Octaphenylcyclotetrasiloxane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial mutagenicity: Negative with and without metabolic activation in S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A (OECD 471) (KTR, 2011)

Cytogenicity in mammalian cells: Negative with and without metabolic activation in Chinese hamster V79 cells (OECD 473) (Eurofins, 2016)

Mutagenicity in mammalian cells: Negative with and without metabolic activation in mouse lumphoma L5178Y cells (OECD 490) (Eurofins, 2017).

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:

2011-08-08 to 2011-09-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

There were minor deviations

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

other: NIER 2010-29

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine operon - Salmonella strains, tryptophan operon - E.coli strain

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 induced rat liver S9

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500 and 5000 µg/plate

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

-MA, TA 1535 (0.5 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

-MA, TA 1537 (80 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

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

Remarks:

-MA, TA 98 (0.1 µg/plate) TA 100 (0.01 µg/plate) WP2 uvrA (0.01 µg/plate)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

+MA, TA 98 (0.5 µg/plate) TA 100 (1.0 µg/plate) TA 1535 (2.0 µg/plate) TA 1537 (2.0 µg/plate) WP2 uvrA (10 µg/plate)

Details on test system and experimental conditions:

ACTIVATION:
Content of 1 mL S9 mix:
- 0.1 mL S9
- 8 µmol MgCl2
- 33 µmol KCl
- 5 µmol Glucose-6-phosphate
- 4 mol NADPH
- 4 µmol NADH
- 100 µmol Sodium phosphate buffer (pH 7.4)

METHOD OF APPLICATION: plate incorporation

DURATION
- Preincubation period:
- Exposure duration:
- Expression time (cells in growth medium):

SELECTION AGENT (mutation assays): histidine tryptophan deficient agar

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: other: clearing of diminution of background lawn, the appearance of micro-colonies, and/or the decrease more than 50% in the number of colonies compared to the vehicle control.

Evaluation criteria:

Results were judged to be positive if there was a reproducible increase in the number of colonies in a dose-dependant manner, at least in one test strain with or without the metabolic system, clearly exceeding 2 times of that of control.

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Key result

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

Positive controls validity:

valid

Remarks on result:

other: No mutagenic potential

Plate incorporation: number of revertant colonies (mean of 3)

Dose (μg/plate)	TA 98		TA 100		TA 1535		TA 1537		WP2 uvrA
	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA	- MA	+ MA
0	13	18	90	88	10	10	6	9	28	33
50	14	16	86	86	8	8	5	9	32	32
100	12	17	83	87	8	12	7	7	29	30
500	14	14	79	88	11	10	5	7	31	31
1000	10	12	85	85	9	11	7	8	28	32
5000	8	12	80	80	6	9	7	7	27	31
Positive control	353	329	416	462	312	200	712	260	316	320

Conclusions:

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

Octaphenylcyclotetrasiloxane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD TG 471, compliant with GLP. No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to cytotoxic/limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 July 2016 to 17 November 2016

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:

2014

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: chromosome aberration in mammalian cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 603152
- Expiration date of the lot/batch: 31 March 2018
- Purity test date: WILL BE INCLUDED IN THE FINAL REPORT

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 soluble and stable in cell culture medium
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: precipitation occurred in the highest tested concentration of 2 mg/mL

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was dissolved in cell culture medium up to the maximum recommended concentration of 2 mg/mL.
- Final dilution of a dissolved solid, stock liquid or gel: From the test item stock solution of 500 µg/mL separate dosing solutions of the test item were prepared for each of the concentrations by serial dilution.
- Final preparation of a solid: The osmolality and pH of the highest concentration were measured. The osmolality was 315 mOsmol/kg and the pH was within the physiological range.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: No data
- Suitability of cells: widely used to examine the ability of chemicals to induce cytogenic changes and thusidentify potential carcinogens or mutagens
- Cell cycle length, doubling time or proliferation index: doubling time is 12 - 14 h, plating efficiency is more than 50 %
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable:
- Modal number of chromosomes: diploid number, 2n = 22
- Normal (negative control) cell cycle time: no data

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: MEM (minimal essential medium) medium supplemented with 10 % fetal bovine serum (FBS), 100 U/ 100 µh/mL penicillin/ streptomycin solution, 2 mM L-glutamine, 2.5 µg/mL amphotericin, 25 mM HEPES. Thawed cultures were set up in 75 cm² cell culture plastic flasks at 37°C in a 5% carbon dioxide atmosphere (95% air)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) induced rat liver metabolic activation system

Test concentrations with justification for top dose:

Pre-experiment: 0.5, 1, 2, 5, 10, 20, 50, 100, 200 and 500 µg/mL were tested with and without metabolic activation. Test concentrations in the main experiment I and II were chosen based on cytotoxity data from the pre-experiment.
Experiment I, with and without metabolic activation: 50, 100, 200 µg/mL
Experiment II, without metabolic activation: 5, 10 and 20 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: cell culture medium
- Justification for choice of solvent/vehicle: a solubility test was performed with different solvents up to the maximum recommended concentration of 2 mg/mL in cell culture medium. According to the results from the solubility test the test item was dissolved in cell culture medium.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: complete culture medium without FBS
- Cell density at seeding: 5 x 10⁵ cells per flask were seeded in 15 mL of MEM (minimal essential medium) supplemented with 10 % FBS and subcultures were made 3-4 days after seeding. Then, 1 x 10⁴ cells/ mL were seeded into cell culture flasks with complete culture medium.

ACTIVATION: The S9 supernatant was thawed and mixed with S9 co-factor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. The final percentage of S9 min in the cell culture medium is 5% (v/v). The added co-factors were: 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.

DURATION
- Exposure duration:
Experiment I: short-term exposure 4 h (with and without metabolic activation)
Experiment II: long-term exposure 21 h (without metabolic activation)
- Expression time (cells in growth medium): 17 hours for Experiment I, no expression time after Experiment II
- Fixation time (start of exposure up to fixation or harvest of cells): at 21 hours post-incubation following Experiment I and immediately after treatment following Experiment II


SPINDLE INHIBITOR (cytogenetic assays): incubation with Colcemid (0.2 µg/ mL culture medium) for 2.5 h

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures were treated at each concentration

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Following incubation with a spindle inhibitor, the cells were trypsinated and resuspended in about 9 mL complete culture medium. Then, the cultures were transferred into tubes and incubated with hypotonic solution for 15-20 min followed by fixation with methanol and glacial acetic acid, and spread onto slides. Then the slides were stained with Giemsa and coverslipped. Afterwards, they were air dried.

NUMBER OF CELLS EVALUATED: 150 metaphases per culture were scored

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells):

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC)


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no

Rationale for test conditions:

Test concentrations in the main experiment I and II were chosen based on cytotoxity data from the pre-experiment.

Evaluation criteria:

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data

Statistics:

Statistical significance (p value); Fischer´s exact test; χ² test for trend

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: Precipitate of the test item was noted at concentrations of 200 and 500 µg/mL after treatment with the test item in Experiment I. In Experiment II without metabolic activation, precipitation was observed at a concentration of 20 µg/mL and higher.
- Definition of acceptable cells for analysis:

RANGE-FINDING/SCREENING STUDIES: Precipitation of the test item was noted at concentrations of 50 µg/mL and higher. The highest dose group evaluated in the pre-experiment was 2000 µg/mL. The relative increase in cell count (RICC) was used as parameter for toxicity. The concentrations evaluated in the main experiment based on the results obtained in the pre-experiment.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within the ranges of the historical control data
- Negative (solvent/vehicle) historical control data: within the ranges of historical control data

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: in Experiment I and II, with and without metabolic activation, no biologically relevant decrease of the relative increase in cell count was noted in all tested concentrations up to 500 µg/mL. These results indicated that the test item has no cytotoxic effects in this test system up to the highest concentration tested.
- Polyploid cells: no biologically relevant increase in the frequencies of polyploid cells was observed after treatment with the test item.

Table 1: Summary: Experiment I and II, with and without metabolic activation

	Dose Group µg/mL	RICC (%)	Mean aberrant cells including gaps (%)	Mean aberrant cells excluding gaps (%)	Precipitation	Statistical significance
Experiment I and II, without metabolic activation
Experiment I 4-hour treatment, 21-hour preparation interval	Negative control (treatment medium) 0	100	3.3	2.3	-	-
	50	100	5.0	3.0	-	-
	100	103	5.0	3.7	-	-
	200	109	3.3	1.7	+	-
	EMS 900	95	10.2	8.9	-	+
Experiment II 21-hour treatment, no preparation interval	Negative control (treatment medium ) 0	100	2.3	1.3	-	-
	5	97	3.0	1.7	-	-
	10	103	2.3	0.7	-	-
	20	98	5.7	1.7	+	-
	EMS 400	92	11.0	7.7	-	+
Experiment I, with metabolic activation
Experiment I 4-hour treatment, 21-hour preparation interval	Negative control (treatment medium) 0	100	2.7	1.7	-	-
	50	107	3.0	1.3	-	-
	100	111	2.7	1.3	-	-
	200	98	3.7	2.7	+	-
	CPA 0.83	99	8.0	5.3	-	+

EMS: ethylmethanesulphonate

CPA: cyclophosphamine

RICC: relative increase in cell count

+ : with precipitation; statistical significance

- : without precipitation; no statistical significance

Conclusions:

Octaphenylcyclotetrasiloxane has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD TG 473 and under GLP. No increase in the number of cells with aberrations was observed either with or without metabolic activation when tested up to a precipitating concentration. Appropriate negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

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

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes

Type of assay:

other: Gene mutation in mammalian cells

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: The test material was stable under the test conditions.
- Solubility and stability of the test substance in the solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Based on the results of the solubility test tetrahydrofuran (THF) was used as solvent.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The solvent was compatible with the survival of the cells and the S9 activity.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Based on the results of the solubility test tetrahydrofuran (THF) was used as solvent. To reach a final concentration of 0.5% solvent v/v the test item stock solution was diluted in RPMI + 5% HS to obtain the final concentration of the different dose groups.
- Preliminary purification step (if any): no preliminary purification step

Target gene:

Tymidine kinase locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins Munich stock cultures
- Suitability of cells: Mouse Lymphoma L5178Y cells (clone TK+/- -3.7.2C) have been used successfully in in vitro experiments for many years.
- Cell cycle length, doubling time or proliferation index: 10-12 h doubling time
- Sex, age and number of blood donors if applicable: not applicable
- Whether whole blood or separated lymphocytes were used if applicable: not applicable
- Number of passages if applicable: not applicable
- Methods for maintenance in cell culture if applicable: To prevent high backgrounds arising from spontaneous mutation, cells lacking TK can be eliminated by culturing cells in RPMI 1640. Large stock cultures of the cleansed L5178Y cell line are stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich. This allows the repeated use of the same cell batch in experiments. Each cell batch is routinely checked for mycoplasma infection. Thawed stock cultures are maintained in plastic culture flasks in RPMI 1640 complete medium and subcultured three times per week.
- Modal number of chromosomes: 40 +/- 2 chromosomes
- Normal (negative control) cell cycle time:

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: in complete culture medium and in 5% CO2/95% humidified air.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) induced rat liver S9.

Test concentrations with justification for top dose:

50,100, 150, 200, 250, 300 and 400 µg/mL, 4-hour exposure without metabolic activation
50, 100, 150, 200, 250, 300, 400 and 500 µg/mL, 4-hour exposure with metabolic actication

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 2 mg/mL. Based on the results of the solubility test tetrahydrofuran (THF) was used as solvent.

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

tatrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 1.6 cells/well

ACTIVATION: An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. The cofactors that were added to the S9 mix to reach the concentrations included 8 mM MgCl2, 33 mM KCl, 5 mM Glucose-6-phosphate, 5 mM NADP in 100 mM sodium-phosphate-buffer pH 7.4.


DURATION
- Exposure duration: 4 hours, with and without metabolic activation
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 12 days at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells):

SELECTION AGENT (mutation assays): selective medium with TFT

NUMBER OF REPLICATIONS: single replication for treatment groups and duplicate replication for negative and solvent controls

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not specified

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): not applicable

Rationale for test conditions:

Test concentrations were selected based on results from a dose-range finding test.

Evaluation criteria:

The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 mutants per 106 cells and
- a dose-dependent increase in mutant frequency is detected.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

400 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

500 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value detected with the test item was within the physiological range.
- Effects of osmolality: Not specified
- Evaporation from medium: Not specified
- Water solubility: Not specified
- Precipitation: In the main experiment without metabolic activation precipitation was noted at concentrations 250 µg/mL and higher, with metabolic activation precipitation was observed at concentrations 400 µg/mL and higher.
- Definition of acceptable cells for analysis: Not specified
- Other confounding effects: Not specified

RANGE-FINDING/SCREENING STUDIES: Precipitation of the test item was noted in the pre-experiment at concentrations of 1000 µg/mL and higher.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: within the range of positive control data
- Negative (solvent/vehicle) historical control data: within the range of positive control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative total growth
- Other observations when applicable: No clastogenic effect was observed when the test item was tested with or without metabolic activation.

Table 1: Main Experiment - Toxicity Data, without metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	314000	861000	1550000	13.3	108.8	97.6	106.2
C2	0	359000	1030000	1470000	15.1	123.5	94.5	116.7
S1	0	304000	689000	1590000	11.0	100.0	100.0	100.0
S2	0	324000	843000	1610000	13.6	100.0	100.0	100.0
1	50	282000	688000	1680000	11.6	94.2	102.4	96.5
2	100	321000	770000	1630000	12.6	102.3	100.8	103.1
3	150	331000	770000	1520000	11.7	95.4	96.0	91.7
4	200	353000	832000	1680000	14.0	114.0	102.4	116.7
5	250 P	274000	739000	1720000	12.7	103.6	96.0	99.5
6	300 P	332000	650000	1750000	11.4	92.8	99.2	92.0
7	400 P	330000	698000	1690000	11.8	96.2	102.4	98.5
EMS	300	340000	788000	1420000	11.2	91.2	83.7	76.4
MMS	10	363000	815000	1350000	11.0	89.7	82.5	74.0

Table 2: Main Experiment - Toxicity Data, with metabolic activation

Test Group	Concen-tration [µg/mL]	Number of Cells 4 h after Treatment	Number of Cells 24 h after Treatment	Number of Cells 48 h after Treatment	SG^a	RSG^b[%]	RCE^c[%]	RTG^d[%]
C1	0	322000	950000	1390000	13.2	102.0	102.9	105.0
C2	0	359000	967000	1420000	13.7	106.1	104.6	110.9
S1	0	329000	903000	1300000	11.7	100.0	100.0	100.0
S2	0	325000	983000	1440000	14.2	100.0	100.0	100.0
1	50	290000	833000	1350000	11.2	86.9	89.7	77.9
2	100	314000	867000	1310000	11.4	87.7	99.8	87.5
3	150	302000	820000	1340000	11.0	84.9	92.4	78.4
4	200	331000	945000	1410000	13.3	102.9	95.3	98.0
5	250	305000	858000	1460000	12.5	96.8	95.3	92.2
6	300	324000	925000	1290000	11.9	92.2	108.0	99.5
7	400 P	301000	880000	1210000	10.6	82.2	115.4	94.9
8	500 P	306000	1060000	1240000	13.1	101.5	119.5	121.3
B[a]P	2.5	325000	332000	776000	2.6	19.9	62.7	12.5

Table 3:Main Experiment - Mutagenicity Data, without metabolic activation

Test Group	Concen-tration [µg/mL]	Cloning Efficiency (CE) Plate 1^e	Cloning Efficiency (CE) Plate 2^e	Cloning Efficiency (CE) CE^f[%]	Mutagenicity Data Number of cultures / 96 wells Plate 1^e	Mutagenicity Data Number of cultures / 96 wells Plate 2^e	Mutagenicity Data Number of cultures / 96 wells Plate 3^e	Mutagenicity Data Number of cultures / 96 wells Plate 4^e	Mutagenicity Data Number of cultures / 96 wells Mean	MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
C1	0	74	78	98.0	12	5	14	6	9.3	52.2	/
C2	0	76	74	95.0	8	14	7	8	9.3	53.6	/
S1	0	74	78	98.0	8	13	12	10	10.8	60.7	/
S2	0	72	83	102.9	12	16	8	14	12.5	68.1	/
1	50	75	80	102.9	9	14	11	11	11.3	60.7	-3.7
2	100	77	77	101.2	11	11	15	11	12.0	66.1	1.7
3	150	74	77	96.5	11	4	12	14	10.3	59.0	-5.4
4	200	81	74	102.9	11	9	10	12	10.5	56.3	-8.1
5	250 P	68	83	96.5	19	10	5	12	11.5	67.0	2.7
6	300 P	72	81	99.6	6	8	5	15	8.5	47.0	-17.3
7	400 P	79	76	102.9	16	13	10	7	11.5	62.4	-2.0
EMS	300	73	69	84.1	79	65	63	73	70.0	796.5	732.1
MMS	10	65	76	82.9	69	57	63	61	62.5	640.6	576.2

Table 4: Main Experiment - Mutagenicity Data, with metabolic activation

Test Group	Concen-tration [µg/mL]	Cloning Efficiency (CE) Plate 1^e	Cloning Efficiency (CE) Plate 2^e	Cloning Efficiency (CE) CE^f[%]	Mutagenicity Data Number of cultures / 96 wells Plate 1^e	Mutagenicity Data Number of cultures / 96 wells Plate 2^e	Mutagenicity Data Number of cultures / 96 wells Plate 3^e	Mutagenicity Data Number of cultures / 96 wells Plate 4^e	Mutagenicity Data Number of cultures / 96 wells Mean	MF^g     [mutants / 10^6cells]	IMF^h      [mutants / 10^6cells]
C1	0	73	78	96.5	9	15	13	10	11.8	67.9	/
C2	0	78	74	98.0	17	10	11	12	12.5	71.4	/
S1	0	75	66	82.9	7	8	5	11	7.8	51.0	/
S2	0	76	80	104.6	5	10	12	12	9.8	51.4	/
1	50	73	69	84.1	14	12	9	8	10.8	70.8	19.6
2	100	71	78	93.5	7	9	14	17	11.8	70.4	19.2
3	150	71	73	86.6	7	8	10	6	7.8	48.7	-2.6
4	200	75	71	89.3	5	11	8	10	8.5	52.1	0.9
5	250	71	75	89.3	18	9	10	16	13.3	83.8	32.5
6	300	81	73	101.2	6	11	7	16	10.0	54.9	3.6
7	400 P	75	83	108.2	10	10	11	12	10.8	54.9	3.7
8	500 P	81	79	112.0	8	15	9	11	10.8	53.2	2.0
B[a]P	2.5	56	61	58.8	51	51	57	39	49.5	625.0	573.8

C: Negative control

S: Solvent control (THF)

P: Precipitation

a: Suspension Growth, SG = [((value 24hx30)/1x107) x ((value 48 h x 20)/(value 24 h*x20))]; * : for value 24h > 3x105then value 24h = 3x105

b: Relative Suspension Growth, RSG = [(value SG / value SG of corresponding controls) x 100]

c: Relative Cloning Efficiency, RCE = [(CEdose group/ CEof corresponding controls) x 100)

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100

d: Relative Total Growth, RTG = (RSG x RCE)/100

B[a]P:  Benzo[a]pyrene

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

Conclusions:

Octaphenylcyclotetrasiloxane has been tested for mutagenicity in Mouse lymphoma L5178Y cells according to OECD TG 490, and under GLP (Eurofins, 2017). No test-substance induced increase in the number of mutations was observed with or without metabolic activation, when tested up to precipitating concentrations. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Octaphenylcyclotetrasiloxane has been tested for mutagenicity to bacteria, in a study which was conducted according to the OECD Test Guideline 471, compliant with GLP (KTR, 2011). No evidence of a test-substance related increase in the number of revertants was observed with or without metabolic activation in Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 or E. coli WP2 uvrA in the initial or the repeat experiments up to cytotoxic/limit concentrations. Appropriate positive and solvent controls were included and gave the expected results. It is concluded that the test substance is negative for mutagenicity to bacteria under the conditions of the test.  

Negative results were also reported in a gene mutation study using S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, which supports the findings of the key study (DCC, 1987).

Octaphenylcyclotetrasiloxane has been tested for ability to cause chromosome aberrations in Chinese hamster V79 cells according to OECD Test Guideline 473 and under GLP (Eurofins, 2016). No increase in the number of cells with aberrations was observed either with or without metabolic activation when tested up to a precipitating concentration. Appropriate negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for the induction of chromosome aberrations under the conditions of the test.  

Octaphenylcyclotetrasiloxane has been tested for mutagenicity in mouse lymphoma L5178Y cells according to OECD Test Guideline 490, and under GLP (Eurofins, 2017). No test-substance induced increase in the number of mutations was observed with or without metabolic activation, when tested up to precipitating concentrations. Appropriate solvent, negative (treatment medium) and positive controls were included and gave expected results. It is concluded that the test substance is negative for mutagenicity to mammalian cells under the conditions of the study.

Justification for classification or non-classification

Based on the available data for octaphenylcyclotetrasiloxane, no classification is required for genetic toxicity according to Regulation (EC) No 1272/2008.