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

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 2008

Deviations:

no

Test guideline 3

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

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

469-070-1

EC Name:

-

Cas Number:

17861-60-8

Molecular formula:

C9H26O2Si3

IUPAC Name:

4-ethyl-2,2,4,6,6-pentamethyl-3,5-dioxa-2,4,6-trisilaheptane

Test material form:

liquid

Remarks:

Clear colourless liquid

Specific details on test material used for the study:

Storage conditions: Room temperature in the dark

Method

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

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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.

Applicant's summary and conclusion

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.