Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation assay / Ames test, conducted using a protocol similar to OECD test guideline 471 and in compliance with GLP, no increase in the number of revertants was observed in any test strain, with or without activation. Therefore, it is concluded that the test substance is negative for bacterial mutagenicity (BioReliance, 2001).

In an in vitro cytogenicity study in mammalian cells, conducted using a protocol similar to OECD test guideline 473 and in compliance with GLP, it was concluded that the test substance was positive in Chinese hamster ovary cells (Bushy Run Research Center, 1985a).

In a mutagenicity study in mammalian cells, conducted using a protocol similar to OECD test guideline 476 and in compliance with GLP, the test substance was negative with and without metabolic activation in Chinese hamster ovary cells (HGPRT gene mutation assay) (Bushy Run Research Center, 1985b).

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:
2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
preliminary toxicity test: 0.13 to 5000 µg/plate; definitive assay: 15 to 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: request of sponsor; compatibility with target cells; solubility of test article
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 98 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 100 and TA 1535 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
TA 1537 without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
WP2 uvrA without activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
All strains with activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation;

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48-72 hours
- Expression time (cells in growth medium): 48-72 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48-72 hours

NUMBER OF REPLICATIONS: triplicate cultures

DETERMINATION OF CYTOTOXICITY
- Method: other: condition of bacterial lawn

METABOLIC ACTIVATION: S9 was prepared from male Sprague-Dawley rats induced with Aroclor 1264. Each batch was assayed for ability to metabolise 2-aminoanthracene and 7,12-dimethylbenz(a)anthracene to forms mutagenic to S. typhimurium TA100. S9 mix contained 10% S9, 5mM glucose-6-phosphate, 4 mM NADP, 8 mM MgCl2 and 33 mM KCl in 100 mM phosphate buffer pH 7.4. 0.5 ml of S9 mix were added to 2.0 ml top agar, 0.1 ml tester strain and 0.05 ml vehicle or test article, giving a final concentration of approximately 2% S9.
Evaluation criteria:
For the test article to be evaluated positive, it must cause a dose-related increase in the mean number of revertants per plate relative to solvent control over a minimum of two concentrations; by at least 2-fold in strains TA 98, TA 100 and E coli WP2 uvrA; by at least 3-fold in strains TA 1535 and 1537.
Statistics:
Means and standard deviations were calculated
Key result
Species / strain:
bacteria, other: Salmonella typhimurium TA98, TA100, TA1535 and TA1537; Escherichia coli WP2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: > 1500 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no information
- Effects of osmolality: no information
- Evaporation from medium: no information
- Water solubility: reacts slowly with water
- Precipitation: no precipitate observed

RANGE-FINDING/SCREENING STUDIES: on all strains

COMPARISON WITH HISTORICAL CONTROL DATA: Control results were within the range of historical data

ADDITIONAL INFORMATION ON CYTOTOXICITY: slightly variable between studies

Table 1: Dose range-finding study. Number of revertants per plate (1 plate per strain)

Conc.
µg/plate

 

TA98

TA100

TA1535

- MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

15

15

no

84

102

no

11

14

no

6.7

16

14

no

88

109

no

10

14

no

10

15

17

no

94

122

no

10

9

no

33

15

15

no

109

110

no

11

12

no

67

19

15

no

93

101

no

13

13

no

100

11

18

no

72

104

no

17

10

no

333

17

17

no

116

117

no

8

13

no

667

15

14

no

86

115

no

12

8

no

1000

11

20

no

60

80

no

6

11

no

3333

8

15

yes**

75

102**

yes

4***

7***

yes

5000

0

13**

yes****

69***

48***

yes

0****

4***

yes

*solvent control with Ethylene glycol dimethyl ether

** Background lawn slightly reduced

***Background lawn severely reduced

****Background lawn extremely reduced

 

Table 2: Dose range-finding study. Number of revertants per plate (1 plate per strain)

 

Concentration (µg/Plate)

 

TA 100

WP2 uvrA

Plate 1

- MA

Plate 2

+ MA

Cytotoxic (Yes/No)

Plate 1

- MA

Plate 2

+ MA

Cytotoxic (Yes/No)

0*

5

5

no

11

11

no

6.7

7

8

no

14

13

no

10

7

6

no

11

8

no

33

3

6

no

12

11

no

67

7

4

no

12

11

no

100

5

6

no

11

11

no

333

3

5

no

10

12

no

667

4

2

no

7

12

no

1000

5

6

no

7

13

no

3333

5***

7**

yes

6

7

no

5000

1****

5***

yes

2**

13

yes

*solvent control with Ethylene glycol dimethyl ether

** Background lawn slightly reduced

***Background lawn severely reduced

****Background lawn extremely reduced

 

Table 3: Experiment 1 Mutagenicity Assay. Number of revertants per plate (mean of 3 plates)

Conc.
µg/plate

 

TA98

TA100

TA1535

- MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

15

19

no

100

103

no

12

10

no

15

17

16

no

995

91

no

11

9

no

50

14

20

no

102

91

no

9

13

no

150

10

18

no

106

94

no

11

12

no

500

9

19

no

116

113

no

12

9

no

1500

7

16

yes

6

100

yes

8

7

no

5000

0

3

yes

0

0

yes

0

0

yes

Positive control

324

499

-

451

458

-

152

74

-

*solvent control with Ethylene glycol dimethyl ether

 

Table 4: Experiment 1 Mutagenicity Assay. Number of revertants per plate (mean of 3 plates)

Conc.
µg/plate

 

TA1537

WP2uvrA

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

6

8

no

11

11

no

15

6

6

no

10

12

no

50

4

6

no

9

10

no

150

5

6

no

10

11

no

500

3

5

no

11

10

no

1500

4

4

no

9

11

no

5000

0

0

yes

4

6

yes

Positive control

20

65

-

227

76

-

  *solvent control withEthylene glycol dimethyl ether

 

Table 5: Experiment 2 Mutagenicity Assay. Number of revertants per plate (mean of 3 plates)

TA98

TA100

TA1535

Conc.
(
µg/plate)

-MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

-

MA

+ MA

Cytotoxic
(yes/no)

0*

15

23

no

116

135

no

12

9

no

15

14

30

no

126

151

no

13

6

no

50

18

22

no

129

147

no

9

9

no

150

18

25

no

111

154

no

10

9

no

500

15

17

no

133

149

no

10

7

no

1500

17

19

no

133

122

no

15

6

no

5000

7

8

yes

77

57

yes

8

8

no

Positive control

589

951

-

509

706

-

228

163

-

*solvent control withEthylene glycol dimethyl ether

 

Table 6: Experiment 2 Mutagenicity Assay. Number of revertants per plate (mean of 3 plates)

TA1537

WP2 uvrA

Conc.
(
µg/plate)

- MA

+ MA

Cytotoxic
(yes/no)

- MA

+ MA

Cytotoxic
(yes/no)

0*

4

7

no

14

12

no

15

4

6

no

13

8

no

50

6

5

no

12

10

no

150

6

7

no

12

6

no

500

8

5

no

15

11

no

1500

6

7

no

11

7

no

5000

7

3

yes

9

8

no

Positive control

935

317

-

219

275

-

Conclusions:
In a valid bacterial reverse mutation assay, conducted according to OECD TG 471 and in compliance with GLP, 3-(Trimethoxysilyl)propyl methacrylate has been tested using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA. No increase in the number of revertants was observed in any test strain, with or without activation. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for bacterial mutagenicity under the conditions of the test.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-03-18 to 1985-04-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
The restrictions were: sampling times; number of cells evaluated.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The restrictions were: sampling times; number of cells evaluated.
Principles of method if other than guideline:
Method: other: Health Effects Test Guidelines. HG-Chromo-In Vitro, EPA, August 1982. Report No. EPA 560/6-82-001
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.2, 0.3, 0.4 mg/ml (8 h exposure, without metabolic activation and 12 h exposure with metabolic activation); 0.3, 0.4, 0.6 mg/ml (12 h exposure without metabolic activation and 8h exposure with metabolic activation).
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: methanol
- Justification for choice of solvent/vehicle: reactivity with water
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with activation
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
triethylenemelamine
Remarks:
without activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium;

DURATION
- Exposure duration: 2 hours (with S9) 8 hours (without S9)
- Expression time (cells in growth medium): 6 hours and 12 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 8 and 14 hours

SPINDLE INHIBITOR (cytogenetic assays): colchicine added 2 hours before harvest
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicate cultures per dose

NUMBER OF CELLS EVALUATED: 50

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index;

OTHER EXAMINATIONS:
- Determination of polyploidy: not evaluated
- Determination of endoreplication: not evaluated

METABOLIC ACTIVATION: S9 liver homogenate, prepared from Aroclor induced male Sprague-Dawley rats, was purchased from Microbiological Associates. It was tested with three different activation-dependent mutagens in S. typhimurium TA100. 1.0 ml of S9 mix was added to 4.0 ml culture medium. S9 mix contained 10 µmoles/ml MgCl, 30 µmoles/ml KCl, 5 µmoles/ml glucose-6-phosphate, 4 µmoles/ml NADP-oxidised, 50 µmoles/ml Na3HPO4, 10 µmoles/ml CaCl2 and 50-200 µg/ml S9 (depending on metabolic activity).
Evaluation criteria:
Not described in report
Statistics:
Fischer's exact test
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
other: 0.6 mg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: reactive with water
- Precipitation: precipitate occurred at concentrations greater than 0.6 mg/ml

RANGE-FINDING/SCREENING STUDIES: carried out

COMPARISON WITH HISTORICAL CONTROL DATA: None presented

ADDITIONAL INFORMATION ON CYTOTOXICITY: a >50% reduction in mitotic index was observed at the highest concentration. Mitotic index was not determined as part of the main chromosome aberration test.

Table 1: Preliminary toxicity assay: percentage of cells in metaphase (500 cells counted)

Concentration (mg/ml)

Without activation

With activation

0*

3.6

5.8

0.08

5.2

2.4

0.1

6.8

5.2

0.2

3.8

1.0

0.4

4.0

1.4

0.6

2.0

1.0

0.8

1.8

2.2

1.0

0.4

1.6

 *Solvent control with methanol

 

Table 2: Results of chromosome analysis Experiment 1 (8 h treatment, 8 h sampling) without activation (total count from 2 cultures / 100 cells)

 

Untreated

Solvent*

Control

Positive

Control

Low dose 0.2 mg/ml

Mid dose 0.3 mg/ml

High dose 0.4 mg/ml

 

Total per 100 cells

Chromatid aberrations

gaps

0

2

0

1

3

2

deletions**

1

1

12

4

6

7

interchanges

0

0

2

0

0

0

Chromosome

gaps

0

0

0

0

0

0

deletions**

0

0

2

1

1

1

interchanges

0

0

0

0

0

0

Cells with aberrations %

2

2

26

4

6

11

Polyploidy

NR

NR

NR

NR

NR

NR

Endo reduplication

0

0

0

0

0

0

 *Solvent control with methanol

 **sum of cells with breaks and fragments

NR not reported

 

Table 3: Results of chromosome analysis Experiment 1 (12 h treatment, 12 h sampling) without activation (total count from 2 cultures / 100 cells)

 

Untreated

Solvent*

Control

Positive

Control

Low dose 0.3 mg/ml

Mid dose 0.4mg/ml

High dose 0.6mg/ml

 

Total per 100 cells

Chromatid aberrations

gaps

0

0

0

3

0

5

deletions**

0

1

10

1

4

8

interchanges

0

0

0

0

0

0

Chromosome

gaps

0

0

0

0

0

0

deletions**

0

2

4

0

0

1

interchanges

0

0

0

0

0

0

Cells with aberrations %

0

2

28

3

4

7

Polyploidy

NR

NR

NR

NR

NR

NR

Endo reduplication

0

1

0

0

0

0

*Solvent control with methanol

 **sum of cells with breaks and fragments

NR not reported

Table 4: Results of chromosome analysis Experiment 2, (2 h treatment, 8 h sampling) with activation (total count from 2 cultures / 100 cells)

 

Untreated

Solvent*

Control

Positive

Control

Low dose 0.3 mg/ml

Mid dose 0.4 mg/ml

High dose 0.6 mg/ml

 

Total per 100 cells

Chromatid aberrations

gaps

0

1

0

1

5

8

deletions**

1

5

7

20

20

26

interchanges

0

0

0

0

0

0

Chromosome

gaps

0

0

0

1

0

0

deletions**

0

0

3

3

9

6

interchanges

0

0

0

0

0

0

Cells with aberrations %

2

5

24

17

13

31

Polyploidy

NR

NR

NR

NR

NR

NR

Endo reduplication

0

0

0

0

0

0

*Solvent control with methanol

 **sum of cells with breaks and fragments

NR not reported

 

Table 5 Results of chromosome analysis Experiment 2, (2 h treatment, 12 h sampling) with activation (total count from 2 cultures / 100 cells)

 

Untreated

Solvent*

Control

Positive

Control

Low dose 0.2 mg/ml

Mid dose 0.3mg/ml

High dose 0.4mg/ml

 

Total per 100 cells

Chromatid aberrations

gaps

1

1

3

2

1

2

deletions**

2

2

3

7

7

5

interchanges

0

0

1

2

0

4

Chromosome

gaps

0

0

0

0

0

0

deletions**

0

3

3

3

4

5

interchanges

0

0

0

0

2

0

Cells with aberrations %

4

5

16

13

13

12

Polyploidy

NR

NR

NR

NR

NR

NR

Endo reduplication

0

0

0

0

0

0

Conclusions:
3-(Trimethoxysilyl)propyl methacrylate produced a statistically significant increase in chromosome aberrations in the absence of metabolic activation only at 0.4 mg/l, the highest concentration tested at the 8 hour sampling period.  In contrast, in the presence of metabolic activation, the test substance produced significant increases in the incidences of chromosome aberrations at both 8 and 12 hours and at several concentrations. Appropriate positive, negative and solvent controls were included and gave the expected results. It is concluded that the test substance is positive for clastogenicity (causing chromosome damage) under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines, HG-Gene Muta-Somatic cells, EPA Report No. 560/6-82-001, August 1982
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor induced rat liver S9
Test concentrations with justification for top dose:
0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 mg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: methanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: dimethylnitrosamine
Remarks:
with activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 2-3 days
- Selection time (if incubation with a selection agent): 9-12 days
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): TG

NUMBER OF REPLICATIONS: duplicate cultures

NUMBER OF CELLS EVALUATED 2 x 10E5 cells/plate in 5 plates/dosed culture (i.e. 1 x 10E6 total cells/dosed culture).

DETERMINATION OF CYTOTOXICITY
- Method: other: clonal assay; growth inhibition
- Preliminary experiments (0.001 to 10 mg/ml without S9; 0.03 to 3 mg/ml with S9) were performed with CHO cells to determine an appropriate range of test concentrations in which the highest concentration would kill no more than (approximately) 90% of the treated cells both in the presence and absence of a rat-liver S9 metabolic activation system.


METABOLIC ACTIVATION: S9 homogenate from Aroclor induced Sprague Dawley rats was purchased from Microbiological Associates or from Litton Bionetics. Concentration of S9 in each study was based on evaluation of relative toxicity and mutant production by the positive control. The S9 mix contained 10 µmoles/ml MgCl, 30 µmoles/ml KCl, 5 µmoles/ml glucose-6-phosphate, 4 µmoles/ml NADP-oxidised, 50 µmoles/ml Na3HPO4, 10 µmoles/ml CaCl2 and 50-200 µl/ml S9. I ml of S9 mix was added to 4.0 ml culture medium.
Evaluation criteria:
A statistically significant, repeatable dose-dependent increase in the mutant frequency is considered to be a positive result.
Statistics:
Student's t-test
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
>1.0 mg/ml
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Preliminary test results with 3-(trimethoxysilyl)propyl methacrylate indicated that concentrations above 1.0 mg/ml were lethal to CHO cells. 

3-(Trimethoxysilyl)propyl methacrylate did not produce any statistically significant increases in the incidence of mutations of CHO cells at concentrations between 0.1 to 0.8 mg/ml in tests with and without an S9 metabolic activation system. The range of doses tested in this study produced cytotoxic effects which indicated suitable concentrations were evaluated for mutagenic potential. The highest dose produced decreases in cell growth assessed 24 hour after exposure. However, the colony forming potential was essentially normal for the cells treated without S9 and only moderately depressed for cells exposed to 3-(trimethoxysilyl)propyl methacrylate together with S9 activation. Positive, negative and solvent controls produced the expected results.

Table 1 Results without metabolic activation: plating efficiency and (mean of 4 plates)

Concentration

mg/ml

Culture

Plating efficiency

Mutant determination

Mean colonies/plate

% of combined solvent control

Mean colonies per plate

Total colonies

Mutation frequency

0.2

A

115.2

94.7

0

0

0

B

148.0

121.7

0

0

0

0.4

A

133.5

109.8

0

0

0

B

113.8

93.6

0

0

0

0.6

A

120.0

98.7

0

0

0

B

126.0

103.6

0

0

0

0.7

A

137.8

113.3

0.2

1

0.7

B

134.5

110.6

0.6

3

2.2

0.8

A

100.2

82.4

0

0

0

B

114.5

94.2

0.8

4

3.5

Solvent*

A

93.0

76.5

2.0

10

10.8

B

150.2

123.5

0.4

2

1.3

Negative**

136.5

112.3

0

0

0

Positive***

128.5

105.7

27.8

139

108.2

*Solvent control with methanol

** Negative control with cell culture medium

*** Positive control with Ethylmethansulfonate

Table 2 Results with metabolic activation: plating efficiency and (mean of 4 plates)

Concentration

mg/ml

Culture

Plating efficiency

Mutant determination

Mean colonies/plate

% of combined solvent control

Mean colonies per plate

Total colonies

Mutation frequency

0.2

A

102.2

101.7

0.2

1

1.0

B

101.8

101.3

0

0

0

0.3

A

126.5

125.9

0.8

4

3.2

B

112.8

112.2

1.8

9

8.0

0.4

A

116.5

115.9

0

0

0

B

117.5

116.9

1.0

5

4.3

0.6

A

1075

107.0

0.4

2

1.9

B

137.5

136.8

0.6

3

2.2

0.8

A

98.5

98.0

0

0

0

B

120.8

120.2

0.6

3

2.5

Solvent*

A

91.8

91.3

0.8

4

4.4

B

109.2

108.7

0.2

1

0.9

Negative**

117.0

116.4

1.0

5

4.3

Positive***

105.2

104.7

28.8

144

136.8

*Solvent control with methanol

** Negative control with cell culture medium

*** Positive control with dimethylnitrosamine

Conclusions:
In a mutagenicity study, conducted according to an US EPA method and in compliance with GLP, 3-(Trimethoxysilyl)propyl methacrylate has been tested in Chinese hamster ovary cells. No test-substance induced increase in the number of mutations was observed. Appropriate solvent, negative (cell culture medium) and positive controls were included and gave expected results. It is concluded that 3-trimethyoxysilylpropyl methacrylate is negative for mutagenicity to mammalian cells under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In a micronucleus assay in mouse, conducted using a protocol similar to OECD test guideline 474 and in compliance with GLP, it is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test (Bushy Run Research Center, 1986).

In a comet assay in rat, conducted using a protocol similar to OECD test guideline 489 and in compliance with GLP, it was concluded that the test substance was negative for DNA damage in liver, lung and nasal tissue in the in vivo Comet Assay (Bioreliance, 2018).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss Webster
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: not stated
- Weight at study initiation: 15-5 g (female); 20-30 g (male)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: shoe-vox type plastic cages.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 to 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled but not specified in report
- Humidity (%): controlled but not specified in report
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours dakr / 12 hours light

Route of administration:
intraperitoneal
Vehicle:
Corn oil; all solutions made just before dosing.
Substance reacted slowly with water.
Duration of treatment / exposure:
30, 48 and 72 hour
Frequency of treatment:
One treatment
Post exposure period:
Samples taken 30,48 and 72 hours after treatment
Dose / conc.:
2 500 mg/kg bw/day
Dose / conc.:
4 000 mg/kg bw/day
Dose / conc.:
5 000 mg/kg bw/day
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance: triethylenemelamine
- Route of administration: ip injection
- Doses / concentrations: 0.3 and 0.5 mg/kg bw
Tissues and cell types examined:
Peripheral blood; 1000 Polychromatic erythrocytes (PCE) examined for micronuclei; PCE/NCE (normochromatic erythrocyte) ratio calculated for approximately 1000 total cells
Evaluation criteria:
Results were considered positive under the following conditions:
- at least one statistically significant increase above the control was obtained and there was a statistically significant indication of a dose-related effect of treatment;
- at least two dose level from the same sample period produced micronucleus frequencies which were significantly above the control values;
- at least two dose level from the same sample period produced micronucleus frequencies which were significantly above the control values and there was an indication of a significant dose-related increase in at least one of the sample intervals
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
25% reduction in proportion of PCEs at MTD 72h post dose
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Table 1 Results of micronucleus assay

Dose mg/kg

Collection interval (hours)

%Micronucleated PCE’s1

Mean ± S.D

Ratio PCE : NCE2

Mean ± S.D

 

 

Males

Females

Males

Females

2500

30

0.46± 0.35

0.34± 0.34

41.4± 11.8

46.4± 20.8

48

0.50± 0.49

0.20± 0.29

38.6± 14.9

34.5± 9.8

72

0.18± 0.05

0.28± 0.38

21.4±4.4

27.2± 2.9

4000

30

0.52± 0.27

0.20± 0.10

43.6± 11.3

53.2± 16.4

48

0.42± 0.33

0.26± 0.23

37.2± 4.8

44.6± 14.7

72

0.14± 0.11

0.18± 0.18

23.2± 6.4

24.6± 8.7

5000

30

0.24± 0.11

0.38± 0.15

43.6± 10.1

40.8± 12.2

48

0.34± 0.15

0.22± 0.05

32.2± 11.1

38.3± 6.2

72

0.26± 0.17

0.18± 0.18

19.8± 5.4

21.0± 12.3

Vehicle (corn oil)

30

0.30±0.12

0.36±0.11

44.6± 12.4

47.6± 11.3

48

0.34± 0.18

0.14± 0.11

42.4± 10.9

44.2± 11.4

72

0.24± 0.17

0.04± 0.06

29.4±6.7

32.0± 8.5

Positive control 0.3 mg/kg

30

2.92± 0.9

2.84± 1.00

34.4± 10.0

29.8± 8.2

48

NE

NE

NE

NE

 

NE

NE

NE

NE

1 Polychromatic erythrocytes

2 Normochromatic erythrocytes

Conclusions:
In a mouse micronucleus assay, conducted in a similar manner to OECD TG 474 and in compliance with GLP, 3-trimethoxysilylpropyl methacrylate did not produce treatment-related or statistically significant increases in the incidence of micronuclei in the peripheral blood polychromatic erythrocytes when administered by intraperitoneal injection up to limit concentrations. A decrease in the PCE/NCE ratio at 72 hours was considered to be evidence that the test substance had reached the target tissue. Positive and vehicle controls produced appropriate responses. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.
Endpoint:
genetic toxicity in vivo, other
Remarks:
Alkaline Comet Assay by Nose-only Inhalation in Sprague Dawley Rats
Type of information:
experimental study
Adequacy of study:
key study
Study period:
28 November 2017 to 06 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was disolved in deionized water/acetic acid 50/50 mixture.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The hydrolysed test substance formulation used for generation of exposure atmospheres was prepared for dosing as a 15% mixture in stock water solution by adding the test substance to the stock water and stirring until clear. Once clear, the pH was checked to ensure that the final pH was between 3.0 to 3.3. The hydrolysed test substance formulation was prepared on each day of use. No analysis of test solution was carried out, however, the hydrolysate preparation would have contained some parent substance.
- Preliminary purification step: none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 7-8 weeks
- Weight at study initiation: 200-320 grams (males) / 150-250 grams (females)
- Assigned to test groups randomly: yes
- Fasting period before study: not specified
- Housing: Housed in groups of 2 to 4 per cage following receipt in clean, solid bottom cages with bedding material in an environmentally controlled room.
- Diet: PMI Nutrition International, LLC Certified Rodent LabDiet® 5002 meal was offered ad libitum during the study, except during exposure periods, acclimation to nose-only restraint tubes, and designated periods of fasting.
- Water: Reverse osmosis-treated water was available, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-26 °C
- Humidity (%): 30-70%
- Air changes (per hr): 10 / hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

Route of administration:
inhalation: aerosol
Vehicle:
- Vehicle(s)/solvent(s) used: deionized water (acetic acid), pH 3. The vehicle, deionized water (pH 3 ± 0.1) and also referred to as stock water solution, was prepared twice using glassware cleaned according to Sponsor-provided instructions and stored at room temperature (18°C to 24°C). Details of the preparation and dispensing of the vehicle for the test substance have been retained in the Study Records.
JUSTIFICATION: not specified
In the substance evaluation final decision, it is stated that “ECHA considers that the in vivo comet assay should be conducted with an aerosolised atmosphere of the registered substance which mimics the worst case test conditions of the aerosol inhalation repeated dose toxicity studies reported in the registration data.” The worst-case test conditions are the highest achievable concentration at the lowest pH used in the repeated dose studies, with the test substance stirred for one hour, therefore the vehicle was chosen to produce a hydrolysate of the test substance at pH 3.
Details on exposure:
TYPE OF INHALATION EXPOSURE: nose only
Test substance preparation and animal exposure tool place in Charles River Test Facility

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: a stainless steel, conventional nose-only exposure system
- Method of holding animals in test chamber: Animals were kept in nose-only restrained tubes.
- Source and rate of air: Exposure system air was supplied from a breathing quality in-house compressed air source and/or a HEPA- and charcoal-filtered air source. Airflow rate through the exposure system was set based on output from the aerosol generator and the dilution airflow, and provided a sufficient volume for the number of animals to be exposed and for exposure atmosphere sampling. The airflow rates for the nose-only system were calculated from calibration curves for the generation device and/or flowmeters.
- Method of conditioning air: not specified
- System of generating particulates/aerosols: not specified
- Temperature, humidity, pressure in air chamber: The target range for average temperature and relative humidity of the exposure atmosphere were 22 ± 3ºC and 50 ± 20%, respectively.
- Air flow rate: not specified
- Air change rate: not specified
- Method of particle size determination: Aerosol particle size determinations were conducted at least twice utilizing a cascade impactor. Pre-weighed collection substrates were loaded into the impactor prior to sample collection. Following sample collection, the substrates were re-weighed and the particle size was calculated based on the impactor stage cut-offs. The aerosol particle size was expressed in terms of the mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD). The target range for MMAD is 1 to 3.0 µm and GSD is 1.5 to 3.0
- Treatment of exhaust air: not specified

TEST ATMOSPHERE
- Brief description of analytical method used: Following sample collection, the sample was re-weighed and the wet weight was recorded. The wet weight was only used to guide the exposures, not to define exposure concentrations. Following wet weight determination, the filter sample was first dried by placing it in an oven set to approximately 120 °C for 20 minutes and then cooled for 10 minutes in a desiccator. Following the drying and cooling period, the filter was re-weighed. The aerosol exposure concentration (mg/m3) was calculated by dividing the gravimetrically determined mass of aerosol by the sample volume. The mass was determined by subtracting the initial filter weight from the final dried weight of the post-sample filter. Sample volume was calculated by multiplying the sample flow rate by the length of the sampling period. The adjusted exposure concentrations (equivalent MPTMS concentration) were calculated by dividing the aerosol exposure concentration by 0.7217. This correction factor represents the ratio of silicone formed on the dried filter relative to the MPTMS.
- Samples taken from breathing zone: yes
Duration of treatment / exposure:
6 hours a day
Frequency of treatment:
once a day for two consecutive days
Post exposure period:
between 2 and 4 hours following last exposure
Dose / conc.:
250 mg/m³ air
Dose / conc.:
500 mg/m³ air
Dose / conc.:
1 000 mg/m³ air
No. of animals per sex per dose:
6 males and 6 females per sex per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate
- Justification for choice of positive control(s): A known substance that induces DNA strand breaks.
- Route of administration: oral (gavage)
- Doses / concentrations: The ethyl methanesulfonate formulation was prepared for dosing as a weight-to-volume mixture in 0.9% saline at a concentration of 200 mg/kg (10 mL/kg volume).
Tissues and cell types examined:
Immediately following euthanasia, the five (5) surviving rats in Groups 1-5 had nasal tissue, lung and liver collected and examined.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: The target exposure concentrations were selected by the Sponsor Representative in consultation with the Study Director based, in part, on the a previous acute inhalation toxicity study in rats. In that previous study, hydrolysed MPTMS aerosol was administered as a single 4-hour exposure at an achieved concentration of 2280 mg/m3. No deaths were observed.
In order to assess the potential for exposure-limiting toxicity, a range-finding study was carried out using a high target adjusted aerosol concentration (equivalent MPTMS concentration) of 1450 mg/m3 was selected in an effort to define the maximum tolerated dose (MTD). During method development, an actual maximal feasible concentration was determined and the target concentration for the high-dose was updated or confirmed. The mid and low target aerosol concentrations were selected to be one-half (1/2) and one-quarter (1/4) of the selected high-dose, respectively. The concentrations achieved in the dose range-finder were 0, 256, 523, and 1043 mg/m3.
All animals survived until the scheduled euthanasia (~2 hours post 2nd exposure).
Following each exposure, there were observations related to respiration in the high-dose group: labored respiration, increased respiration, gasping, and/or rales. These findings were not considered to be dose limiting.
The exposure levels for the definitive phase were selected based on data from the dose range finding phase (mortality/moribundity, body weights, etc.). The high exposure concentration was the highest achievable non-lethal exposure concentration (maximum tolerated dose) based on the results of the dose range-finding phase. The mid and low target aerosol concentrations were selected to be one-half (1/2) and one-quarter (1/4) of the selected high dose, respectively.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): treatment and euthanasia of animals was carried out at Charles River Test Facility by Charles River technicians, who removed head, liver and lungs. Samples of the nasal tissue, right lung and liver were collected by BioReliance staff at the Charles River Test Facility.

DETAILS OF SLIDE PREPARATION: A section of each of the tissues were placed in 3 mL of chilled mincing solution, then minced with fine scissors to release the cells. The cell suspensions were strained into a pre-labelled conical polypropylene tube through a Cell Strainer and were kept on wet ice during preparation of the slides.
From each liver, lung and nasal tissue cell suspension, an aliquot of 2.5 µL was mixed with 75 µL (0.5%) of low melting agarose. The cell/agarose suspension was applied to microscope slides. Commercially purchased pre-treated, multi-well slides were used and these slides have 20 individual circular areas, referred to as wells in the text below. The slides were kept at 2 - 8°C for at least 15 minutes to allow the gel to solidify. Sliders were identified with a random code that reflects the study number, group, animal number, and organ/tissue. At least two Trevigen, Inc 20-well slides were prepared per animal per tissue. Three slides/wells were used in scoring and the other wells were designated as a backup. Following solidification of agarose, the slides were placed in jars containing lysis solution.
Slides of the processed nasal tissue, lung, and liver prepared by BioReliance personnel at the Charles River Test Facility were stored at room temperature with desiccant. Slides were shipped on the first available Monday, Tuesday, or Wednesday at ambient temperature to BioReliance by overnight shipment.

METHOD OF ANALYSIS: Analysis of slides was carried out at the BioReliance Test site. Three wells per organ/animal were used. Fifty randomly selected, non-overlapping cells per slide/well were scored resulting in a total of 150 cells evaluated per animal for DNA damage using the fully validated automated scoring system Comet Assay IV.
The following endpoints of DNA damage were assessed and measured:
• Comet Tail Migration; defined as the distance from the perimeter of the Comet head to the last visible point in the tail.
• % Tail DNA; (also known as % tail intensity or % DNA in tail); defined as the percentage of DNA fragments present in the tail.
• Tail Moment (also known as Olive Tail moment); defined as the product of the amount of DNA in the tail and the tail length [(% Tail DNA x Tail Length)/ 100].
Each slide/well was also examined for indications of cytotoxicity. The rough estimate of the percentage of “clouds” was determined by scanning 150 cells per animal, when possible (percentage of “clouds” was calculated by adding the total number of clouds for all slides scored, dividing by the total number of cells scored and multiplying by 100). The “clouds”, also known as “hedgehogs”, are a morphological indication of highly damaged cells often associated with severe genotoxicity, necrosis or apoptosis. A “cloud” is produced when almost the entire cell DNA is in the tail of the comet and the head is reduced in size, almost nonexistent. “Clouds” with visible gaps between the nuclei and the comet tail were excluded from comet image analysis.
Evaluation criteria:
All conclusions were based on sound scientific judgment. As a guide to interpretation of the data, the following were considered:
• The test substance was considered to induce a positive response in a particular tissue if the mean % tail DNA (or other parameters of DNA damage) in one or more test substance groups (doses) was statistically elevated relative to the concurrent negative control group.
• The test substance was judged negative for induction of DNA damage if no statistically significant increase in the mean % DNA damage (or other parameters) in the test substance groups relative to the concurrent negative control group was observed.
However, the results of the statistical analysis may not be the only criterion in determination of the test substance potential to induce DNA damage. The following may be taken in consideration:
• The historical vehicle control data; a statistically significant increase in the mean % DNA (or other parameters) was not considered biologically relevant if the values did not exceed the range of historical vehicle control.
• Because cells undergoing necrosis or degeneration are prone to DNA degradation, independent of direct genotoxic effects of the test substance, doses that were found to be cytotoxic, by histopathology evaluation, were not considered as relevant doses and were not taken in consideration during the generation of the study conclusions. Accordingly, any statistically significant increase in DNA damage occurring at a cytotoxic dose was not considered as a positive finding.
• A dose-dependent increase in the mean % tail DNA (or other parameters) across the dose levels tested; if there was evidence of a dose-response with no evidence of a statistically significant increase, additional testing, including histopathology evaluation of the tissue, was considered.
Statistics:
In order to quantify the test substance-related effects on DNA damage, the following statistical analysis was performed:
• The use of parametric or non-parametric statistical methods in evaluation of data was based on the variation between groups. The group variances for % tail DNA (or other parameters of DNA damage) generated for the negative control (filtered air control) and test substance-treated groups were compared using Levene’s test (significant level of p = 0.05). If the differences and variations between groups were found not to be significant, a parametric one-way ANOVA followed by a Dunnett’s post-hoc test were performed (significant level of p < 0.05).
• Linear regression analysis was used to determine a dose-response relationship (p < 0.01).
• A pair-wise comparison (Student’s T-test p¿ 0.05) was used to compare the data from the positive control group against the negative control group.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY:
- Dose range: A dose range finding study has been completed using mean concentrations of 0, 256, 523, and 1043 mg/m3.
- Solubility: not specified
- Clinical signs of toxicity in test animals: Following each exposure, there were observations related to respiration in the high-dose group: labored respiration, increased respiration, gasping, and/or rales. While present, these type of findings were not considered to be dose limiting. All animals survived until the scheduled euthanasia.


RESULTS OF DEFINITIVE STUDY

Liver cells

Males: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the test substance groups was = 1.4%, which was comparable with the % of clouds in the negative control group (0.8%).

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the negative control group (Student’s t test, p = 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the liver

Females: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for liver cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the test substance groups was = 2.4%, which was higher than the % of clouds in the negative control group (1.0%).

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in liver cells as compared to the negative control group (Student’s t test, p = 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the liver

Lung cells

Males: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for lung cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the low and mid test substance groups was 0.4%, which was comparable with the % of clouds in the negative control group (0.4%). The high test substance group was 1.6% which was higher than the % clouds in the negative control group.  

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• A statistically significant increase response in the % Tail DNA (DNA damage) was observed in the test substance group (500 mg/m3) relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05); however this increase was not biologically significant and was within the Test Site’s historical control for this tissue, thus it was not considered to be biologically relevant.  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the negative control group (Student’s t test, p = 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the lung

Females: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for lung cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the test substance groups was = 3.2%, which was lower than the % of clouds in the negative control group (4.2%).

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• A statistically significant decrease response in the % Tail DNA (DNA damage) was observed in the test substance group (1000 mg/m3) relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p < 0.05).  This was not considered to be biologically relevant, since the study was designed to detect and evaluate increase in the DNA damage, which would be evident by an increase in % tail DNA.

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in lung cells as compared to the negative control group (Student’s t test, p = 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the lung

Nasal cells

Males: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for nasal cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the low and high test substance groups was = 12.4 %, which was higher than the % of clouds in the negative control group (8.4%). The mid test substance group was 6.8% which was lower than the % clouds in the negative control group.

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in nasal cells as compared to the negative control group (Student’s t test, p = 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the nasal

Females: Median values for the % Tail DNA, Tail moment and Tail migration (µm) for nasal cells are calculated per 150 cells for each animal

• The presence of ‘clouds’ in the low and mid test substance groups was = 34.8%, which was higher than the % of clouds in the negative control group (24.4%). The high test substance group was 17.4% which was lower than the % of clouds in the negative control group.

• Group variances for mean of medians of the % Tail DNA in the negative and test substance groups were compared using Levene’s test.  The test indicated that there was no significant difference in the group variance (p > 0.05); therefore, the parametric approach, ANOVA followed by Dunnett’s post-hoc analysis, was used in the statistical analysis of data.

• No statistically significant response in the % Tail DNA (DNA damage) was observed in the test substance groups relative to the concurrent negative control group (ANOVA followed by Dunnett’s post-hoc analysis, p > 0.05).  

• No dose-dependent increase in the % Tail DNA was observed across three test substance doses (regression analysis, p > 0.01).  

• The positive control, EMS, induced a statistically significant increase in the % Tail DNA in nasal cells as compared to the negative control group (Student’s t test, p = 0.05).  

• In the negative control group, % Tail DNA was within the historical vehicle control range for the nasal

Conclusions:
In an in vivo Comet Assay, conducted according to OECD test guideline 489 and in compliance with GLP, 3-trimethoxysilylpropyl methacrylate (aerosolized, hydrolysed) was tested for the ability to induce DNA damage in Sprague-Dawley rats. No test-substance induced DNA-damaging response was observed in nasal tissue, lung and liver from male and female rats following 6-hour nose-only inhalation exposure with the test substance for two consecutive days. None of the test substance-treated animal slides had significant increases in the % Tail DNA compared to the respective negative control. The negative control % Tail DNA was within the laboratory’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the negative control. Thus, all criteria for a valid assay were met for liver, lung and nasal tissue. It was concluded that the test substance was negative for DNA damage in liver, lung and nasal tissue in the in vivo Comet Assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Information is available from reliable studies for all the required in vitro endpoints. Where there was more than one result for an endpoint, the most reliable study available was chosen as key study. Where there was more than one reliable study, the most recent study was selected. The results of most of the studies were in agreement. There was evidence for clastogenicity (causing chromosomal aberrations) in the presence of metabolic activation and for weak clastogenicity in the absence of metabolic activation in vitro. An in vivo micronucleus study did not support this finding, so it is concluded that the in vitro result does not reflect an ability to cause chromosome aberrations in vivo.

3-Trimethoxysilylpropyl methacrylate has been tested in a valid bacterial reverse mutation assay, conducted to a protocol similar to OECD test guideline 471 and in compliance with GLP, using Salmonella typhimurium TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA (BioReliance, 2001). No increase in the number of revertants was observed in any test strain, with or without metabolic activation. Appropriate positive and solvent controls were added and gave expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test. This result is supported by other studies including a more recent study using E. coli WP2 uvrA only (Stockhausen GmbH, 2006), and older studies which did not include a bacterial strain capable of detecting cross-linking mutagens (Microtest Research Ltd., 1988; Isquith AJ, Whaley DP, 1977; Bushy Run Research Centre, 1985b). The key study was selected because a fully-compliant range of strains was tested.

3-Trimethoxysilylpropyl methacrylate has been tested for ability to cause chromosome aberrations in Chinese hamster ovary cells according to a protocol that is similar to OECD test guideline 473 and in compliance with GLP (Bushy Run Research Centre, 1985c). In this study, in the absence of metabolic activation, the test substance produced a statistically significant increase in chromosome aberrations only at 0.4 mg/ml, the highest concentration tested at the 8 hour sampling period. In contrast, in the presence of metabolic activation, the test substance produced significant increases in the incidences of chromosome aberrations at both 8 and 12 hours and at several concentrations. It is concluded that the test substance is positive for clastogenicity (causing chromosome damage) under the conditions of the test. In addition, the registered substance has been tested for the induction of sister chromatid exchanges in Chinese hamster ovary cells (Bushy Run Research Centre, 1985a). No evidence for the induction of SCEs was observed.

3-Trimethyoxysilylpropyl methacrylate has been tested for mutagenicity in Chinese hamster ovary cells, conducted according to an US EPA method and in compliance with GLP (Bushy Run Research Centre, 1985a). No test-substance induced increase in the number of mutations was observed. Appropriate solvent, negative (cell culture medium) and positive controls were included and gave expected results. It is concluded that 3-trimethyoxysilylpropyl methacrylate is negative for mutagenicity to mammalian cells under the conditions of the test.

3-Trimethoxysilylpropyl methacrylate has been tested in a reliable, valid in vivo mouse micronucleus assay, conducted in a similar manner to a protocol that is similar to OECD test guideline 474 and in compliance with GLP (Bushy Run Research Centre, 1986). The test substance did not produce treatment-related or statistically significant increases in the incidence of micronuclei in the peripheral blood polychromatic erythrocytes when administered by intraperitoneal injection up to limit concentrations. A decrease in the PCE/NCE ratio at 72 hours was considered to be evidence that the test substance had reached the target tissue. Positive and vehicle controls produced appropriate responses. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.

3-Trimethoxysilylpropyl methacrylate (aerosolized, hydrolysed) has been tested for ability to induce DNA damage in Sprague-Dawley rats, conducted according to OECD test guideline 489 and in compliance with GLP. No test-substance induced DNA-damaging response was observed in nasal tissue, lung and liver from male and female rats following 6-hour nose-only inhalation exposure with the test substance for two consecutive days. None of the test substance-treated animal slides had significant increases in the % Tail DNA compared to the respective negative control.  The negative control % Tail DNA was within the laboratory’s historical range, and the positive control had a statistically significant increase in % Tail DNA compared to the negative control.  Thus, all criteria for a valid assay were met for liver, lung and nasal tissue. It was concluded that the test substance was negative for DNA damage in liver, lung and nasal tissue in the in vivo Comet Assay.

Justification for classification or non-classification

Based on the available data, 3-trimethoxysilylpropyl methacrylate does not require classification for genotoxicity according to Regulation (EC) No. 1272/2008.