acrylic acid, 3-(trimethoxysilyl)propyl ester

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity for 3-(trimethoxysilyl)propyl acrylate (CAS No. 4369-14-6, EC No. 419-560-6): Negative (conducted according to OECD Test Guideline 471 and in compliance with GLP) (reliability score 1, Hita Research Laboratories, 1994c).

 

Cytogenicity in mammalian cells for 3-(trimethoxysilyl)propyl acrylate: Positive (conducted according to EEC Test Guideline (1991; similar to OECD Test Guideline 473) and in compliance with GLP) (reliability score 1, Hita Research Laboratories, 1994d).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6th April 1994 - 9th June 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine deficient S. typhimurium strains and tryptophan deficient E. coli strains

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:

Type and composition of metabolic activation system:
- Source of S9: Phenobarbital (30 mg/kg x 1, 60 mg/kg x 3) and 5,6-benzoflavon (80 mg/kg x 1) induced rat liver S9 from Sprague-Dawley rats, male, 7 weeks old prepared by Hita Research Laboratories
- Method of preparation of S9 mix: In 1 mL of S9 mix, there was 8 µmol MgCl2, 33 µmol KCl, 5 µmol glucose-6-phosphate, 4 µmol NADPH, 4 µmol NADH, 100 µmol 0.2 M sodium phosphate buffer (pH 7.4), 0.1 mL S9
- Concentration or volume of S9 mix and S9 in the final culture medium: 0.5 mL of S9 mix

Test concentrations with justification for top dose:

- Dose range-finding test: 5000, 2000, 1000, 500, 200, 100 and 50 µg/plate.
- Main test doses: The highest dose chosen for the main test 5000 µg/plate was based on the findings of the dose range-finding study and four more doses of 2500, 1250, 625 and 313 µg/plate were chosen by dilution with a geometric progression of two in two independent tests.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: No denaturation of the test solution was observed until 2 hours after preparation which demonstrated that the test substance was stable within the solvent.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-(2-furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-methoxy-6-chloro-9-[3-(2-chloroethyl)-aminopropylamino]acridine (ICR-191)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2AA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Dose range-finding test was tested in triplicate for the negative control and in duplicate for the test and positive control groups; triplicate cultures were used for the main test.
- Number of independent experiments : 2

METHOD OF TREATMENT/ EXPOSURE:
- The tests were carried out using the preincubation method

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: 20 minutes
- Exposure duration/duration of treatment: 48 hours
- Harvest time after the end of treatment (sampling/recovery times): After 48-hour incubation period.

FOR GENE MUTATION:
- Method used: Agar

METHODS FOR MEASUREMENT OF CYTOTOXICITY :
- Method: Background growth inhibition

Rationale for test conditions:

Based on a dose range-finding study.

Evaluation criteria:

The test substance was judged to be positive, when the number of revertant colonies is more than twice that of the negative control and a dose relationship and reproducibility are obtained.

Statistics:

Means with standard deviation, and statistical significance

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity was seen at 5000 µg/plate without S9 mix; no increase in the number of revertants was seen.

STUDY RESULTS :
- Concurrent vehicle negative and positive control data : See attachment for study data tables. Positive and negative controls were within the range of the background data for the laboratory.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : There was no concentration driven increase in the number of revertants seen.
- Statistical analysis: Statistical analysis was performed but no statistically significant increase in the number of revertants was seen.

Ames test:
- Signs of toxicity : Yes, at 5000 µg/plate with and without S9 mix
- Individual plate counts : Yes, see attachment for study data tables
- Mean number of revertant colonies per plate and standard deviation : Yes, see attachment for study data tables

Conclusions:

3-(Trimethoxysilyl)propyl acrylate has been tested in a bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 (1983) and in compliance with GLP, using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2. No increase in the number of revertants was observed in any test strain, with or without metabolic activation, when tested up to cytotoxic concentration. 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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29th June 1994 to 27th September 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: EEC Test Guideline, similar to OECD Test Guideline 473

Version / remarks:

1991

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Details on mammalian cell type (if applicable):

CELLS USED:
- Type and source of cells: Chinese hamster lung fibroblasts CHL cells, clone No. 11
- Suitability of cells: Highly sensitive to chemical mutagens
- Normal cell cycle time (negative control): Not specified

For cell lines:
- Absence of Mycoplasma contamination: Not specified
- Number of passages if applicable: Not specified
- Methods for maintenance in cell culture: In culture medium
- Cell cycle length, doubling time or proliferation index: 15 hours
- Modal number of chromosomes: 25
- Periodically checked for karyotype stability: Not specified
- Periodically ‘cleansed’ of spontaneous mutants: Not specified

MEDIA USED:
- Type and composition of media, CO2 concentration, humidity level, temperature: MEM solution prepared by dissolving 9.4 mg Eagle's minimum essential medium in 500 mL of pure water. To make 5 mL of 10%/NCS/MEM which included 10 v/v % of newborn calf serum 4.5 mL of the stock MEM solution was diluted with the same volume of the pure water and added with 0.5 mL of NCS. Throughout the study, the incubation of the cells was conducted in CO2 incubator controlled at 37+/-0.5°C, 5% CO2 and 100% relative humidity.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- Source of S9: phenobarbital (30 mg/kg x 1, 60 mg/kg x 3) and 5,6-benzoflavon (80 mg/kg x 1) induced rat liver S9 from Sprague-Dawley rats, male, 7 weeks old prepared by Hita Research Laboratories
- Method of preparation of S9 mix: 1 mL of S9 mix consisted of 0.3 mL of the S9 fraction, 0.2 mL of 20 mM HEPES Buffer with pH 7.2, 0.1 mL of 50 mM MgCl2, 1 mL of 330 mM KCl, 0.1 mL of 50 mM glucose-6-phosphate, 0.1 mL of 40 mM NADPH and 0.1 mL of pure water. 3 mL of S9 mix/MEM for the metabolic activation method was prepared by adding 0.5 mL of S9 mix to 2.5 mL 10% NCS/MEM.
- Concentration or volume of S9 mix and S9 in the final culture medium: 3 mL of S9 mix/MEM.

Test concentrations with justification for top dose:

- The concentrations of the test substance for the main chromosomal aberration tests were based on two kinds of tpreliminary tests, the cell growth inhibition tests and the cell division inhibition tests. The highest concentration possible to observe the cells in the mitotic metaphase should be included in the chromosomal aberration tests.
- The test concentration in the preliminary cell growth inhibition tests and the cell division inhibition tests are: 0, 30, 50, 70, 90 and 100 µg/mL for 24 and 48-hour treatment without metabolic activation and 0, 100, 200, 300, 350 and 400 µg/mL for the 6-hour treatment with metabolic activation.
- Test concentrations used in the main chromosome aberration test: 0, 50, 70, 90 for 24 and 48-hour treatment without metabolic activation and 0, 100, 200 and 300 µg/mL for the 6-hour and 18-hour recovery treatment with and without metabolic activation and 0, 150, 250 and 350 µg/mL for the 6-hour and 42-hour recovery treatment with metabolic activation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was demonstrated to be stable in the vehicle for up to 4 hours.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

0.05 µg/mL without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

10 µg/mL with metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: Duplicate dishes
- Number of independent experiments: Two main chromosomal aberration experiments were conducted.

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 mL of 10% NCS/MEM containing 1.5 x 10^4 or 0.5 x 10^4 cells/mL was placed in a 60 mm diameter Petri dish and cultured for 2 or 3 days. After the preculture, the mono-layer cells in logarithmic growth phase were used for exposure tests.
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 24 and 48 hours without metabolic activation for experiment 1 and 2; 6 hours with metabolic activation for experiment 1 and 2.
- Harvest time after the end of treatment: Sampling time straight after treatment without metabolic activation and sampling time after 18 and 42-hour recovery time after 6-hour treatment with metabolic activation.

FOR CHROMOSOME ABERRATION AND MICRONUCLEUS:
- Spindle inhibitor (cytogenetic assays): 0.1 µg/mL colcemid added 2 hours prior to the end of the respective incubation period.
- Methods of slide preparation and staining technique used including the stain used: In the cell division inhibition tests and the chromosomal aberration tests, the cells after whole incubation period were detached from each dish using 2 mL of 0.25% trypsin and collected using centrifugation. The cells were suspended in 0.075 M KCI for 15 minutes at 37°C and fixed 3 times using methanol-acetic acid (3:1) solution by repetition of centrifugation and resuspension. The cell suspensions were placed on microscopic slides, air-dried and stained with 2% Giemsa solution. One slide per one dose was prepared in the cell division inhibition tests and two slides were prepared in the chromosomal aberration tests.
- Number of cells spread and analysed per concentration: 100 well-spread metaphases for each dish
- Criteria for scoring chromosome aberrations: Each number of cells with each of numerical aberrations (polyploids and endoreduplication) and structural aberrations of chromatid type and chromosome type (gap, break, exchange, etc.) was counted by observing 100 well-spread metaphases for each dish, and each incidence of the cells with each of the numerical and/or structural aberration was recorded too.
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

METHODS FOR MEASUREMENT OF CYTOTOXICITY:
- Method: Background growth inhibition, mitotic index.

Rationale for test conditions:

Based on preliminary tests.

Evaluation criteria:

The test substance was judged to be positive for induction of chromosomal aberration in this in vitro study, when there was an incidence of numerical aberration or structural aberration including gap not less than 10% in a test condition that was found dose-related and/or reproducible.

Statistics:

Fisher's exact probaility test (P<0.05 or P<0.01)

Key result

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Data on pH: Not specified
- Data on osmolality: Not specified
- Possibility of evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation and time of the determination: Not specified
- Definition of acceptable cells for analysis: Not specified
- Other confounding effects: Not specified

RANGE-FINDING/SCREENING STUDIES: In the growth inhibition tests, IC50 were determined to be about 74 µg/mL in the 24 hours treatment group and about 52 µg/mL in the 48 hours treatment group in the direct method, and about 210 µg/mL in the 18 hours recovery group and about 200 µg/mL in the 42 hours recovery group in the metabolic activation method.

The relative ratios of the mitotic indices in the test substance groups were calculated in percentage to that in the solvent control group, and the concentrations to result 50 % inhibition ratios were about 65 µg/mL in the 24 hours and the 48 hours treatment groups in the direct method and about 240 µg/mL in the 18 hours recovery group and about 370 µg/mL in the 42 hours recovery group in the metabolic activation method. Mitotic metaphases of chromosomes sufficient for evaluating chromosomal aberration were observed at doses of 90 µg/mL or less in the 24 hours and the 48 hours treatment groups in the direct method and 300 µg/mL or less in the 18 hours recovery group and 350 µg/mL or less in the 42 hours recovery group in the metabolic activation method. Therefore, the first trial of the chromosomal aberration tests was conducted by observing the following dose levels of microscopic slides from the cell division inhibition tests: 50, 70 and 90 µg/mL for 24 and 48 hours treatments in the direct method, 100, 200 and 300 µg/mL for the 18 hours recovery group and 150, 250 and 350 µg/mL for the 42 hours recovery group in the metabolic activation method. The second trial of the chromosomal aberration tests were repeatedly conducted at the same doses as the first.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: Valid, see attached data tables.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: Yes
- Statistical analysis: Yes, calculated (P<0.05, P<0.01)


Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements: In the growth inhibition tests, IC50 were determined to be about 74 µg/m] in the 24 hours treatment group and about 52 µg/mL in the 48 hours treatment group in the direct method, and about 210 µg/mL in the 18 hours recovery group and about 200 µg/mL in the 42 hours recovery group in the metabolic activation method.

- The relative ratios of the mitotic indices in the test substance groups were calculated in percentage to that in the solvent control group, and the concentrations to result 50 % inhibition ratios were about 65 µg/mL in the 24 hours and the 48 hours treatment groups in the direct method and about 240 µg/mL in the 18 hours recovery group and about 370 µg/mL in the 42 hours recovery group in the metabolic activation method.

- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excluding gaps:

EXPERIMENT 1
- 24-hour treatment without metabolic activation: The incidences of cells with structural aberration including gap were 4.5% at 50 µg/mL, 39.0% at 70 µg/mL and 79.5% at 90 µg/mL. Therefore, the inductivity of the chromosomal aberration was positive and dose-dependent. The incidence in the solvent control group was 0% within a normal range, while it was 60.5% in the positive control (MMC) group, showing high induction of chromosomal aberration. The incidence of cells with structural aberration without gap in the 50 µg/mL test substance group was not significantly higher than that in the solvent control group, while those in the 70 and 90 µg/mL test substance groups were significantly higher (P<0.01). The incidence of numerical aberration cells in the 70 µg/mL test substance group was positive as 15.0%, but those in other two groups (50 and 90 µg/mL) were negative. The incidences of numerical aberration cells in the 50 and 90 µg/mL test substance groups were not significantly different from that in the solvent control group, but the incidence in 70 µg/mL test substance group was significantly higher (P <0.01).

- 48-hour treatment without metabolic activation: The incidences of cells with structural aberration including gap were 2.0% at 50 µg/mL, 10% at 70 µg/mL and 14% at 90 µg/mL. Therefore, the inductivity of the chromosomal aberration was positive and dose-dependent. The incidence in the solvent control group was 0.5% within a normal range, while that in the positive control group (MMC) was 76% showing high induction of chromosomal aberration. The incidence of cells with structural aberration without gap in the 50 µg/mL test substance group was not significantly higher than that in the solvent control group, while the incidences in the 70 and 90 µg/mL test substance groups were significantly higher (P<0 01). The incidence of numerical aberration cells in the 90 µg/mL test substance group was 9.5%, suspected as positive, but the other two groups (50 and 70 µg/mL) were negative. There was no significant difference in the 50 µg/mL test substance group, but there were significant differences at 70 µg/mL (P <0.05) and 90 µg/mL (P<0 01).

- 6-hour treatment and 18-hour recovery period with metabolic activation: The incidences of cells with structural aberration including gap were 3.5% at 100 µg/mL, 27.5% at 200 µg/mL and 30% at 300 µg/mL. Therefore, the inductivity of the chromosomal aberration was positive and dose-dependent The incidence in the solvent control group was 0% within a normal range, while that in the positive control group (CPA) was 26%, showing high induction of chromosomal aberration. The incidence of cells with structural aberration without gap in the 100 µg/mL test substance group was significantly higher than that in the solvent control group (P<0.05), moreover the significant level decreased to P<0.01 in the 200 and 300 µg/mL test substance groups. Numerical aberration cells appeared in all test substance groups by less than 5%, and the inductivity of the chromosomal aberration was negative. The incidences of numerical aberration cells were not significantly different between the test substance groups and the solvent control group.

- 6-hour treatment and 42-hour recovery period with metabolic activation: The incidences of cells with structural aberration were 1.0% in the 150 µg/mL test substance group, 6.0% at 250 µg/mL and 8.5% at 350 µg/mL, and the respective inductivities of chromosomal aberration were negative, suspected as positive and suspected as positive. The incidences of these cells were 1% in the solvent control group and 4% in the positive control group (CPA), both were within a normal range. The incidences of structural aberration without gap in the 150 µg/mL test substance group were not significantly different from that in the solvent control group, but the incidence at 250 µg/mL was significantly higher than that in the solvent control group (P<0.05), and that in the 350 µg/mL was further elevated. The incidence of cells with numerical aberration in the 350 µg/mL test substance group was 7%, suspected as positive, but the other two groups (150 and 250 µg/mL) were negative. There were no significant differences in the 150 µg/mL test substance group from the solvent control group, though there were significant differences at 250 and 350 µg/mL (P<0.01).

EXPERIMENT 2
- 24-hour treatment without metabolic activation: The incidences of cells with structural aberrations were 3.5% at 50 µg/mL, 24% at 70 µg/mL and 82% at 90 µg/mL, and the inductivity of the chromosomal aberration was positive and dose-dependent. The incidence in the solvent control was 1.5% within a normal range, while that in the positive control group (MMC) was 69% showing high induction of the chromosomal aberration. The incidence of cells with structural aberration without gap in the 50 µg/mL test substance group was not significantly higher than that in the solvent control group, while those in the 70 and 90 µg/mL test substance groups were significantly higher (P<0.01). The incidence of cells with numerical aberration was 9.5% in the 70 µg/mL test substance group, suspected as positive, but other two groups (50 and 90 µg/mL) were negative. The incidences of these cells in the 50 and 90 µg/mL test substance groups were not significantly different from that in the solvent control group, but that in the 70 µg/mL test substance group was significantly higher (P<0.01).

- 48-hour treatment without metabolic activation: The incidences of cells with structural aberrations were 0.5% in the 50 µg/mL test substance group, 7.0% at 70 µg/mL and 19 5% at 90 µg/mL, which were respectively negative, suspected as positive and positive in the inductivity of chromosomal aberrations. The incidence in the solvent control group was 1.5% within a normal range, and that in the positive control group MMC was 64.5%, showing high induction of chromosomal aberration. The incidence of cells with structural aberration without gap in the 50 µg/mL test substance group was not significantly higher than that in the solvent control group, while incidences in the 70 and 90 µg/mL test substance groups were significantly higher (P<0.01). The incidence of cells with numerical aberration in the 90 µg/mL test substance group was positive as 11%, but the other two groups (50 and 70 µg/mL) were negative. In the 50 and 70 µg/mL test substance groups, there was no significant difference from the solvent control group, though there were significant differences in the 90 µg/mL test substance group (P <0.01).

- 6-hour treatment and 18-hour recovery period with metabolic activation: The incidences of cells with structural aberration including gap were 3% at 100 µg/mL, 25.5% at 200 µg/mL and 59.5% at 300 µg/mL. Therefore, the inductivity of the chromosomal aberration was positive and dose-dependent. The incidence in the solvent control group was 1.5% within the normal range, and that in the positive control group (CPA) was 34%, showing high induction of chromosomal aberrations. The incidence of cells with structural aberration without gap in the 100 µg/mL test substance group was not significantly higher than that in the solvent control group, while the incidences at 200 and 300 µg/mL were significantly higher (P<0.01). Numerical aberration cells appeared in all test substance groups by less than 5% and the inductivity of the chromosomal aberration was negative. There was no significant difference in the 100 and 300 µg/mL groups from the solvent control group, though there was significant difference in the 200 µg/mL test substance group (P<0.01).

- 6-hour treatment and 18-hour recovery period without metabolic activation: To make clear the effects of S9 Mix in the metabolic activation method, the examinations using culture mediums without S9 mix were conducted at the same concentrations of the test substance, the positive control and the solvent control. The incidence of cells with structural aberration including gap at 100 µg/mL was 31.5%, positive in the inductivity of chromosomal aberrations, while any cell division was unable to be observed due to cytotoxicity at 200 or 300 µg/mL. The incidences in the solvent control and the positive control groups were respectively 2.5% and 0.5%, which were within a normal range. The incidences of cells with structural aberration without gap in the 100 µg/mL test substance group was significantly higher than that in the solvent control group (P<0.01). The incidences of cells with numerical aberration in the 100 µg/mL test substance group was 10%, and the induction of chromosomal aberrations was positive. The incidence of numerical aberration cells in the 100 µg/mL test substance group was significantly different from the solvent control group (P <0.01).

- 6-hour treatment and 42-hour recovery period with metabolic activation: The incidences of cells with structural aberration including gap were 3% at 150 µg/mL, 4 at 250 µg/mL and 14% at 350 µg/mL, which was positive at only the maximum concentration. The incidence was 2% in the solvent control group and 4% in the positive control group (CPA), which were within a normal range. The incidences of cells with structural aberration without gap in the 150 and 250 µg/mL test substance groups were not significantly different from the solvent control group, while that in the 350 µg/mL group was significantly different (P<0.01). Numerical aberration cells appeared in all test substance groups by less than 5%. Among them, the incidences of these cells in the 150 and 350 µg/mL test substance groups were significantly different from the solvent control group (P<0.05) and that in the 250 µg/mL group was significantly higher (P<0.01).

- Changes in ploidy (polyploidy cells and cells with end reduplicated chromosomes): Yes

Executive summary:

3-(Trimethoxysilyl)propyl acrylate has been tested for its ability to cause chromosome aberrations in Chinese hamster lung fibroblast cells (CHL) in an in vitro study conducted according to EEC Test Guideline (1991; similar to OECD Test Guideline 473) and in compliance with GLP (reliability score 1). Statistically significant, reproducible, and dose-dependent increases in chromosomal aberrations were seen following 24 and 48-hour treatment without metabolic activation and 6-hour treatment with 18 and 42-hour recovery periods with metabolic activation when tested up to cytotoxic concentration. Appropriate negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations under the conditions of this in vitro mammalian cell study.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Micronucleus assay for 3-(trimethoxysilyl)propyl acrylate: Negative (conducted according to OECD Test Guideline 474 and in compliance with GLP) (reliability score 2, SafePharm Laboratories Limited, 1995f).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14th November 1994 - 7th December 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was conducted according to an older (1981) version of OECD Test Guideline 474, with only a single dose used at the initial sampling time and only 1000 peripheral erythrocytes scored for micronuclei.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian bone marrow chromosome aberration test

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Manston, Kent
- Age at study initiation: 5-8 weeks old
- Weight at study initiation: males 22-29 g (males, females 20-24 g
- Assigned to test groups randomly: Yes
- Fasting period before study: Not specified
- Housing: In groups of 5 per sex
- Diet (e.g. ad libitum): Rat and Mouse Expanded diet No. 1, ad libitum
- Water (e.g. ad libitum): Mains drinking water, ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22
- Humidity (%): 51-53
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Arachis oil
- Justification for choice of solvent/vehicle: Not specified
- Concentration of test material in vehicle: 100 mg/mL
- Dose volume: 10 mL/kg
- Lot/batch no. (if required): 010699
- Purity: Not specified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test material was freshly prepared as required as a suspension at the appropriate concentration in arachis oil.

Duration of treatment / exposure:

Single intraperitoneal dose

Frequency of treatment:

Single exposure

Post exposure period:

24, 48 or 72 hours

Dose / conc.:

1 000 mg/kg bw (total dose)

No. of animals per sex per dose:

5M, 5F

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide Monohydrate
- Justification for choice of positive control(s): The substance is known to produce micronuclei under the conditions of the test.
- Route of administration: Oral dose
- Doses / concentrations: Dose 50 mg/kg bw; concentration 5 mg/mL; dose volume 10 mL/kg

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Based on the findings of a dose range-finding study. The dose tested in the micronucleus test was the maximum tolerated dose.

TREATMENT AND SAMPLING TIMES:
Three groups of 5 male and 5 female mice where each animals was dosed via the intraperitoneal route with the test material at 1000 mg/kg bw. One group of mice was killed at 24 hours following the treatment, the second group was killed at 48 hours and the third at 72 hours post-treatment.

DETAILS OF SLIDE PREPARATION:
Immediately following sacrifice (at 24, 48 and 72 hours), one femur was dissected from each animal, aspirated with foetal calf serum and bone marrow smears prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol and stained in May-Grunwald/Giemsa.

METHOD OF ANALYSIS:
Stained bone marrow smears were coded and examined blind using light microscopy at x1000 magnification. The incidence of micronucleated cells per 1000 PCEs (blue stained immature cells) per animal was scored. In addition, the NCEs (pink stained mature cells) associated with 1000 polychromatic erythrocytes were counted; these cells are also scored for incidence of micronuclei. The ratio of NCEs to PCEs was calculated together with appropriate group mean values for males and females separately and combined.

Evaluation criteria:

A positive mutagenic response is demonstrated when a statistically significant increase in the number of micronucleated polychromatic erythrocytes is observed for either the 24, 48 or 72-hour kill times. If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test. A positive response for bone marrow toxicity is demonstrated when the dose group mean normochromatic to poluchromatic ratio is shown to be statistically significant from the concurrent vehicle control group.

Statistics:

All data were statistically analysed, withg group means, standard deviation, and NCE: PCE ratios calculated and statistical significance determined,

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY:
- Dose range: 400, 800, 1250, 2500 mg/kg bw
- Solubility: Not assessed
- Clinical signs of toxicity in test animals: Clinical signs were observed in animals dosed at and above 1250 mg/kg bw and included lethargy, decreased respiratory rate, laboured respiration, ataxia, pallor of the extremities, ptosis and hunched posture. Premature deaths were seen in one animal dosed at 1250 mg/kg bw and in two animals at 2500 mg/kg bw. Since no clinical signs were seen at 800 mg/kg bw and one premature death seen at 1250 mg/kg bw, the dose level selected as the maximum tolerated dose was 1000 mg/kg bw.
- Evidence of cytotoxicity in tissue analyzed: Not specified
- Rationale for exposure: To determine the suitable dose level for the micronucleus study. The dose level selected should be the maximum tolerated dose level or that which produces some evidence of cytotoxicity up to a maximum dose of 5000 mg/kg bw.
- Harvest times: No tissue harvest performed
- High dose with and without activation: The high dose was 2500 mg/kg bw and metabolic activation was not used in the study.

RESULTS OF DEFINITIVE STUDY:
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not specified
- Induction of micronuclei (for micronucleus assay): There was no statistically significant increase in the frequency of micronucleated PCEs in any of the test material dose groups when compared to the control group.
- Ratio of NCE/PCE (for micronucleus assay): There was no statistically significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent controls, although the presence of clinical signs indicated that systemic absorption had occurred.
- Appropriateness of dose levels and route: Premature deaths were seen for one animal at 48 hours and two at 72 hours. Clinical signs were seen in the dosed animals and included lethargy, decreased respiratory rate, laboured respiration, ataxia and tiptoe gait.
- Statistical evaluation: No statistically significant changes were seen in the dosed groups.

Conclusions:

3-(Trimethoxysilyl)propyl acrylate has been tested for its ability to induce micronuclei in vivo in male and female CD-1 mice in a study conducted according to OECD Test Guideline 474 and in compliance with GLP (reliability score 2). There was no statistically significant increase in the frequency of micronucleated PCEs in any of the test material dose groups when compared to the control group. There was no statistically significant change in the NCE/PCE ratio in any of the test material dose groups when compared to their concurrent controls, although the presence of clinical signs indicated that systemic absorption had occurred. Appropriate negative (treatment medium) and positive 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 this in vivo study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

3-(Trimethoxysilyl)propyl acrylate has been tested in vitro in a bacterial reverse mutation assay, conducted according to OECD Test Guideline 471 (1983) and in compliance with GLP (reliability score 1, Hita Research Laboratories, 1994c), using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2. No increase in the number of revertants was observed in any test strain, with or without metabolic activation, when tested up to cytotoxic concentration. 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 this in vitro test.

 

3-(Trimethoxysilyl)propyl acrylate has been evaluated in vitro for its ability to cause chromosome aberrations in Chinese hamster lung fibroblast cells (CHL) in a study conducted according to EEC Test Guidelines (1991; similar to OECD Test Guideline 473) and in compliance with GLP (reliability score 1, Hita Research Labratories, 1994d). Statistically significant, reproducible, and dose-dependent increases in chromosomal aberrations were seen following 24 and 48-hour treatment without metabolic activation and 6-hour treatment with 18 and 42-hour recovery periods with metabolic activation when tested up to cytotoxic concentration. Appropriate negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is positive for the induction of chromosome aberrations under the conditions of this in vitro mammalian cell study.

 

3-(Trimethoxysilyl)propyl acrylate has been assessed for its ability to induce micronuclei in vivo in male and female CD-1 mice in a study conducted according to OECD Test Guideline 474 and in compliance with GLP (reliability score 2, SafePharm Laboratories Limited, 1995f). There was no statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (PCEs) in any of the test material dose groups when compared to the control group. There was no statistically significant change in the normochromatic erythrocytes (NCE): PCE ratio in any of the test material dose groups when compared to their concurrent controls, although the presence of clinical signs indicated that systemic absorption had occurred. Appropriate negative (treatment medium) and positive 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 this in vivo study.

 

Overall, and based on the findings in the in vivo OECD Test Guideline 474 study, 3-(trimethoxysilyl)propyl acrylate is considered negative for genetic toxicity.

Justification for classification or non-classification

Based on the available data, 3-(trimethoxysilyl)propyl acrylate does not require classification for genetic toxicity according to Regulation (EC) No. 1272/2008.