 

 

 

1

A

B

11.81

9.97

4.58

7.84

5.33

6.62

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

 

2

A

B

11.14

11.62

5.12

6.41

5.15

5.77

NP

NP

NP

NP

 

 

 

 

NP

NP

NP

NP

 

 

 

 

 

Positive control EMS (µg/mL)

150

A

B

9.34

9.05

4.45

5.03

4.07

5.12

57

62

71

61

 

 

 

 

82

90

77

82

 

 

 

 

 

Summary Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

Concentration

(µg/mL)

 

SG

%RSG

%V

RTG

MF§

0

 

102.51

100

80.34

1.00

168.32

0.015

 

88.01

87

84.40

0.91

172.49

0.03

 

97.28

98

85.11

1.01

171.05

0.06

 

93.37

88

89.59

1.02

137.78

0.13

 

91.85

88

83.01

0.92

158.82

0.25

 

97.77

92

83.70

0.99

163.62

0.5

 

77.53

76

85.83

0.84

169.62

1

Ø

67.35

61

 

 

 

2

Ø

59.70

57

 

 

 

Positive control EMS

Concentration

(µg/mL)

SG

%RSG

%V

RTG

MF§

150

 

33.38

26

53.01

0.22

1867.74

 GEF =126, therefore MF threshold for a positive response = 294.32

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 24-Hour Exposure

 

Concentration

(µg/mL)

Viability #

after day 2

Small colonies #

after day 2

Large colonies #

after day 2

0

A

B

72

73

78

81

78

77

79

76

7

10

11

12

8

11

9

12

15

12

11

12

12

14

13

13

0.015

A

B

75

81

78

79

 

 

 

 

12

12

12

11

 

 

 

 

10

12

14

14

 

 

 

 

0.03

A

B

80

76

81

77

 

 

 

 

10

8

8

10

 

 

 

 

15

15

16

15

 

 

 

 

0.06

A

B

82

80

79

79

 

 

 

 

8

7

9

12

 

 

 

 

16

11

11

10

 

 

 

 

0.13

A

B

79

80

80

72

 

 

 

 

9

10

8

10

 

 

 

 

11

13

14

14

 

 

 

 

0.25

A

B

73

76

82

81

 

 

 

 

8

12

6

13

 

 

 

 

14

8

18

13

 

 

 

 

0.5

A

B

80

80

76

79

 

 

 

 

12

15

11

14

 

 

 

 

14

9

14

8

 

 

 

 

150 EMS

A

B

57

62

71

61

 

 

 

 

48

46

51

51

 

 

 

 

34

44

26

31

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Mutation frequencies

Concentration

(µg/mL)

 

Small colonies

Large colonies

Proportion

small

colony

mutants

 

Viable

Mutants

 

Mutants

 

 

 

Yv

Nv

Ym

Nm

MF§

Ym

Nm

MF§

 

0

 

154

768

688

768

68.5

666

768

88.7

0.44

0.015

 

71

384

337

384

77.3

334

384

82.6

0.48

0.03

 

70

384

348

384

57.8

323

384

101.6

0.37

0.06

 

64

384

348

384

54.9

336

384

74.5

0.43

0.13

 

73

384

347

384

61.0

332

384

87.6

0.42

0.25

 

72

384

345

384

64.0

331

384

88.7

0.42

0.5

 

69

384

332

384

84.8

339

384

72.6

0.54

150 EMS

 

133

384

188

384

673.6

249

384

408.6

0.59

 

Historical Vehicle and Positive Control Mutation Frequencies

Experiments ‑S9

Vehicle control

Positive control (EMS)

MF

MF

151.16

792.66

193.22

2131.98

130.62

1293.51

140.54

1147.39

168.04

1059.80

165.45

1324.26

120.34

1320.11

144.63

1120.42

166.40

1383.36

160.23

1131.52

150.04

1242.43

143.67

1832.91

119.09

913.73

135.00

1168.73

158.36

1133.94

167.39

1203.85

158.52

1594.01

148.54

950.32

114.37

1343.40

150.65

1427.33

Mean: 149.31

Mean: 1275.78

SD: 19.34

SD: 308.21

Minimum: 114.37

Minimum: 792.66

Maximum: 193.22

Maximum: 2131.98

 

Experiments +S9 

Vehicle control

Positive control (CP)

MF

MF

160.56

603.47

147.97

907.26

129.03

641.72

85.13

880.23

179.29

714.89

127.77

620.07

153.14

1228.33

146.31

924.98

127.77

1230.16

131.38

534.63

183.21

1028.90

134.37

872.82

152.45

1377.33

121.81

752.27

148.30

566.49

134.66

1972.34

159.07

1000.00

116.93

731.43

137.21

873.07

117.83

876.29

Mean: 139.71

Mean: 916.83

SD: 22.50

SD: 338.79

Minimum: 85.13

Minimum: 534.63

Maximum: 183.21

Maximum: 1972.34

SD       =Standard deviation

Conclusions:
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group

Concentration of Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide (µg/mL) plated for viability and mutant frequency

4-hour without S9

0.015, 0.03, 0.06, 0.13, 0.25, 0.5

4-hour with S9 (2%)

24-hour without S9

Results

The maximum dose levels in the Mutagenicity Test were limited by test item precipitate in all three of the exposure groups, asrecommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames test (OECD TG 471):

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 typhimuriumstrains TA1535, TA1537, TA98 and TA100 andEscherichia colistrain WP2uvrAwere 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

Mg-SiO was considered to be non-mutagenic under the conditions of this test.

Chromosome Aberration test (OECD TG 473):

Introduction

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al., 1991). 

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. In this study, three exposure conditions were investigated; 4 hours exposure in the presence of an induced rat liver homogenate metabolizing system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period, 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period and a 24-hour exposure in the absence of metabolic activation.

The dose levels used in the Main Experiment were selected using data from the Preliminary Toxicity Test where the results indicated that the maximum concentration should be limited on precipitate. The dose levels selected for the Main Experiment were as follows:

Group

Final concentration of test item Mg-SiO(µg/mL)

4(20)-hour without S9

0, 0.31, 0.63, 1.25, 5, 10, 20

4(20)-hour with S9 (2%)

0, 0.31, 0.63, 1.25, 5, 10, 20

24-hour without S9

0, 0.31, 0.63, 1.25, 5, 10, 20

Results

All vehicle (MEM) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes.

All the positive control items induced statistically significant increases in the frequency of cells with aberrations. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with aberrations, using a dose range that included a dose level that was the lowest precipitating dose level.

Conclusion

The test item, Mg-SiO was considered to be non-clastogenic to human lymphocytes in vitro.

MLA (OECD TG 490):

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guideline for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, and the US EPA OPPTS 870.5300 Guideline.

Methods

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at eight dose levels in duplicate, together with vehicle (R0 medium), and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated for viability and expression of mutant colonies were as follows:

Mutagenicity Test

Group

Concentration of Mg-SiO (µg/mL) plated for viability and mutant frequency

4-hour without S9

0.015, 0.03, 0.06, 0.13, 0.25, 0.5

4-hour with S9 (2%)

24-hour without S9

Results

The maximum dose levels in the Mutagenicity Test were limited by test item precipitate in all three of the exposure groups, asrecommended by the OECD 490 guideline. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

Based on the negative results of the available test data , the substance does not have to be classified for mutagenicity in accordance with Regulation (EC) No. 1272/2008.