2,4-Hexadienoic acid, 3-(trimethoxysilyl)propyl ester, (2E,4E)-

Endpoint summary

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

Reference

Reference 1

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

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

Key result

Species / strain:

S. typhimurium TA 1537

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

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

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

Key result

Species / strain:

E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

positive

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 (NaN₃)	-	-	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 (NaN₃)	-	-	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

NaN₃      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.

Reference 2

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.

Reference 3

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).

 

The analogous substance CAS 2530-85-0 comet assay study in rat: negative (similar to OECD 489) (Bioreliance, 2018).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

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

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 or test system 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.

Reference 2

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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

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 read across from available data for structural analogue, 3-trimethoxysilylpropyl methacrylate (CAS 2530-85-0 EC No. 219-785-8). Both the source substance and the target substance have rapid hydrolysis rates, producing similar hydrolysis products, 3-(trihydroxysilyl)propyl-(2E,4E)-2,4-hexadienoate (for the target) and 3-(trihydroxysilyl)propyl methacrylate (for the source), and methanol. Please see the read-across justification document attached in Section 13 of the IUCLID dataset for details of the read-across.

 

The structural analogue, 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 in vitro and in vivo genotoxicity data for the submission substance and in vivo genotoxicity data for the structural analogue, 3-(trimethoxysilyl)propyl-(2E,4E)-hexa-2,4 -dienoate  does not require classification for mutagenicity according to Regulation (EC) No 1272/2008.