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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro:
Gene mutation (Bacterial reverse mutation assay / Ames test): negative with and without metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 (OECD 471, 1997) (BSL Bioservice, 2012e).

Cytogenicity in mammalian cells: negative with and without metabolic activation in cultured human lymphocytes (OECD 473, 1997) (BSL Bioservice, 2013b).

Mutagenicity in mammalian cells: positive with metabolic activation in L5178Y mouse lymphoma cells (OECD 476, 1997) (BSL Bioservice, 2013c).

Link to relevant study records

Referenceopen allclose all

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:
23 January - 14 May, 2012
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:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
All Salmonella strains contain mutation, in the histidine operon, thereby imposing a requirement for histidine in the growth medium. They contain the deep rough (rfa) mutation which deletes the polysaccharide side chain of the lipopolysaccharides of the bacterial cell surface. This increases cell permeability of larger substances. The other mutation is a deletion of the uvrB gene coding for a protein of the DNA nucleotide
excision repair system resulting in an increased sensitivity in detecting many mutagens. This deletion also includes the nitrate reductase (chl) and biotin (bio) gene. (Bacteria require biotin for growth).
The tester strains TA 98 and TA 100 contain the R·factor plasmid, pkM101. These strains are reverted by a number of mutagens that are detected weakly or not at all with the non R-factor parent strains. pkM101 increases chemical and spontaneous mutagenesis by enhancing an error-prone DNA repair system which is normally present in these organisms.
The tester strain E. coli WP2 uvrA carries the defect in one of the genes for tryptophan biosynthesis. Tryptophan-independent mutants (revertants) can arise either by a base change at the site of the original alteration or by a base change elsewhere in the chromosome so that the original defect is suppressed. This second possibility can occur in several different ways so that the system seems capable of detecting all types of mutagen, which substitute one base for another. Additionally, the strain is deficient in the DNA nucleotide excision repair system.

The properties of the S. typhimurium and E. coli strains, with regard to membrane permeability, ampicillin- and tetracycline-resistance as well as normal spontaneous mutation rates are cheeked regularly according to Ames et al. In this way it is ensured that the experimental conditions set up by Ames are fulfilled.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9.
Test concentrations with justification for top dose:
Exposure Concentrations
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 5000 µg/plate was selected as the maximum concentration. The concentration range covered two logarithmic decades. Two independent experiments were performed at the following concentrations:

Experiment I (plate incorporation method):
31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Experiment II (pre-incubation method):
10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

As the results of the pre-experiment were in accordance with the criteria described above, these were reported as a part of the main Experiment I.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: miscible and stable in DMSO
Untreated negative controls:
yes
Remarks:
A. dest., BSL Bioservice Lot No, 120203, 120221
Negative solvent / vehicle controls:
yes
Remarks:
DMSO, AppliChem Lot No. IP008076, I Y009099
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-nitro-o-phenylene-diamine; 2-aminoanthacene
Remarks:
Pos. control substances assigned to different tester strains.
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9. S9 mix included 5% S9, with MgCl₂, KCl, glucose-6-phosphate and NADP. 500 μl of S9 mix were included in 2.7 ml agar, test solution and bacterial suspension, giving a final concentration of approximately 1% S9 in the plates.

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period (second experiment): 60 minutes
- Exposure duration: at least 48 hours

SELECTION AGENT (mutation assays): histidine or tryptophan deficient agar

NUMBER OF REPLICATIONS: triplicate plates. The initial plate incorporation assay was repeated in an independent experiment using the pre-incubation method.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: clearing or diminution of background lawn or reduction in number of revertants to ≤ 0.5 relative to solvent control

Evaluation criteria:
A test item is considered mutagenic if a clear dose-related increase in the number of revertants occurs, and/or there is a biologically relevant response for at least on dose group in at least one strain with or without metabolic activation.
Statistics:
Not required.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
reduction in background lawn was observed at concentrations of 1000 μg/plate and above. A reduction in the number of revertants to 50% of solvent control was observed in several strains. This was not considered biologically relevant.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Results of Experiment 1 (plate incorporation) and Experiment 2 (pre-incubation). Revertants per plate (mean of three plates)

 

Experiment 1 (plate incorporation)

Concentration μg/plate (solvent or control substance)

TA98

TA100

TA1535

TA1537

E. coli WP2

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0 (distilled water)

22

27

101

115

5

7

9

10

41

50

0 (DMSO)

18

25

90

113

6

6

6

7

33

47

31.6

19

26

98

112

7

12

6

10

38

31

100

19

32

92

115

9

8

7

11

44

45

316

21

34

91

126

2

9

10

13

42

55

1000

23

42

80*

110

4*

10

7*

8

39*

58

2500

22

25

59*

104

4*

7

6*

9

38*

49

5000

22

28

70*

82

3*

8

3*

8

27*

47

10 (4-NOPD)

390

-

-

-

-

-

140

-

-

-

10 (NaN)

-

-

850

-

803

-

-

-

-

-

1 μl (MMS)

-

-

-

-

-

-

-

-

340

-

2.5 (2-AA)

-

2659

-

2075

-

126

-

213

-

133

 

Experiment 2 (pre-incubation)

Concentration μg/plate (solvent or control substance)

TA98

TA100

TA1535

TA1537

E. coli WP2

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

-MA

+MA

0 (distilled water)

23

29

128

125

7

5

6

7

77

60

0 (DMSO)

19

24

107

110

5

9

4

7

51

49

10

23

34

113

93

8

8

7

3

46

55

31.6

20

25

114

110

9

6

5

5

55

56

100

23

28

109

98

5

4

7

6

53

63

316

21

28

85

108

6

7

7

3

50

46

1000

21

29

62*

106

2*

7

5*

6

52

56

2500

17

31

55*

102*

1*

8

4*

10

40

56

5000

19

28

67*

83*

3*

5*

4*

8*

38

49

10 (4-NOPD)

446

-

-

-

-

-

86

-

-

-

10 (NaN)

-

-

839

-

577

-

-

-

-

-

1 μl (MMS)

-

-

-

-

-

-

-

-

710

-

2.5 (2-AA)

-

1985

-

1543

-

57

-

185

-

161

* Reduction in background lawn

4-NOPD  4-nitro-o-phenylene-diamine

NaN      sodium azide

MMS      methylmethanesulfonate

2-AA      2-aminoanthacene

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frame shifts in the genome of the tester strains used. Appropriate positive, solvent and negative controls were included and gave expected results. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay under the conditions of the test.
Executive summary:

The test item was investigated for its potential to induce gene mutations according to the plate incorporation test (Experiment I) and the pre-incubation test (Experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA. In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments: Experiment I (plate incorporation method): 31.6, 100, 316, 1000, 2500 and 5000 μg/plate Experiment II (pre-incubation method): 10.0, 31.6, 100, 316, 1000, 2500 and 5000 μg/plate

No precipitation of the test item was observed in any tester strain used in Experiment I and II (with and without metabolic activation). Cytotoxicity related to clearing or rather diminution of the background lawn and a reduction in the number of revertants down to a mutation factor equal and less than 0.5 in relation to the solvent control was noted in both experiments. Cytotoxicity was noted in four tester strains used in Experiment I and in three tester strains used in Experiment II. In Experiment I, reduced background lawn was observed in four tester strains (TA 100, TA 1535, TA 1537 and WP2 uvrA) at concentrations greater than or equal to 1000 μg/plate (without metabolic activation). In strain TA 1537, the mutation factor was 0.5 at 5000 μg/plate. One other strain TA1535 (without metabolic activation) had one concentration, 316 μg/plate, with a mutation factor less than 0.5. However, the finding is not considered to be biologically relevant, because it did not appear to be attributable to the test item. In Experiment II, reduced background lawn was observed in three tester strains (TA 100, TA 1535, and TA 1537) at concentrations of 1000 μg/plate and higher (without metabolic activation). With metabolic activation, two of same three strains (TA 1535, and TA 1537) had reduced background lawn at a concentration of 5000 μg/plate. The remaining strain (TA 100) had reduced background lawn concentrations greater than or equal to 2500 μg/plate. A mutation factor of 0.5 and less was noted at various concentrations for TA 100 (without metabolic activation), TA 1535 (with and without metabolic activation), and TA 1537 (with metabolic activation). However with the exception of strain TA 1535 (without metabolic activation) where the mutation factor was equal to or less than 0.5 at the three highest concentrations tested, the finding was not considered to be biologically relevant due to its sporadic incidence. No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 2,4 -hexadienoic acid, 3-(trimethoxysilyl) propyl ester at any concentration level, neither in the presence nor absence of metabolic activation in Experiment I and II. The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, the test item is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
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)
Version / remarks:
1997
Deviations:
no
Remarks:
however, the number of cells scored for aberrations was lower than required in the 2014 guideline
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
The cells were obtained from blood samples from healthy donors not receiving medication.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and β-naphthoflavone induced rat liver S9.
Test concentrations with justification for top dose:
Experiment I: tested concentrations: 0.25-5.00 mM (-MA), 1.00-10.00 mM (+MA); evaluated concentrations: 0.50, 1.00, 1.75 mM (-MA), 1.00 2.50, 7.50 mM (+MA)
Experiment II: tested concentrations: 0.10-10.00 mM (-MA), 0.40-10.00 mM (+MA); evaluated concentrations 0.19, 0.47, 1.0 mM (-MA), 2.00, 6.00, 8.00 mM (+MA)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle: DMSO was selected as solvent following a solubility test and as a result of further formulation analysis.
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Remarks:
without metabolic activation 400 and 900 µg/ml
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation 5 µg/ml
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital and β-naphthoflavone induced rat liver S9, protein concentration 47.7 mg/ml. S9 mix included 5% S9, with MgCl₂, KCl, glucose-6-phosphate and NADP. The final protein concentration in the cultures was 0.75 mg/ml S9.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (with and without metabolic activation, experiment I; with metabolic activation, experiment II); 24 hours (without metabolic activation, experiment II).
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giesma

NUMBER OF REPLICATIONS: Cultures were duplicated. The initial experiment with 4 hour treatment was repeated using 24 hours exposure time without metabolic activation.

NUMBER OF CELLS EVALUATED: 200 cells per concentration were evaluated for aberrations. The mitotic index was determined in 1000 cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
OTHER:
Evaluation criteria:
Criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations.
- a biologically relevant for at least one of the dose groups, higher than the laboratory negative control range.
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Experiment I: 1.75 mM (-MA), 7.5 mM (+MA); Experiment II: 1.00 mM (-MA), 6.00 mM (+MA)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES: A pre-experiment for toxicity was used to select concentrations for the main experiment

COMPARISON WITH HISTORICAL CONTROL DATA: control data were within the range of historical controls

ADDITIONAL INFORMATION ON CYTOTOXICITY: A reduction in mitotic index was observed
Remarks on result:
other: a negative result was determined

Summary of results

Dose Group

Concentration [mM]

Relative Mitotic Index [%]

Proliferation

Index

Mean % Aberrant Cells

Historical Laboratory Negative Control Range

Precipitation

incl. Gaps

excl. Gaps

Experiment I without metabolic activation 4 h treatment, 24 h preparation interval

C

0

75

1.68

2.5

0.00

0.0% - 4.0% aberrant cells

-

S

0

100

1.61

0.5

0.5

 

2

0.50

79

1.56

0.5

0.0

-

3

1.00

81

1.66

1.5

0.5

-

6

1.75

52

1.14

2.5

0.5

-

EMS

900 μg/ml

62

-

13.5

13.0

-

Experiment II without metabolic activation 24 h treatment, 24 h preparation interval

C

0

102

1.66

1.5

0.5

0.0% - 4.0% aberrant cells

-

S

0

100

1.52

1.0

0.5

-

3

0.40*

72

1.66

1.0

0.5

-

4

0.60**

90

1.32

1.5

0.0

-

6

1.00

58

1.44

2.0

1.0

-

EMS

400 μg/ml

43

-

23.5

20.0

-

Experiment I with metabolic activation 4 h treatment, 24 h preparation interval

C

0

129

1.16

3.0

1.0

.0% - 4.0% aberrant cells

-

S

0

100

1.39

3.0

1.5

 

1

1.00

117

1.23

2.5

1.5

-

3

2.50

88

1.03

2.5

1.5

-

5

7.50

40

1.06

6.8

2.7

-

EMS

5 μg/ml

87

-

14.5

12.5

-

Experiment II with metabolic activation 4 h treatment, 24 h preparation interval

C

0

105

1.38

2.5

1.0

0.0% - 4.0% aberrant cells

-

S

0

100

1.23

1.5

0.5

 

3

2.00

87

1.27

1.0

0.5

-

6

6.00

67

1.28

1.5

1.0

+

8

8.00

93

1.16

3.0

1.5

+

EMS

5 μg/ml

98

-

11.0

10.0

-

C:       Negative Control (Culture Medium)

S:       Solvent Control (DMSO)

EMS:  Ethylmethanesulfonate

CPA:   Cyclophosphamide

*          The measured concentration was 0.19 mM with regard to the recovery rate of the GC analysis (47.8%)

**        The measured concentration was 0.47 mM with regard to the recovery rate of the GC analysis (77.6%)

Conclusions:
3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in an in vitro chromosome aberration study conducted in accordance with OECD 473 (1997) and in compliance with GLP. No test substance-related increase in the number of cells with aberrations was observed up to cytotoxic concentrations in either the initial or the independent repeat experiment. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for cytogenicity under the conditions of the test.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase operon
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9
Test concentrations with justification for top dose:
Without metabolic activation: 0.2, 0.5, 1.0, 1.2, 1.7, 2.0, 2.3, 2.5 mM
With metabolic activation: 0.3, 0.6, 1.3, 2.5, 5.0, 8.0, 9.0, 10.0 mM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility study indicated that the test substance was not soluble in RPMI, but was soluble in DMSO. The solvent was compatible with the survival of the cells and S9 activity.
Untreated negative controls:
yes
Remarks:
treatment medium
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
ACTIVATION: Phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver S9. S9 mix included MgCl₂, KCl, glucose-6-phosphate and NADP, with S9 to a final protein concentration of 0.75 mg/ml in the cultures.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 14 days

SELECTION AGENT (mutation assays): TFT

NUMBER OF REPLICATIONS: no replicates

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER EXAMINATIONS:
- Other: occurrence of small colonies
Evaluation criteria:
The test item is considered mutagenic if the following criteria are met:
1) The induced mutant frequency meets or exceeds the Global Evaluation Factor of 126 mutants per 10⁶ cells;
2) A dose-dependent increase in mutant frequency is detected.

Combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) was considered an indication for potential clastogenic effects and/or chromosomal aberrations.
Statistics:
Statistical significance was investigated by use of the non-parametric Mann-Whitney test.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
The Global Evaluation Factor (GEF) was exceeded by the mutant frequency at some concentrations; a dose-response relationship was observed.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
RTG at 10.00 mM: 24.8%
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
The RTG was 19.0% at 2.5 mM, the highest concentration evaluated
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: precipitation was observed but did not interfere with scoring the colonies.

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity was observed in the pre-experiment for toxicity, with and without metabolic activation

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

 Summary of mutagenicity results

Concentration (mM)

RTG

MF

IMF

GEF exceeded

Statistical significance

Precipitate

Without metabolic activation

0 (negative control)

107.7/94.9

88.6

/

/

/

-

0 (solvent control)

100

88.5

/

/

/

-

0.2

72.3

80.1

-8.4

-

-

-

0.5

87.9

82.8

-5.7

-

-

-

1.0

68.8

90.9

2.4

-

-

-

1.2

68.0

61.9

-26.6

-

-

+

1.7

70.5

80.3

-8.2

-

-

+

2.0

39.4

128.0

39.5

-

-

+

2.3

27.7

99.3

10.7

-

-

+

2.5

19.0

70.8

-17.7

-

-

+

EMS 300 μg/ml

57.9

886.0

797.5

+

+

-

MMS 10 μg/ml

80.3

425.2

336.5

+

+

-

With metabolic activation

0 (negative control, duplicates)

106.1/1.5.1

72.5

/

/

/

-

0 (solvent control, duplicates)

100

91.3

/

/

/

-

0.3

95.7

101.1

9.8

-

-

-

0.6

108.3

80.2

-11.1

-

-

-

1.3

91.8

81.8

-9.5

-

-

-

2.5

77.2

81.5

-9.8

-

-

+

5.0

53.5

178.2

87.0

-

+

+

8.0

35.4

247.2

156.0

+

+

+

9.0

27.8

222.8

131.5

+

+

+

10.0

24.8

231.4

140.1

+

+

+

B[aP] 1.5 μg/ml

62.2

568.0

476.7

+

+

-

RTG: Relative total growth = (relative suspension growth/relative cloning efficiency)/100

MF: mutant frequency

IMF: induced mutant frequency

GEF: Global Evaluation Frequency

EMS: ethylmethanesulphonate

MMS: methylmethanesulfonate

B[aP]: benzo(a)pyrene

Colony sizing results

Without metabolic activation

Test group

Concentration mM

% small colonies

C1

0

7.5

C2

0

5.0

S1

0

15.6

S2

0

1.8

14 (P)

2.0

14.6

15 (P)

2.3

7.8

16

2.5

8.0

MMS

10 μg/ml

46.8

With metabolic activation

Test group

Concentration

% small colonies

C1

0

3.2

C2

0

5.8

S1

0

4.7

S2

0

8.5

10 (P)

8.0

34.7

11 (P)

9.0

40.6

12 (P)

10.0

49.3

B[a]P

1.5 μg/ml

41.1

C: Negative control

S: Solvent control

(P): Precipitation

MMS: methylmethanesulfonate

B[aP]: benzo(a)pyrene

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation

3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in a mammalian gene cell mutation assay conducted in accordance with OECD 476 (1997) and in compliance with GLP. A dose-related increase in excess of the Global Evaluation Frequency was observed in mouse lymphoma L5178Y cells in the presence but not in the absence of metabolic activation. An increase in the percentage of small colonies was observed in the presence of metabolic activation. It is concluded that the test substance is mutagenic and clastogenic to mammalian cells in the presence of metabolic activation under the conditions of the test.
Executive summary:

3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate was investigated for its ability to induce gene mutations in mammalian cells using mouse lymphoma L5178Y cells. The cells were exposed for four hours in the presence and the absence of metabolic activation; as a positive result was obtained, a repeat experiment was not required. The following concentrations of the test item were prepared and used in the experiments:

Without metabolic activation: 0.2, 0.5, 1.0, 1.2, 1.7, 2.0, 2.3, 2.5 mM

With metabolic activation: 0.3, 0.6, 1.6, 2.5, 5.0, 8.0, 9.0, 10.0 mM

Non-interfering precipitation was observed at concentrations of 1.2 mM and higher (without metabolic activation) and 2.5 mM and higher (without metabolic activation).

Growth inhibition was observed with and without metabolic activation: Relative total growth (RTG) was 19.0 at the highest concentration (2.5 mM) without metabolic activation and 24.8 at the highest concentration (10 mM) with metabolic activation.

A dose-related increase in the mutant frequency was observed in the main experiment with metabolic activation, and an increase in the percentage of small colonies was recorded. These effects were considered to be biologically relevant.

The reference mutagens induced a distinct increase of mutant frequency indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item caused gene mutations and an increase in small colonies in mouse lymphoma L51874Y cells. Therefore, the test item is considered to be mutagenic and clastogenic in this mammalian cell gene mutation assay.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In vivo:
Micronucleus assay oral (gavage) study in rat: negative (OECD 474, 1997) (BioReliance, 2013).

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan, Frederick, MD
- Age at study initiation: 7 weeks
- Weight at study initiation: males: 171.3-174.5 g; females: 140.8-143.3 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: Micro-Barrier cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 1 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: none given in study report
- Concentration of test material in vehicle: 100, 200 and 400 mg/ml
- Amount of vehicle (if gavage or dermal): 5 ml
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Dosing solutions were prepared on the day prior to the study. Test material and vehicle where vortexed and stirred until homogenous.
Duration of treatment / exposure:
Twenty four and forty eight hours
Frequency of treatment:
Single dose
Post exposure period:
Twenty four and forty eight hours
Remarks:
Doses / Concentrations:
500, 1000, 2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
Five rats per sex at each dose level (24 h sampling time), five rats per sex at high dose (48 h sampling time).
Control animals:
yes, concurrent vehicle
Positive control(s):
- Positive control substance: cyclophosphamide
- Justification for choice of positive control(s): none given in study report
- Route of administration: oral (gavage)
- Doses / concentrations: 40 mg/kg bw
Tissues and cell types examined:
Bone marrow: polychromatic erythrocytes and normochromatic erythrocytes
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: based on toxicity information and guideline recommended high dose

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling times: 24 and 48 hours after dosing

DETAILS OF SLIDE PREPARATION: suspensions of bone marrow cells were spread on a clean glass slides and air dried then fixed in methanol. Two slides were prepared from each rat. One set of slides was stained with acridine orange and used in microscopic evaluation. The second set of slides was frozen

METHOD OF ANALYSIS: Bone marrow was evaluated by fluorescent microscopy. The initial scoring was deemed invalid because of out-of-range historical control values for the vehicle. The slides were therefore re-scored and this data was presented in the study report. Cells were scored under high power. At least 2000 polychromatic erythrocytes (PCE) were scored per animal for micronuclei: the number of cells with micronuclei was recorded. In addition, at least 1000 total erythrocytes (PCEs plus normochromatic erythrocytes (NCE)).

Evaluation criteria:
The test substance is considered positive if it induces a significant increase in micronucleated PCE frequency at any dose level or sampling time compared to the concurrent vehicle control. Other criteria may be used in reaching a conclusion including magnitude of the increase, comparison to historical control values, biological significance.
Statistics:
The frequency of micronucleated PCEs and proportion of PCEs to total erythrocytes was determined for each animal and treatment group. Statistical significance, p ≤ 0.05, was determined using Kastenbaum-Bowman tables.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
No mortality or clinical signs; no effect on PCE/NCE ratio
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no induction of micronuclei observed.
- Ratio of PCE/total erythrocytes (for Micronucleus assay): between 0.422 (low dose males) and 0.464 (high dose, 48 h sampling time males).
- Appropriateness of dose levels and route: dose levels and route were appropriate.
- Statistical evaluation: Only the positive control results were statistically significant.

Summary of bone marrow micronucleus assay following single oral administration of test substance

Concentration mg/kg bw

Sex

Time

Number of animals

PCE/Total erythrocytes

Number of mnPCE/1000 PCE

Number of mnPCE/PCE scored

0 (corn oil)

M

24

5

0.479

0.6

6/10000

F

24

5

0.444

0.8

8/10000

500

M

24

5

0.422

0.4

4/10000

F

24

5

0.427

0.6

6/10000

1000

M

24

5

0.427

0.4

4/10000

F

24

5

0.436

0.6

6/10000

2000

M

24

5

0.438

0.5

5/10000

F

24

5

0.436

0.4

4/10000

Cyclophosphamide (40)

M

24

5

0.337

26.0*

260*/10000

F

24

5

0.337

23.6*

236*/10000

Corn oil

M

48

5

0.460

0.8

8/10000

F

48

5

0.457

0.3

3/10000

2000

M

48

5

0.464

0.5

5/10000

F

48

5

0.436

0.5

5/10000

PCE: polychromatic erythrocytes

mnPCE: micronucleated PCE

* statistically significant increase compared to the vehicle control, p≤ 0.05

Conclusions:
3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in an in vivo micronucleus assay conducted in accordance with OECD 474 and in compliance with GLP. No evidence for systemic toxicity, toxicity to bone marrow or induction of micronuclei was observed at any test concentration in bone marrow sampled 24 hours and 48 hours following oral administration by gavage of a single dose of test substance. The animals were dosed with 500, 1000 and 2000 mg/kg bw (24 h sampling time) and 2000 mg/kg bw (48 hour sampling time), Appropriate positive and vehicle controls were included and gave expected results. It is concluded that the test substance is negative for the induction of micronuclei under the conditions of the test.
Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study planned
Study period:
Planned: after approval by ECHA
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
- Name of the substance on which testing is proposed to be carried out: 3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate


CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies in vitro studies in bacterial and mammalian cells; results negative except for in vitro gene mutation with mammalian cells where a positive response exceeding the Global Evaluation Factor was observed in the presence of metabolic activation. Absence of effects in vivo in a micronucleus assay is not sufficient to address the concerns about potential mutagenicity arising from the positive result.
- Available non-GLP studies: none.
- Historical human data: no relevant data available
- (Q)SAR: in silico methods are available which are able to identify potential genotoxic substances based on the presence of alerts, but (Q)SAR cannot be used to follow up a positive in vitro result.
- In vitro methods: the GLP studies mentioned above include a positive mutagenic response in mammalian cells.
- Weight of evidence: there is only one study per endpoint.
- Grouping and read-across: no suitable read-across data are available.
- Substance-tailored exposure driven testing: not applicable.
- Approaches in addition to above: not applicable.
- Other reasons: not applicable.

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Column 2 of Annex VIII Section 8.4 states that "Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII." The REACH Guidance on Information requirements, Part R.8a indicates that an in vivo micronucleus assay is not an appropriate study to consider in the case of positive mutagenicity results in vivo.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
- Details on study design / methodology proposed [if relevant]
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in a bacterial mutagenicity study conducted in accordance with OECD 471 and in compliance with GLP (BSL Bioservice, 2012e). No increase in the number of revertants was observed when tested with and without metabolic activation in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2. Solvent controls and reference mutagens produced results within the range of historical controls. It is concluded that the text substance is negative for mutagenicity to bacterial cells under the conditions of the test.

3-(Ttrimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in an in vitro chromosome aberration study conducted in accordance with OECD 473 (1997) and in compliance with GLP (BSL Bioservice, 2013b). No test substance-related increase in the number of cells with aberrations was observed up to cytotoxic concentrations in either the initial or the independent repeat experiment. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for cytogenicity under the conditions of the test.

3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in an in vitro mammalian gene cell mutation assay conducted in accordance with OECD 476 (1997) and in compliance with GLP (BSL Bioservice, 2013c). A dose-related increase in excess of the Global Evaluation Frequency was observed in mouse lymphoma L5178Y cells in the presence but not in the absence of metabolic activation. An increase in the percentage of small colonies was observed in the presence of metabolic activation. Solvent controls and reference mutagens produced results within the range of historical controls. It is concluded that the test substance is mutagenic and clastogenic to mammalian cells in the presence of metabolic activation under the conditions of the test.

3-(Trimethoxysilyl)propyl-(2E,4E)-hexa-2,4-dienoate has been tested in an in vivo micronucleus assay conducted in accordance with OECD 474 and in compliance with GLP (BioReliance, 2013). No evidence for systemic toxicity, toxicity to bone marrow or induction of micronuclei was observed at any test concentration in bone marrow sampled 24 hours and 48 hours following oral administration by gavage of a single dose of test substance. The animals were dosed with 500, 1000 and 2000 mg/kg bw (24 h sampling time) and 2000 mg/kg bw (48 hour sampling time). Appropriate positive and vehicle controls were included and gave expected results. It is concluded that the test substance is negative for the induction of micronuclei in vivo under the conditions of the test.

Although there was no evidence for toxicity to bone marrow and therefore for the test substance reaching the target tissue, data are available from an OECD 417 study (Dow Corning Corporation 2014), which demonstrated that the test substance is systemically available subsequent to oral gavage dosing, therefore it is concluded that the test substance is not clastogenic in vivo.

No data are available to investigate in vivo the potential for mutagenicity to mammalian cells observed in vitro, therefore an in vivo comet assay is proposed.

Justification for classification or non-classification

Based on the available in vitro and in vivo genotoxicity data, 3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4 -dienoate does not require classification for mutagenicity according to Regulation (EC) No 1272/2008, however further information on mutagenicity is needed.