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Genetic toxicity: in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
between 18 June 2008 and 08 July 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of 3-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-2-hydroxypropyl methacrylate and 1-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-3-hydroxypropan-2-yl methacrylate.
EC Number:
700-334-3
Molecular formula:
C17H38O6Si3
IUPAC Name:
Reaction mass of 3-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-2-hydroxypropyl methacrylate and 1-[3-(1,1,1,3,5,5,5-heptamethyltrisiloxan-3-yl)propoxy]-3-hydroxypropan-2-yl methacrylate.
Test material form:
other: liquid
Details on test material:
identification: SiMAA2
Description : Clear colourless liquid
Lot number : 003068
Date received : 25 March 2008
Storage conditions: Approximately 4°C in the dark

Method

Target gene:
Histidine (for Salmonell), tryptophan (for E.coli).
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone/beta-naphthoflavone induced rat liver (S9 mix)
Test concentrations with justification for top dose:
Preliminary Toxicity Test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Mutation Test (Experiments 1 and 2): 50, 150, 500, 1500 and 5000 µg/plate
Vehicle / solvent:
The test material was immiscible in sterile distilled water at 50 mg/ml but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Controls
Untreated negative controls:
yes
Remarks:
Spontaneous mutation rates
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Remarks:
ENNG, 9AA, 4NQO used without S9 mix. BP and 2AA used with S9 mix.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: 48 hours
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours


NUMBER OF REPLICATIONS: Triplicate plates


NUMBER OF CELLS EVALUATED: 10-9 cells per ml


DETERMINATION OF CYTOTOXICITY
- Method: other: Evalaution of background lawn, frequency of revertant colonies.
Evaluation criteria:
There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation.  Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS (5) can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
A test material 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 a definitive judgement about the test material activity.  Results of this type will be reported as equivocal.
Statistics:
Standard deviation

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Precipitation: An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.


Any other information on results incl. tables

Preliminary Toxicity Test

The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-).  The test material formulation and S9-mix used in this experiment were both shown to be sterile. The number of revertant colonies for the toxicity assay were;

With (+) or without (-) S9-mix

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

79

72

81

80

99

77

89

77

80

112

72P

+

TA100

70

81

67

73

73

76

82

89

61

73

80P

-

WP2uvrA-

29

20

28

29

27

21

17

22

23

20

36P

+

WP2uvrA-

28

21

23

22

25

32

28

35

34

31

42P

Mutation test:

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.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable.  These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations for the test material, vehicle and positive controls both with and without metabolic activation, are presented in Table 2 to Table 5.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.

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

All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains.

Table1:Spontaneous Mutation Rates (Concurrent Negative Controls)

Range finding test: Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

67

15

30

13

7

79

(79)

12

(14)

23

(24)

19

(17)

7

(9)

90

15

19

18

13

Main test: Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA-

TA98

TA1537

117

20

29

18

7

129

(130)

22

(20)

34

(29)

15

(17)

6

(6)

143

19

25

17

5

Table 2: Range finding test-without metabolic activation.

Test Period

From: 28 June 2008

To: 01 July 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

-

0

86

70

70

(75)

9.2#

18

21

18

(19)

1.7

30

34

25

(30)

4.5

18

19

11

(16)

4.4

14

15

11

(13)

2.1

-

50

71

75

70

(72)

2.6

23

16

16

(18)

4.0

19

41

24

(28)

11.5

24

20

12

(19)

6.1

13

12

14

(13)

1.0

-

150

70

71

71

(71)

0.6

16

23

16

(18)

4.0

23

20

24

(22)

2.1

20

16

22

(19)

3.1

15

12

13

(13)

1.5

-

500

71

79

79

(76)

4.6

18

19

21

(19)

1.5

24

29

20

(24)

4.5

15

19

13

(16)

3.1

12

11

13

(12)

1.0

-

1500

87

70

71

(76)

9.5

19

13

25

(19)

6.0

19

31

30

(27)

6.7

15

20

16

(17)

2.6

14

13

12

(13)

1.0

-

5000

79 P

75 P

78 P

(77)

2.1

20 P

24 P

19 P

(21)

2.6

31 P

24 P

20 P

(25)

5.6

13 P

15 P

18 P

(15)

2.5

11 P

14 P

12 P

(12)

1.5

Positive

controls

S9-Mix

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

514

556

511

(527)

25.2

255

151

150

(185)

60.3

716

668

647

(677)

35.4

222

203

218

(214)

10.0

2113

2210

1974

(2099)

118.6

Table 3: Range finding test-with metabolic activation.

Test Period

From: 28 June 2008

To: 01 July 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

+

0

71

74

74

(73)

1.7#

11

14

14

(13)

1.7

30

38

30

(33)

4.6

20

19

18

(19)

1.0

16

15

11

(14)

2.6

+

50

71

70

71

(71)

0.6

13

10

10

(11)

1.7

33

31

27

(30)

3.1

19

20

18

(19)

1.0

15

11

12

(13)

2.1

+

150

71

70

76

(72)

3.2

13

13

10

(12)

1.7

23

33

22

(26)

6.1

14

16

16

(15)

1.2

14

11

13

(13)

1.5

+

500

78

73

76

(76)

2.5

12

11

12

(12)

0.6

30

31

35

(32)

2.6

16

20

16

(17)

2.3

11

14

12

(12)

1.5

+

1500

73

77

81

(77)

4.0

11

12

12

(12)

0.6

29

31

35

(32)

3.1

19

16

18

(18)

1.5

13

10

13

(12)

1.7

+

5000

74 P

73 P

79 P

(75)

3.2

13 P

13 P

12 P

(13)

0.6

32 P

26 P

36 P

(31)

5.0

20 P

20 P

15 P

(18)

2.9

13 P

16 P

14 P

(14)

1.5

Positive

controls

S9-Mix

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

1306

1178

1426

(1303)

124.0

277

376

345

(333)

50.6

299

352

293

(315)

32.5

511

387

483

(460)

65.0

260

398

389

(349)

77.2

Table 4: Maint test- without metabolic activation:

Test Period

From: 05 July 2008

To: 08 July 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

-

0

95

99

98

(97)

2.1#

20

14

20

(18)

3.5

22

14

25

(20)

5.7

26

22

22

(23)

2.3

5

11

3

(6)

4.2

-

50

84

93

125

(101)

21.5

13

24

18

(18)

5.5

31

30

16

(26)

8.4

21

16

34

(24)

9.3

12

4

7

(8)

4.0

-

150

112

108

124

(115)

8.3

8

21

9

(13)

7.2

30

24

16

(23)

7.0

19

25

24

(23)

3.2

10

4

5

(6)

3.2

-

500

99

104

93

(99)

5.5

15

11

7

(11)

4.0

29

31

23

(28)

4.2

18

26

21

(22)

4.0

9

4

10

(8)

3.2

-

1500

119

100

103

(107)

10.2

19

16

9

(15)

5.1

36

20

30

(29)

8.1

20

18

18

(19)

1.2

3

7

7

(6)

2.3

-

5000

99 P

97 P

133 P

(110)

20.2

13 P

10 P

21 P

(15)

5.7

27 P

21 P

20 P

(23)

3.8

24 P

15 P

18 P

(19)

4.6

2 P

7 P

4 P

(4)

2.5

Positive

controls

S9-Mix

-

Name

Concentration

(μg/plate)

No. colonies

per plate

ENNG

ENNG

ENNG

4NQO

9AA

3

5

2

0.2

80

539

547

509

(532)

20.0

264

196

214

(225)

35.2

709

818

823

(783)

64.4

178

136

183

(166)

25.8

997

970

1262

(1076)

161.4

Table 5: Main test-with metabolic activation:

Test Period

From: 05 July 2008

To: 08 July 2008

With or without

S9-Mix

Test

substance

concentration

(µg/plate)

Number of revertants (mean number of colonies per plate)

Base-pair substitution type

Frameshift type

TA100

TA1535

WP2uvrA‑

TA98

TA1537

+

0

91

108

114

(104)

11.9#

9

13

13

(12)

2.3

23

35

25

(28)

6.4

14

18

21

(18)

3.5

9

10

8

(9)

1.0

+

50

123

92

97

(104)

16.6

21

22

10

(18)

6.7

32

29

42

(34)

6.8

18

18

19

(18)

0.6

10

4

10

(8)

3.5

+

150

84

88

119

(97)

19.2

13

13

19

(15)

3.5

29

23

40

(31)

8.6

13

22

19

(18)

4.6

10

7

8

(8)

1.5

+

500

107

95

103

(102)

6.1

13

15

7

(12)

4.2

29

29

29

(29)

0.0

31

19

21

(24)

6.4

13

8

12

(11)

2.6

+

1500

101

99

101

(100)

1.2

20

9

14

(14)

5.5

30

25

25

(27)

2.9

25

31

16

(24)

7.5

5

9

9

(8)

2.3

+

5000

80 P

106 P

103 P

(96)

14.2

15 P

9 P

8 P

(11)

3.8

33 P

31 P

29 P

(31)

2.0

22 P

11 P

18 P

(17)

5.6

5 P

7 P

11 P

(8)

3.1

Positive

controls

S9-Mix

+

Name

Concentration

(μg/plate)

No. colonies

per plate

2AA

2AA

2AA

BP

2AA

1

2

10

5

2

723

882

811

(805)

79.7

272

274

227

(258)

26.6

465

475

459

(466)

8.1

344

326

317

(329)

13.7

431

322

368

(374)

54.7

Results of range finding test without metabolic activation.

Applicant's summary and conclusion

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

Introduction.

The method was designed to conform to the guidelines for bacterial mutagenicity testing published by the major Japanese Regulatory Authorities including METI, MHLW and MAFF. It also meets the requirements of the OECD Guidelines for Testing of Chemicals No. 471 "Bacterial Reverse Mutation Test", Method B13/14 of Commission Directive 2000/32/EC and the, EPA (TSCA) OPPTS harmonised guidelines.

Methods.

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA-were treated with the test material using the Ames plate incorporation method at five dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the range-finding test was determined in a preliminary toxicity assay and was 50 to 5000 µg/plate. The experiment was repeated on a separate day using the same dose range as the range-finding test, fresh cultures of the bacterial strains and fresh test material formulations.

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 or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. An oily precipitate was initially observed under an inverted microscope at 1500 µg/plate and at 5000 µg/plate by the naked eye. These observations did not prevent the scoring of revertant colonies.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Conclusion.

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