Octaphenylcyclotetrasiloxane

Endpoint summary

Description of key information

In the first key in vitro skin irritation study with octaphenylcyclotetrasiloxane (CAS 546-56-5) using EpiDermTM tissue, the reported mean relative tissue viability (% negative control) was > 50 % (91.4 %) after 60 minutes treatment and 42 hours post-incubation (Eurofins, 2017).

In the second key in vitro skin irritation study with octaphenylcyclotetrasiloxane (CAS 546-56-5) using EpiSkinTM tissue, the reported mean relative tissue viability (% negative control) was > 50 % (104.9 %) after 15 minutes treatment and 42 hours post-incubation (Eurofins, 2017a).

In the key in vitro Bovine Corneal Opacity and Permeability Assay, conducted according to an appropriate OECD test guideline and in compliance with GLP, the in vitro irritation score reported for the test substance, octaphenylcyclotetrasiloxane, was < 3. The test substance was concluded to be not irritating to eyes (Eurofins, 2017).  

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 March 2017 to 24 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 25 µL of sterile DPBS was applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item was applied directly atop the EpiDermTM tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by gently shaking the inserts or by using a bulb-headed Pasteur pipette.
- Preliminary purification step (if any): not specified

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): 25086
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 +/- 1 °C, 5.0% CO2
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C, 5.0% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface.
- Observable damage in the tissue due to washing: No damage specified.
- Modifications to validated SOP: Not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL MTT
- Incubation time: 3 h +/- 5 min at 37 +/- 1 °C, 5.0% CO2, humidified to 95%.
- Spectrophotometer: plate spectrophotometer using isopropanol as a blank.
- Wavelength: 570 nm
- Filter: OD was measured at 570 nm with a filter band pass of maximum ± 30 nm without reference wavelength.
- Filter band width: Not specified
- Linear OD range of spectrophotometer: yes

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.581+/-0.238, acceptance criteria OD (540-570 nm) [1.0-3.0]
- Barrier function: 5.58 h, acceptance criteria ET-50 [4.77-8.72 h]
- Morphology: not specified
- Contamination: No contamination was detected.
- Reproducibility: Not specified.

NUMBER OF REPLICATE TISSUES: Triplicate tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: killed tissues
- Procedure used to prepare the killed tissues (if applicable): not specified
- N. of replicates : duplicate
- Method of calculation used: NSMTT (non-specific MTT) was then calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula:
TODTT = ODTM – (ODKT – ODKU)

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean relative tissue viability after 1 hour exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin if the mean relative tissue viability after 1 hour exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable

Control samples:

yes, concurrent positive control

other: Negative control: Dulbecco’s phosphate buffered saline (DPBS);

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL DPBS

NEGATIVE CONTROL
- Concentration (if solution): 30 µL DPBS

POSITIVE CONTROL
- Concentration (if solution): 30 µL 5% SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate tissues were used.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

No 1 expressed in percentage of negative control

Value:

> 50

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none observed
- Direct-MTT reduction: The test item showed no non-specific reduction of MTT. Therefore, no additional controls were necessary.
- Colour interference with MTT: The test item showed no colouring after mixture with aqua dest. and isopropanol.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Mean Absolute OD570 nm Negative control (NC): 2.038, cut off 0.8 ≤ NK ≤ 2.8
- Relative Viability [%] Positive control (PC): 3.5, cut off ≤ 20%
- SD Viability [%]: 0.2 – 4.9, cut off ≤ 18%


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro skin irritation study with octaphenylcyclotetrasiloxane (CAS 546-56-5) using EpiDermTM tissue, the reported mean relative tissue viability (% negative control) was > 50 % (91.4 %) after 60 minutes treatment and 42 hours post-incubation.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 March 2017 to 07 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 5 µL distilled water (aqua dest.) was applied to the epidermal surface in order to improve further contact between the powder and the epidermis. The water was gently spread on the surface. Afterwards, approximately 10 ± 2 mg (26.3 mg/cm²) of the powder was applied to the epidermis surface.

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Details on animal used as source of test system:

Not applicable

Justification for test system used:

This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™
- Tissue batch number(s): 17-EKIN-014
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature for 15 +/- 0.5 min
- Temperature of post-treatment incubation (if applicable): 37 +/- 1 °C, 5.0% CO2 for 42 +/- 1 h.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The tissues were washed with DPBS to remove any residual test item. Excess DPBS was removed by blotting bottom with blotting paper.
- Observable damage in the tissue due to washing: None reported
- Modifications to validated SOP: None reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 3 mg/mL MTT
- Incubation time: 3 hours
- Spectrophotometer: yes
- Wavelength: at 570 nm
- Filter: filter band
- Filter bandwidth: filter band pass of maximum ± 30 nm in a plate spectrophotometer
- Linear OD range of spectrophotometer: yes

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: IC50 determination (SDS concentration, MTT test, n = 14): Specification ≥ 1.5 mg/mL; Result: 2.2 mg/mL
- Morphology: Specification ≥ 19.5; Result: 22.3 ± 1.0, CV = 4.7%; Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: No contamination was reported.
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: Triplicate tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: Killed tissues
- Procedure used to prepare the killed tissues (if applicable): not specified
- N. of replicates : triplicate
- Method of calculation used: NSMTT (non-specific MMT) was calculated relative to the negative control of living tissues (NK) per treatment period according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100
If non-specific MTT reduction was ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) was corrected according to the following formula:
TODTT = ODTM - (ODKT - ODKU)


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure is less than 50%.
- The test substance is considered to be non-corrosive to skin if the viability after 15 minutes exposure is greater than or equal to 50%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: not applicable

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µL aqua dest

NEGATIVE CONTROL
- Concentration (if solution): 10 µL DPBS

POSITIVE CONTROL
- Concentration (if solution): 10 µL 5% SDS solution

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment I

Value:

> 50

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: The test item showed no non-specific MTT reducing potential, therefore no additional controls for correction of results were necessary.
- Colour interference with MTT: The test item showed no colouring potential, therefore no additional controls for correction of results were necessary.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Mean OD570 nm Blank: value 0.044; cut off < 0.1;
- Mean Absolute OD570 nm NK: 0.610, cut off 0.6 ≤ NK ≤1.5;
- Mean Relative Viability [%] PC: 9.8; cut off ≤ 40%;
- Max. SD of % Viability [%]: 8.1, cut off ≤ 18%

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro skin irritation study with octaphenylcyclotetrasiloxane (CAS 546-56-5) using EpiSkinTM tissue, the reported mean relative tissue viability (% negative control) was > 50 % (104.9 %) after 15 minutes treatment and 42 hours post-incubation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th May 2017 to 07th June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: the test substance was stable in corn oil
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test material was suspended with corn oil to give 20% concentration.
- Preliminary purification step (if any): not specified

Species:

other: animals freshly slaughtered at the abattoir

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories.
- Time interval prior to initiating testing: Immediately after arrival of the eyes, cornea preparation was initiated.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: Not specified

Vehicle:

other: corn oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 750 µL of the test item preparation

VEHICLE
- Amount(s) applied (volume or weight with unit):
- Concentration (if solution): 750 µL

Duration of treatment / exposure:

4 hours ± 5 minutes incubation at 32 ± 1 °C.

Duration of post- treatment incubation (in vitro):

90 minutes at 32 ± 1 °C

Number of animals or in vitro replicates:

3 corneas for test item

Details on study design:

SELECTION AND PREPARATION OF CORNEAS: The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS.The corneas were mounted in corneal holders with the endothelial side against the O-ring of the posterior chambe. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS: The corneas were visually examined for defects and any defective cornea had been discarded.

NUMBER OF REPLICATES: Triplicate

NEGATIVE CONTROL USED: Yes, physiological saline 0.9% NaCl

SOLVENT CONTROL USED (if applicable): Yes, corn oil

POSITIVE CONTROL USED: Yes, imidazole 20% in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 4 hours ± 5 minutes incubation at 32 ± 1 °C.

TREATMENT METHOD: closed chamber method

POST-INCUBATION PERIOD: yes, 90 minutes at 32 ± 1 °C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test substance or the control substance was removed and the epithelium washed at least three times with MEM
- POST-EXPOSURE INCUBATION: yes, 90 minutes at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The following formula was used to calculate the opacity: Opacity= (Io/Iᵦ)/a. The value Io is the illuminance through a holder without cornea, but with windows and liquid. This value is determined by taking the mean for a set of cornea holders and is reevaluated periodically.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of OD490 using a spectrophotometer
- Others (e.g, pertinent visual observations, histopathology): (please specify)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

bovine corneal opacity and permeability assay

Value:

1.55

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay was considered to be valid.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

In the in vitro Bovine Corneal Opacity and Permeability Assay, conducted according to an appropriate OECD test guideline and in compliance with GLP, the in vitro irritation score reported for the test substance, octaphenylcyclotetrasiloxane, was < 3. The test substance was concluded to be not irritating to eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of octaphenylcyclotetrasiloxane (CAS 546-56-5) to induce skin irritation was analysed by using a three-dimensional human epidermis model EpiDermä(MatTek) comprising a reconstructed epidermis with a functional stratum corneum (Eurofins, 2017).

The test substance was applied topically to the EpiDermä tissue for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no non-specific reduction of MTT and no colouring after mixture with aqua dest. and isopropanol. Therefore, no additional controls were necessary. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 50% (91.4%) after 60 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.

The potential of octaphenylcyclotetrasiloxane (CAS 546-56-5) to induce skin irritation was also analysed by using the three-dimensional human skin model EPISKIN-SM comprising a reconstructed epidermis with a functional stratum corneum (Eurofins, 2017a). The test substance was applied topically to the EPISKIN-SM for 15 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay.

Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS.

The test item showed no non-specific MTT reducing potential and no colouring potential, therefore no additional controls for correction of results were necessary. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was> 50% (104.9%) after 15 min treatment and 42 h post-incubation. The controls confirmed the validity of the study.

The eye irritancy potential of octaphenylcyclotetrasiloxane (CAS 546-56-5) was investigated in the bovine corneal opacity and permeability assay. The test item was suspended with corn oil to give a 20% concentration. 750 µL of the test item preparation or the control substance was introduced into the anterior chamber (closed-chamber method). After 4 hours ± 5 minutes incubation at 32 ± 1 °C either the test substance or the control substance was removed and the epithelium washed at least three times with MEM. Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an illuminance measurement was performed. Also, each cornea was observed visually and pertinent observations were recorded. Then the corneas were incubated for 90 minutes at 32±1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.

The calculated in vitro irritation score was calculated to be 1.55. The in vitro irritation score obtained with the positive control fell within the two standard deviations of the current historical mean and therefore this assay is considered to be valid. The negative control responses should result in opacity and permeability values that are less than the established upper limits for background bovine corneas treated with the respective negative control.

Justification for classification or non-classification

Based on the available data for octaphenylcyclotetrasiloxane (CAS 546-56-5), no classification for skin and eye irritation is required according to Regulation (EC) No 1272/2008.