Trisiloxane, 3-ethyl-1,1,1,3,5,5,5-heptamethyl-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three reliable vitro studies and one reliable in vivo study are available. Based on a weight of evidence approach taking all data available into account, the substance is concluded to have no potential for genotoxicity.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

24 May 2021 to 28 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Specific details on test material used for the study:

Storage conditions: Room temperature in the dark

Target gene:

thymidine kinase, TK

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared by mixing S9 with 100 mM phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% S9-mix concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Tests.

Test concentrations with justification for top dose:

Mutagenicity test (+S9,-S9/both 4 hours): 0; 3.91; 7.81; 15.63; 31.25; 62.5; 125; 250; 500
Due to the expected precipitate of the test item not being observed at the end of the exposure period, this experiment was abandoned prior to plating.

Mutagenicity Test Repeat I (+S9, -S9): 0, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000
Due to the mutant frequency of mid-range dose levels exceeding the Global Evaluation Factor (GEF) in the 4-hour exposure group in the absence of metabolic activation, a repeat of this exposure group was performed as Mutagenicity Test Repeat III to confirm or disprove the responses observed. A repeat of the 4-hour exposure group in the presence of metabolic activation was performed as Mutagenicity Test Repeat II in error. Initially it was considered that the acceptability criteria had not been met when in fact it had.

Mutagenicity Test Repeat II (+S9/4hours): 0,7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000
Due to the experiment being performed in error, this experiment was abandoned prior to plating.

Mutagenicity Test Repeat III (-S9): 0, 15.63, 31.25, 62.5, 93.75, 125, 187.5

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Absence of S9 mix

Details on test system and experimental conditions:

Preliminary toxicity test:
A preliminary toxicity test was performed on cell cultures at 5 x 10E5 cells/mL, using a 4-hour exposure period both with and without metabolic activation (S9). The dose range used in the preliminary toxicity test was 3.91 to 1000 µg/mL. The maximum concentration was the maximum practical concentration. Following the exposure periods the cells were washed twice with R10, resuspended in R20 medium, counted and then serially diluted to 2 x 10E5 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 10E5 cells/mL. After a further 24 hours, the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post exposure toxicity, and a comparison of each exposure SG value to the concurrent solvent control performed to give a percentage Relative Suspension Growth (%RSG) value. Results from the preliminary toxicity test were used to set the test item concentrations for the mutagenicity experiments.

Mutagenicity Tests:
Several days before starting each experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. The cells were counted and processed to give 1 x 10E6 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals for the 4-hour exposure groups in both the absence and presence of metabolic activation, where applicable. The exposures were performed in duplicate (A + B), both with and without metabolic activation (2% S9 final concentration), where applicable, at eight concentrations of the test item and solvent and positive controls. To each universal was added 2 mL of S9-mix if required, 0.1 mL of the exposure dilutions, (0.2 mL or 0.15 mL for the positive controls), and sufficient R0 medium to bring the total volume to 20 mL.
The concentrations of test item used were 3.91 to 500 µg/mL in the Main Experiment, 7.81 to 1000 µg/mL for the Main Experiment Repeat I and Repeat II, and 7.81 to 250 µg/mL in the Main Experiment Repeat III. During exposure to the test item cultures were shaken continuously.

Evaluation criteria:

Assessments:
- Measurement of Survival, Cloning Efficiency and Mutant Frequency;
- Plate scoring;
- Calculation of Percentage Relative Suspension Growth (%RSG);
- Calculation of Cloning Efficiency (%V);
- Calculation of Relative Total Growth (RTG);
- Calculation of Mutant Frequency (MF).

Statistics:

Not performed.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main Experiment Repeat I: Precipitate of test item was observed at and above 250 µg/mL at the end of the exposure period in both the absence and presence of metabolic activation. The test item did not induce any increases in the mutant frequency at any of the concentrations in the presence of metabolic activation test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating concentration, and at least four analysable concentrations, as recommended by the OECD 490 guideline. The mean mutant frequencies for the test item treated cultures were all within the laboratory 95% control limits of historical solvent control data. The results observed in the presence of metabolic activation were considered to fulfil the criteria for a clearly negative outcome. Increases in mutant frequency that did exceed the GEF were observed mid-dose range in the absence of metabolic activation. The increases in mutant frequency observed exceeded 95% control limits of historical solvent control data, and were statistically significant but not concentration-related when evaluated with a trend test.

Main Experiment Repeat III: Precipitate of test item was observed at and above 187.5 µg/mL at the end of the exposure period in the absence of metabolic activation. The test item did not induce any increases in the mutant frequency at any of the concentrations in the absence of metabolic activation test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating concentration, and at least four analysable concentrations, as recommended by the OECD 490 guideline. The statistically significant increases observed in the previous experiment were therefore considered to be spurious and of no toxicological significance.

Conclusions:

In an In Vitro Mammalian Cell Gene Mutation Test, performed according to OECD/EC guidelines and GLP principles, the test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

The mutagenicity potential of 3-Ethylheptamethyltrisiloxane was assessed according to the OECD 490 guideline “In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene”.

 

The test item concentrations ranged from 0 to 1000 μg/mL in the two main experiments. Acetone was used as a solvent and negative control. Cells were exposed to the test item and negative control for a period of 4 hours in both presence and absence of S9 mix.

 

The solvent control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive controls produced marked increases in the mutant frequency per viable cell achieving the acceptability criterion.

The test item was tested up to and exceeding precipitating concentrations and did not induce any increases in the mutant frequency at any of the concentrations in the absence of metabolic activation test that exceeded the Global Evaluation Factor (GEF), using a dose range that included the lowest precipitating concentration, and at least four analysable concentrations. The mean mutant frequencies for the test item treated cultures were all within the laboratory 95% control limits of historical solvent control data. The results observed in the absence of metabolic activation were considered to fulfil the criteria for a clearly negative outcome.

In main experiment I, increases in mutant frequency that did exceed the GEF were observed mid-dose range in the absence of metabolic activation. The increases in mutant frequency observed were statistically significant but were not concentration-related when evaluated with a trend test.

The statistically significant increases observed in the Main experiment I were considered to be spurious and of no toxicological significance as the same was not observed in the repeat experiment.

 

Under the conditions of this study, 3-Ethylheptamethyltrisiloxane did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 March 2003 to 25 April 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

No confirmatory experiment conducted, but there is no indication of increases in the number of revertants in the data presented.

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced S9

Test concentrations with justification for top dose:

Concentration range in the absence and in the presence of metabolic activation: 0, 62, 185, 556, 1667 and 5000 µg/plate

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Remarks:

Ethanol

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

ethylnitrosurea

other: 2-aminoanthracene

Details on test system and experimental conditions:

The mutagenicity assay was carried out as follows. To 2 mL molten top agar (containing 0.6% agar, 0.5% NaCI and 0.05 mM L-histidine HCI/0.05 mM biotin for the Salmonella typhimurium strains, and supplemented with 0.05 mM tryptophan for the E coli WP2 uvrA strain), maintained at ca 46°C, were added subsequently: 0 1 mL of a fully grown culture of the appropriate strain, 0.1 mL of the appropriate test substance solution, or of the negative or positive control substance solution, and 0.5 mL S9-mix for treatments with metabolic activation or 0.5 mL sodium phosphate 100mM (pH 7.4) for treatments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37°C for 48-72 hours, Subsequently, the his+ and trp+ revertants were counted. The background lawn of bacterial growth was examined microscopically to detemine any growth-diminishing or growth enhancing effects by the test substance, if a two-fold or greater increase in the mean number of his+ or trp+ revertants was observed. Cytotoxicity is defined as a reduction in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth.

Rationale for test conditions:

Highest concentration tested (5000 µg/plate) as per OECD guideline.

Evaluation criteria:

The mutagenicity study was considered valid if the mean colony counts of the control values of the strains were within acceptable ranges, if the results of the positive controls meet the criteria for a positive response, and if no more than 5 % of the plates were lost through contamination or other unforeseen events.

A test substance was considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates showed concentration-related increases or if a reproducible two-fold or more increase was observed compared to that on the negative control plates.

A test substance was considered to be negative in the bacterial gene mutation test if it produced neither a dose~related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

In case of an inconclusive first assay, a second independent assay was conducted. The first mutagenicity assay was regarded inconclusive if a positive or equivocal response at only one concentration was observed or if a positive or equivocal responses at several concentrations without a concentration-related increase were observed.

Omission of the second assay under these conditions is considered to be acceptable as a single assay does not or hardly results in false negative conclusions (TNO historical data and Kirkland and Dean, 1994).

Statistics:

No statistical analysis performed

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

not valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The original data for strain TA98 in the presence of metabolic activation were presented in the report but since the number of revertant colonies exceeded the historical control ranges, these treatments were repeated.

Results in the absence of metabolic activation

Concentration (µg/plate)	Mean revertant count±standard deviation
	Strain
	TA98	TA100	TA1535	TA1537	WP2 uvrA
0 (ethanol)	49± 7	164± 10	28± 3	14± 4	42± 5
62	44± 6	162± 2	28± 2	15± 3	46± 6
185	43± 10	176± 13	31± 11	13± 4	48± 5
556	52± 6	185± 18	27± 7	12± 3	52± 6
1667	57± 8	170± 12	29± 3	10± 4	45± 15
5000	47± 14	176± 8	33± 6	15± 8	40± 9
Positive control	1646± 72	631± 21	524± 24	1605± 375	229± 33

 

Results in the presence of metabolic activation

Concentration (µg/plate)	Mean revertant count±standard deviation
	Strain
	TA98	TA100	TA1535	TA1537	WP2 uvrA
0 (ethanol)	69± 12	172± 3	28± 7	19± 4	47± 1
62	71± 1	162± 21	31± 5	20± 8	47± 5
185	72± 1	164± 8	39± 20	18± 5	43± 4
556	78± 11	186± 4	34± 4	17± 4	50± 9
1667	75± 9	191± 17	29± 7	24± 7	39± 9
5000	73± 8	192± 23	39± 4	16± 4	45± 6
Positive control	1476± 185	3115± 150	572± 45	310± 42	1837± 188

Strain TA98 treatments in the presence of metabolic activation are from a repeat experiment.

Conclusions:

Based on the results of an AMES study with the test substance in five Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and one Escherichia coli strain (WP2 uvrA), in both the absence and the presence of the S9-mix, it is conclued that Heptamethylethyltrisiloxane is not mutagenic when tested up to a concentration of 5000 µg/plate (the maximum recommended dose in accordance with current in vitro genotoxicity regulatory guidelines) under the conditions employed in this study.

Executive summary:

A GLP compliant study has been conducted with Heptamethylethyltrisiloxane based on OECD test guideline 471 and TA1537 and Escherichia coli strain WP2 uvrA were tested in the absence and in the presence of Acroclor 1254 induced S9 at concentrations of 62, 185, 556, 1667 and 5000 µg/plate using a plate incorporation methodology. There was no clear indication of toxicity (reduction in the number of revertants) and there were no increases in the number of revertant colonies for any concentration tested for any strain in the absence or in the presence of metabolic activation when compared to the concurrent controls.

It is concluded that the results obtained with the test substance in five Salmonella typhimurium strains (TA1535, TA1537, TA98 and TA100) and one Escherichia coli strain (WP2 uvrA), in both the absence and the presence of the S9-mix, indicate that Heptamethylethyltrisiloxane was not mutagenic when tested up to a concentration of 5000 µg/plate (the maximum recommended dose inaccordance with current in vitro genotoxicity regulatory guidelines) under the conditions employed in this study.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 March 2008 to 09 April 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Principles of method if other than guideline:

The number of metaphases scored for each concentration was 200, as per the guideline at the time of study conduct. This has been since been revised to 300 metaphases per concentration.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Storage: Room temperature; protected from light under desiccant

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

Human cells obtained from a healthy non-smoking 27-year-old adult female

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced S-9, rat

Test concentrations with justification for top dose:

Concentrations of 0.005,0.01,0.025,0.05,0.075,0.1,0.2 µL/mL were tested in the absence of metabolic acitvation for 4 hours with 16 hours recovery
Concentrations of 0.005,0.01,0.025,0.05,0.075,0.1,0.2 µL/mL were tested in the absence of metabolic acitvation for 20 hours with no recovery
Concentrations of 0.01, 0.025, 0.05, 0.1, 0.15, 0.2 were tested in the presence of metabolic acitvation for 4 hours with 16 hours recovery

Vehicle / solvent:

Ethanol

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Peripheral blood lymphocytes were obtained from single healthy non-smoking female donor for the preliminary and main experiments.

An initial preliminary toxicity test was performed. Approximately 0.6 mL heparinized blood was inoculated into centrifuge tubes containing 9.4 mL RPMI-1640 complete medium supplemented with 1% phytohemagglutinin
(PHA). The tubes were incubated at 37±1 °C in a humidified atmosphere of 5±1% carbon dioxide in air for 44-48 hours. At the time of test article treatment, the culture tubes were centrifuged, the supernatant was aspirated, and the cells were resuspended in either 10 mL of fresh RPMI-1640 complete medium for the treatments in the absence of metabolic activation or 10 mL S9 reaction mixture (8 mL serum free medium + 2 mL of S9 cofactor pool) for treatments in the presence of metabolic activation, to which was added 50 µL test article dosing solution in solvent or solvent alone (vehicle control). The osmolality in treatment medium of the solvent and of the highest test article concentration were measured. The pH of the highest concentration of dosing solution in the treatment medium was measured using test tape.

In the preliminary test, the cells were exposed to solvent alone and to nine concentrations of the test article for 4 hours in both the presence and absence of S9 activation, and for 20 hours continuously in the absence of S9 activation. The cells were incubated at 37±1°C in a humidified atmosphere of 5±1% carbon dioxide in air. For the 4 hour treatments, at the completion of the 4-hour exposure period, the treatment medium was removed, the cells washed with calcium and magnesium-free phosphate buffered saline, refed with RPMI-1640 complete medium and returned to the incubator for an additional 16 hours. For all treatments, two hours prior to the scheduled cell harvest, Colcemid® was added to the cultures at a final concentration of 0.1 µg/mL and the cultures were returned to the incubator until cell collection. Cells were collected by centrifugation, treated with hypotonic potassium chloride (0.075M KCI), fixed with methanol:glacial acetic acid (3:1 v/v), stained with 5% Giemsa and the number of cells in mitosis per 500 cells scored was determined in order to evaluate test article effect on mitotic index.

In the main experiment, the treatments were conducted as per the preliminary experiment, with the exception that duplicate cultures were used, concentrations were adjusted and positive controls were also included.

For the analysis of chromosome aberrations, slides were coded using random numbers by an individual not involved with the scoring process. Metaphase cells with 46 centromeres were examined under oil immersion without prior knowledge of treatment groups. Whenever possible, a minimum of 200 metaphase spreads (100 per duplicate treatment condition) were examined and scored for chromatid-type and chromosome-type aberrations. The number of metaphase spreads that were examined and scored per duplicate flask was reduced when the percentage of aberrant cells reached a significant level (at least 10%) before 100 cells are scored. Chromatid-type aberrations include chromatid and isochromatid breaks and exchange figures such as
quadriradials (symmetrical and asymmetrical interchanges), triradials, and complex rearrangements. Chromosome-type aberrations include chromosome breaks and exchange figures such as dicentrics and rings. Fragments (chromatid or acentric) observed in the absence of any exchange figure were scored as a break (chromatid or chromosome). Fragments observed with an exchange figure were not scored as an aberration but instead were considered part of the incomplete exchange. Pulverized chromosome(s), pulverized cells and severely damaged cells (= 10 aberrations) were also recorded. Chromatid and isochromatid gaps were recorded but not included in the analysis. The XY coordinates for
each cell with chromosomal aberrations were recorded using the microscope stage. The percent polyploid and endoreduplicated cells was evaluated per 100 cells.

Evaluation criteria:

The toxic effects of treatment are based upon mitotic inhibition relative to the solvent-treated control and are presented for the preliminary toxicity test and the chromosome aberration assay. The number and types of aberrations per cell, the percentage of structurally and numerically damaged cells (percent aberrant cells), and the frequency of structural aberrations per cell (mean aberrations per cell) in the total population of cells examined was calculated and reported for each treatment group. Chromatid and isochromatid gaps were not included in the total percentage of cells with one or more aberrations or in the
frequency of structural aberrations per cell.

All conclusions were based on sound scientific judgement; however, as a guide to interpretation of the data, the test article was considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner with one or more concentrations being statistically elevated relative to the solvent control group (p= 0.05). However, values that are statistically significant but do not exceed the range of historical solvent controls may be judged as not biologically significant. Test articles not demonstrating a statistically significant increase in aberrations will be concluded to be negative.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test article dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Species / strain:

lymphocytes: Human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The chromosome aberration assay indicates no significant increase in structural and numerical chromosome aberrations in the test article-treated groups relative to the respective solvent controls in both presence and absence of metabolic activation system when tested up to a concentration . The percent aberrant cells in the test article-treated groups were within the historical solvent control range. The results for the positive and negative controls indicate that all criteria for valid assay were met. Based on these criteria, the negative result is justified and does not require a repeat of any portions of the study.

Preliminary test: Toxicity in the absence and in the presence of metabolic activation

	4-hours in the absence of metabolic activation		20 hours in the presence of metabolic activation		4-hours in the presence of metabolic activation
Concentration (µL/mL)	Mitotic cells/500 cells	Percent change (%)	Mitotic cells/500 cells	Percent change (%)	Mitotic cells/500 cells	Percent change (%)
0 - ethanol	6.6	-	7.6	-	7.6	-
0.0005	6.0	-9	6.8	-11	7.0	-8
0.0015	5.8	-12	6.8	-11	6.6	-13
0.005	6.2	-6	5.8	-24	6.8	-11
0.015	6.4	-3	6.2	-18	6.2	-18
0.05	2.4	-64	1.0	-87	6.6	-13
0.15	1.6	-76	1.8	-76	3.6	-53
0.5	0.4	-94	1.0	-87	1.2	-84
1.5	0.2	-97	1.6	-79	1.4	-82
5	3.2	-52	6.6	-13	4.2	-45

 

Main experiment: 4 hours in the absence of metabolic activation

Concentration (µL/mL)	Replicate	Mitotic index (%)	Toxicity (%)	Number of cells scored for numerical aberrations	Number of cells scored for structural aberrations	% numerical aberrations	% structural aberrations	gaps	ctb	cte	csb	dic	ring	Multi	Mean aberrations per cell
0 - ethanol	A	11.2	-	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	11.6		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	11.4		200
0.01	A	9.6	12	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	10.4		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	10.0		200
0.025	A	9.0	22	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	8.8		100	100	0	1	0	1	0	0	0	0	0	0.010
	total	8.9		200
0.1	A	5.4	54	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	5.0		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	5.2		200
Positive control	A	4.6	59	100	50	0	18	0	7	5	0	0	0	0	0.240
	B	4.8		100	50	0	16	0	8	5	0	0	0	0	0.260
	total	4.7		200	100

ctb: Chromatid break
cte: Chromatid exchange
csb: Chromosome break
dic: Dicentric chromosome
ring: Chromosome ring
multi: Multiple aberrations (>10 aberrations in a metaphase)

Main experiment: 4 hours in the absence of metabolic activation

Concentration (µL/mL)	Replicate	Mitotic index (%)	Toxicity (%)	Number of cells scored for numerical aberrations	Number of cells scored for structural aberrations	% numerical aberrations	% structural aberrations	gaps	ctb	cte	csb	dic	ring	Multi	Mean aberrations per cell
0 - ethanol	A	7.2	-	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	7.0		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	7.1		200
0.025	A	5.8	17	100	100	0	0	1	0	0	0	0	0	0	0.000
	B	6.0		100	100	0	1	0	0	1	0	0	0	0	0.010
	total	5.9		200
0.05	A	5.2	25	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	5.4		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	5.3		200
0.1	A	3.2	54	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	3.4		100	100	1	0	0	0	0	0	0	0	0	0.000
	total	3.3		200
Positive control	A	4.8	35	100	50	0	20	1	7	6	0	0	0	0	0.260
	B	4.4		100	50	0	18	1	7	3	0	0	0	0	0.200
	total	4.6		200	100

ctb: Chromatid break
cte: Chromatid exchange
csb: Chromosome break
dic: Dicentric chromosome
ring: Chromosome ring
multi: Multiple aberrations (>10 aberrations in a metaphase)

Main experiment: 20 hours in the absence of metabolic activation

Concentration (µL/mL)	Replicate	Mitotic index (%)	Toxicity (%)	Number of cells scored for numerical aberrations	Number of cells scored for structural aberrations	% numerical aberrations	% structural aberrations	gaps	ctb	cte	csb	dic	ring	Multi	Mean aberrations per cell
0 - ethanol	A	12.6	-	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	12.4		100	100	0	0	1	0	0	0	0	0	0	0.000
	total	12.5		200
0.01	A	11.6	11	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	10.6		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	11.1		200
0.025	A	11.0	10	100	100	0	0	1	0	0	0	0	0	0	0.000
	B	11.4		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	11.2		200
0.05	A	5.4	55	100	100	0	0	0	0	0	0	0	0	0	0.000
	B	5.8		100	100	0	0	0	0	0	0	0	0	0	0.000
	total	5.6		200
Positive control	A	4.8	58	100	50	0	18	0	8	2	0	0	0	0	0.200
	B	5.6		100	50	0	18	0	12	1	0	0	1	0	0.280
	total	5.2		200	100

ctb: Chromatid break
cte: Chromatid exchange
csb: Chromosome break
dic: Dicentric chromosome
ring: Chromosome ring
multi: Multiple aberrations (>10 aberrations in a metaphase)

Conclusions:

In a chromosome aberration study, performed according to the OECD guideline and under GLP principles, 3-ethylheptamethyltrisiloxane did not show an increase in the incidence of structural or numerical chromosomal aberrations in isolated human lymphocytes. These conditions included treatments that were limited by an acceptable level of toxicity in the absence (4 and 20 hours) or presence (4 hours) of S9 fraction of a rat liver metabolic activation system.

Executive summary:

A GLP compliant study has been conducted with 3-ethylheptamethyltrisiloxane based on OECD test guideline 471 using human peripheral blood lymphocytes. Cells were treated with a range of concentrations up to 5 µL/mL for 4 hours in the absence and in the presence of metabolic activation with 16 hours recovery and for 20 hours in the absence of metabolic activation with no recovery. Concentrations of 0.01, 0.025 and 0.1 µL/mL were analysed for aberrations in the absence of metabolic activation for the 4 hour treatments; concentrations of 0.025, 0.05 and 0.1 µL/mL were analysed for aberrations in the absence of metabolic activation for the 20 hour treatments; concentrations of 0.025, 0.05 and 0.1 µL/mL were analysed for aberrations in the presence of metabolic activation. Toxicity was observed in all test conditions. No increases in the number of aberrations were observed for any of the treatment regimes. The positive control gave statistically significant increases in the number of aberrant cells, demonstrating the sensitivity of the test system. The overall conclusion of the study is that 3-ethylheptamethyltrisiloxane is not clastogenic in the absence or in the presence of metabolic activation following testing in human peripheral blood lymphocytes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Three reliable vitro and one reliable in vivo studies are available. Based on a weight of evidence approach taking all data available into account, the substance is concluded to have no potential for genotoxicity.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 March 2008 to 13 April 2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Deviations:

yes

Remarks:

refer to "Overall Remarks"

Principles of method if other than guideline:

Current guideline requirements require 150 cells to be scored/animal and that the historical control ranges presented should use C-charts, C-bar charts to identify the variability in the data set. This has not been undertaken.

GLP compliance:

yes

Type of assay:

mammalian comet assay

Specific details on test material used for the study:

Stability of test compound: Confirmed stable for the duration of the study

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Commonly used strain that has been used for many years

Sex:

male

Details on test animals or test system and environmental conditions:

Test animals:
Species: rat
Strain: Wistar
Age: 7-9 wks at dosing
Weight at dosing: 218-236g
Source: Harlan, Frederick
Acclimation period: 5 days
Diet: Harlan 2018C Certified Global Rodent Diet, ad libitum
Water: Municipal water, ad libitum
Housing: Five animals of the same sex/cage

Environmental conditions:
Temperature: 19-25°C
Humidity: 30-70%
Air changes: not stated
Photoperiod: 12h light/dark cycle

Route of administration:

oral: gavage

Vehicle:

Corn oil (dose volume 10 mL/kg bw)

Details on exposure:

Preparation of dosing solutions:
The test article was prepared and diluted in corn oil before treatment.

Duration of treatment / exposure:

Single oral dose

Frequency of treatment:

single

Post exposure period:

sampling of liver and stomach 4 and 24 h post a single oral dose

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Sacrifice time: 4 hours: 0, 1000, 15000, 2000 mg/kg bw, MMS: 50 mg/kg bw; No. of animals: 5/gp
Sacrifice time: 24 hours: 0, 1000, 1500, 2000 mg/kg bw; No. of animals: 5/gp

Control animals:

yes, concurrent vehicle

Positive control(s):

Methylmethanesulphonate (MMS) a widely used postive control for comet induction was used. A single oral dose, via gavage at 50 mg/kg bw (employing a dose volume of 10 mL/kg bw) was used

Tissues and cell types examined:

Hepatocytes and stomach epithelial cells were isolated and a single cell preparation was prepared.

Details of tissue and slide preparation:

Details of tissue preparation:
- Liver:
The hepatocytes were isolated from the liver and the cell suspension was strained using a cell strainer and then kept on ice until slide preparation. The number of isolated living cells was determined by the trypan blue dye exclusion test.

- Stomach:
Stomachs were removed and cut open and washed free from food using mincing buffer. The forestomach was cut, removed and discarded. The surface epithelia of the glandular portion of the stomach was gently scraped using a scrapper. This layer was discarded and the gastric mucosa was rinsed with the cold mincing buffer. Then the stomach epithelium was carefully scraped 4-5 times with a scrapper. The cell suspension was then strained using a cell strainer and kept on ice until preparation of slides. The number of isolated living cells was determined by the trypan blue dye exclusion test.

Slide preparation:
From each liver and stomach cell suspension an aliquot of 5 uL and 10 uL, respectively were mixed with low melting agar and the cell/agarose suspension was applied to glass microscope slides previously coated with normal melting agarose.

Lyses:
After the agarose gel has solidified, the slides were placed overnight at 2-8 °C, in a lysis solution consisting of high salts and detergents. The Iysing solution was chilled prior to use, primarily to maintain the stability of the agarose gel.

Unwinding:
After cell lysis two slides for each organ/animal were washed with neutralisation buffer and place in the electrophoresis chamber. The chamber was filled with alkaline buffer and the slides remained in the buffer for 20 minutes to allow DNA to unwind.

Electrophoresis:
After alkali unwinding, the single-stranded DNA in the gels was electrophoresed under alkaline conditions to produce comets. The electrophoretic conditions were 0.7 V/cm for 30 minutes at room temperature (15-30°).

Neutralization and dehydration of slides:
After electrophoresis, the alkali in the gels were neutralized by rinsing the slides with neutralisation buffer and then dehydrated with 100% ethanol, air dried and stored at room temperature.

Staining:
Slides were stained with Sybr-gold prior to scoring.

Scoring:
Fifty randomly selected, non-overlapping cells/slide were scored for DNA damage using the Comet Assay IV v.4.11 Perceptive Instruments software.

DNA damage was assessed by the software system by measuring:
- Comet migration: distance from the perimeter of the comet head to the last visible point in the tail
- %Tail DNA: % tail intensity, the % of DNA present in the tail
- Olive tail moment: product of tail length and %DNA in tail
Each slide was also assessed for possible indications of cytotoxicity, i.e. presence of clouds (termed hedgehogs). The rough estimate of the %clouds was recorded for each slide.

DNA damage evaluation:
Median of 100 counts of %tail DNA, Olive tail moment and tail migration were determined and presented for each animal in each treatment group for each organ. The mean and standard deviation of the median values for %tail DNA and Olive tail moment were presented for each treatment group for each organ.

Evaluation criteria:

The test article was considered to induce DNA damage if:
- A least one of the test doses exhibited a statistically significant increase in tail intensity, compared with the concurrent vehicle control
- The increase was dose related
The test article was considered negative in this assay if neither of the above criteria were met and target tissue was confirmed.

Statistics:

Levene's test was performed to determine heterogenous group variances (p<0.05). Where evidence of heterogeneity was present, the data were rank transformed prior to analysis. Linear regression was used to determine the dose response relationship for the significance level (p<0.01).

Sex:

male

Genotoxicity:

negative

Remarks:

Liver

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

not determinable because of methodological limitations

Remarks:

No evidence of target organ exposure demonstrated

Sex:

male

Genotoxicity:

negative

Remarks:

Stomach

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Analytical determinations:
Analysis of Y-14877 in the dosing formulations met the acceptance criteria of 85-115% of target concentration and an RSD of <5%. No test article was detected in the vehicle control.

 Comet assay:

1. Clinical observations: No test article related clinical signs of toxicity were observed.

2. Body weight: No test article related effects on body weight were observed.

3. Toxicity: No test article related toxicity, as evident by hedgehog occurrence on comet slides were observed.

4. Clinical chemistry: not undertaken

5. Necropsy and pathology: Both macroscopic and microscopic observations were deemed to be unremarkable.

6. Target tissue exposure: Concurrent target organ (liver) exposure was not demonstrated. Whilst target exposure to the stomach was not demonstrated, it was not considered necessary as the stomach was the first site of contact following oral gavage dosing directly into the stomach.

7. Comet analysis:

- Liver: The presence of hedgehogs was <3% and <4% for the liver at 4 and 24 hour time points, respectively. Group mean %tail intensity and tail moment values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail intensity (or tail moment values) between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range (refer to Table CA 7.6.2/03-1).



-Stomach: The presence of hedgehogs was <27% for the stomach at both time points. The increase in hedgehog values is typical for the stomach due to the mechanical harvesting of stomach epithelium cells required (i.e. scraping of the stomach mucosal epithelia, prior to epithelium cell harvesting).. Group mean %tail intensity values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range.

 

Group mean tail moment values of animals treated with Y-14877 showed a statistically significant decrease at the 4 h sample time point. Typically, decreases in comet parameters are deemed indicative of a cross-linking effects, however the standard comet assay needs to be modified to detect such effects, with such effects not being robustly determined in a standard assay. The statistically significant decrease in tail moment values were limited to the 4 h sample time point, were not replicated at the 24 h nor replicated in the %tail intensity values and were not dose related. Therefore, it can be concluded that the decrease in tail moment values were deemed not biologically relevant (refer to Table CA 7.6.2/03-2).

 

Cell viability (assessed by trypan blue exclusion) for the test article-treated liver and stomach cells was <97% for both time points.

 The positive controls induced an acceptable increase in %tail intensity in both liver and stomach, thereby demonstrating the sensitivity and specificity of the test system.

Table CA 7.6.2/03-1:
Group mean and individual animal liver data

Dose level (mg/kg bw)	No. of animals	No .of cells scored	Mean tail intensity^a(% ±SD)		Mean tail moment^a(% ±SD)		Mean % hedgehogs
4 hour sample time
0	5	500	1.89 ±1.18		0.42 ±0.23		---^b
500	5	500	1.71 ±0.89		0.39 ±0.20		---^b
1000	5	500	3.42 ±1.84		0.62 ±0.32		---^b
2000	5	500	1.90 ±1.18		0.40 ±0.22		---^b
MMS, 50	5	500	60.74 ±4.46*		23.13 ±4.30*		---^b
24 hour sample time
0	5	500	0.70 ±0.41		0.15 ±0.08		---^b
500	5	500	1.07 ±0.82		0.23 ±0.13		---^b
1000	5	500	0.7 ±0.25		0.17 ±0.04		---^b
2000	5	500	1.38 ±0.42		0.30 ±0.10		---^b
Historical control data ranges for rat liver comet assay data collected between 2004 - 2007
Vehicle	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			4.40 ±4.23	Mean:	1.03 ±1.07
	Observed range:			0.14 – 10.49	Observed range:	0.02 – 3.33
Positive	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			50.13 ±22.18	Mean:	18.94 ±13.01
	Observed range:			16.86 – 78.68	Observed range:	3.08 – 38.82
*: p<0.05, statistically significant increase +ve control: EMS – ethyl methylsulphonate a   median values of each slide calculated. The mean of the slide medians were calculated to give the individual mean animal value. The individual mean animal values were averaged to provide group means b   no numerical values presented for each animal / group

Table CA 7.6.2/03-2:
Group mean and individual animal stomach data

Dose level (mg/kg bw)	No. of animals	No .of cells scored	Mean tail intensity^a(% ±SD)		Mean tail moment^a(±SD)		Mean % hedgehogs
4 hour sample time
0	5	500	24.37 ±3.90		7.54 ±1.41		---^b
500	5	500	17.55 ±3.62		4.89 ±1.14**		---^b
1000	5	500	15.44 ±1.63		3.94 ±0.61**		---^b
2000	5	500	17.47 ±6.02		4.42 ±1.69**		---^b
MMS, 50	5	500	77.59 ±4.47*		40.80 ±5.26*		---^b
24 hour sample time
0	5	500	5.83 ±2.18		1.39 ±0.51		---^b
500	5	500	12.45 ±12.39		3.57 ±4.06		---^b
1000	5	500	10.00 ±5.03		2.32 ±1.09		---^b
2000	5	500	6.48 ±1.75		1.61 ±0.35		---^b
Historical control data ranges for rat liver comet assay data collected between 2004 - 2007
Vehicle	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			14.11 ±7.65	Mean:	4.63 ±3.48
	Observed range:			7.41 – 26.90	Observed range:	1.75 – 11.40
Positive	Mean tail intensity(% ±SD)				Olive tail moment (±SD)
	Mean:			50.15 ±22.99	Mean:	20.06 ±16.86
	Observed range:			30.61 – 75.53	Observed range:	7.22 – 43.24
*: p<0.05, statistically significant increase, **: p<0.05, statistically significant decrease +ve control: MMS – methyl methanesulphonate a   median values of each slide calculated. The mean of the slide medians were calculated to give the individual mean animal value. The individual mean animal values were averaged to provide group means b   no numerical values presented for each animal / group

Conclusions:

Based on the results of a reliable Comet assay it is concluded that 3-ethylheptamethyltrisiloxane does not induce DNA damage in stomach and liver cells of male rats following a single oral gavage dose of 2000 mg/kg bw/day, the regulatory maximum recommended dose in accordance with currently regulatory guidelines for short-term in vivo toxicity testing. Although systemic exposure was not demonstrated in this study. based on the full data-set it is reasonable to assume that systemic exposure took place.

Executive summary:

In a liver and stomach comet assay, Sprague Dawley rats (5/group) received a single oral dose of Y-14877 suspended in corn oil (employing a dose volume of 10 mL/kg bw). Doses selected for the comet assay were 0, 1000, 1500 and 2000 mg/kg bw. Liver and stomach were sampled at 4 and 24 hours post dose. A further group of animals (5/sex/group) received a single oral dose of methyl methane sulphonate, with liver and stomach sampled at 4 hour post dosing.

 

The presence of hedgehogs was <3% and <4% for the liver at 4 and 24 hour time points, respectively. Group mean %tail intensity and tail moment values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail intensity (or tail moment values) between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range

 

The presence of hedgehogs was <27% for the stomach at both time points. The increase in hedgehog values is typical for the stomach due to the mechanical harvesting of stomach epithelium cells required (i.e. scraping of the stomach mucosal epithelia, prior to epithelium cell harvesting).. Group mean %tail intensity values for all groups of animals treated with Y-14877 were similar to the group mean vehicle control data. There were no statistically significant differences in %tail between treated and the control group at either time point. All individual animal data at all dose levels were consistent with the vehicle control animals and were generally comparable with the laboratory’s historical control observed range.

 

Group mean tail moment values of animals treated with Y-14877 showed a statistically significant decrease at the 4 h sample time point. Typically, decreases in comet parameters are deemed indicative of a cross-linking effects, however the standard comet assay needs to be modified to detect such effects, with such effects not being robustly determined in a standard assay. The statistically significant decrease in tail moment values were limited to the 4 h sample time point, were not replicated at the 24 h nor replicated in the %tail intensity values and were not dose related. Therefore, it can be concluded that the decrease in tail moment values were deemed not biologically relevant (refer to Table CA 7.6.2/03-2).

 

Cell viability (assessed by trypan blue exclusion) for the test article-treated liver and stomach cells was <97% for both time points.

 

The positive controls induced an acceptable increase in %tail intensity in both liver and stomach, thereby demonstrating the sensitivity and specificity of the test system.

It is concluded that Y-14877 did not induce DNA damage in the stomach of male rats following a single oral gavage dose, with harvesting of stomach tissue at 4 and 24 hours later. The maximum dose administered was 2000 mg/kg bw/day, the regulatory maximum recommended dose in accordance with currently regulatory guidelines for short-termi n vivo toxicity testing.

 

Although systemic exposure was not demonstrated in this study. based on the full data-set it is reasonable to assume that systemic exposure took place.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to CLP (1272/2008/EC) classification criteria for genetic toxicity, no classification is warranted.