Tetrakis(2-ethylbutyl) orthosilicate

Endpoint summary

Description of key information

Skin Irritation-not irritating in in-vitro EpiDerm SIT assay (OECD TG 439)

Skin Corrosion- not corrosive in in-vitro EpiDerm SCT assay (OECD TG 431)

Eye Irritation- meets the criteria for not requiring classification and labeling as a substance causing eye irritation or serious eye damage in an in-vitro reconstructed human corneal-like epithelium (RhCE) assay (OECD TG 492)

Eye Corrosion- not corrosive in an in-vitro bovine corneal opacity & permeability (BCOP) assay (OECD TG 437)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Aug 2017 - 31 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Source: Gelest, Inc. (Morrisville, PA)
Lot #8E-33-134
Expiration date: May 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in dessicator; room temperature
- Solubility and stability of the test substance in the solvent/vehicle: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

not applicable

Justification for test system used:

The EpiDerm™ Skin Model closely parallels human skin, and is recommended by test guidelines. Validation studies have reported that tests employing human skin models are able to reliably discriminate between known skin corrosives and non-corrosives.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek Corporation (Ashland, MA) EpiDerm™ tissues
- Tissue batch number(s): Lot No. 26787 Kit I
- Shipping date: 28 August 2017
- Delivery date: 29 August 2017
- Date of initiation of testing: 30 August 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): not applicable

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: not specified
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL Dulbecco's Modified Eagle's Medium (DMEM)
- Incubation time: 3 hours
- Spectrophotometer: μQuant Plate Reader (Bio-Tek Instruments, Winooski, VT).
- Wavelength: 540 nM


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was assessed with an MTT QC assay. Application of tissue culture water to the tissues for four hours produced an MTT OD value of 1.7 for Lot No. 26787 Kit I. This value met the criteria of >1.0 but < 3.0 indicating adequate viability of tissues. The historical tissue viability values from the 1996 EpiDerm Database (N=184) for water are mean= 1.47; standard deviation = 0.13; Range ± 2 standar deviatons = 1.21 - 1.73.
- Barrier function: Tissues were exposed to 1% Triton X-100 for 4, 6, 8 and 10 hours. The time of exposure required to reduce the tissue viability (ET-50) using the MTT viability assay is determined (See MatTek EpiDerm MTT ET-50 Protocol) for each lot of tissue. ET-50’s must fall within the range of the 1996 EpiDerm database of 4.77 – 8.72 hours. The ET50 for Lot No. 26787 Kit I was 7.57 hours.
- Morphology: Tissue viability and barrier function tests are wtihin the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a function stratum corneum, a viable basal cell layer and intermediate spinous and granular layers.
- Contamination: The cells used to produced EpiDerm tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants as follows: HIV (oligonucleotide-directed amplification), Hepatitis-B (oligonucleotide-directed amplification), Hepatitis-C (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture)
- Reproducibility: Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness. Tissues were used consistently on the same day, i.e. following overnight storage at 4°C (Wednesday morning) to maintain reproducibility.


NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 microliters of test article was applied to the top of each tissue
- Concentration (if solution): not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 microliters of test article was applied to the top of each tissue
- Concentration (if solution): not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 microliters of test article was applied to the top of each tissue
- Concentration (if solution): 8N

Duration of treatment / exposure:

Each test article remained in contact with the EpiDerm™ tissue for 3 and 60 minutes.

Duration of post-treatment incubation (if applicable):

N/A

Number of replicates:

Duplicate EpiDerm™ tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

104.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes

Value:

91.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Viability differences between the two identically treated tissues in all samples and controls at 3 minutes were 0.7% to 5.4%. Viability differences between the two identically treated tissues at 60 minutes were 0.8% to 8.9%. Inter-tissue viability differences at both time points met the acceptance criterion (<30%). Calibration of the uQuant microplate reader passed all acceptance criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

Skin corrosion is defined as the production of irreversible tissue damage in skin following the application of test material. The percent viability following a 3 minute and 60 minute exposure was used to determine corrosivity potential according to OECD TG 431. For mean viability ≥ 50% at 3 minutes and >15%, the test article is determined to be non-corrosive, and ≥ 50% at 3 minutes but <15% at 60 minutes, the test article is determined to be corrosive. In this case, the mean viability of Tetrakis(2-ethylbutyl) Orthosilicate was 91.7 % following a 3 minute exposure and 104.7% following a 60 minute exposure. This finding does not warrant classification of Tetrakis(2-ethyl butyl) Orthosilicate as a Category 1 skin corrosive substance under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

The skin corrosive potential of Tetrakis (2 -ehtylbutyl) Orthosilicate was evaluated using the MatTek EpiDerm™ Skin Corrosivity Test (OECD TG 431). MatTek EpiDerm™ tissues were treated in duplicate with the test articles, negative control and positive control for 3 minutes and 60 minutes according. Following treatment, the viability of the tissues was determined using methyl thiazole tetrazolium (MTT) uptake and conversion, and the absorbance of each tissue was measured at 540 nm. The mean viability was then expressed as a percent of control values. The percent viability was used to determine corrosivity potential. The percent viability following a 3 minute and 60 minute exposure was used to determine corrosivity potential. For mean viability ≥ 50% at 3 minutes and >15%, the test article is determined to be non-corrosive, and ≥ 50% at 3 minutes but <15% at 60 minutes, the test article is determined to be corrosive. In this case, the mean viability of Tetrakis(2 -ethylbutyl) Orthosilicate was 91.7% following a 3 minute exposure and 104.7% following a 60 minute exposure. This finding does not warrant classification of Tetrakis(2 -ethylbutyl) Orthosilicate as a Category 1 skin corrosive substance under the  Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Sep 2017 - 23 Oct 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:

28 July 2015

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Source: Gelest, Inc. (Morrisville, PA)
Lot #8E-33-134
Expiration date: May 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored in dessicator; room temperature
- Solubility and stability of the test substance in the solvent/vehicle: not applicable

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received

Test system:

human skin model

Remarks:

MatTek EpiDerm SIT

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The 3-Dimensional Human Dermal Epithelial Model (EpiDerm™, MatTek, Ashland, MA) is made up of normal human keratinocytes in serum free medium. The cells form an epithelial tissue that consists of organized basal, spinous, granular, and cornified layers analogous to those found in vivo. The EpiDerm™ model also contains epidermis-specific differentiation markers such as pro-filaggrin, the K1/K10 cytokeratin pair, involucrin, and type I epidermal transglutaminase, as well as keratohyalin granules, tonofilament bundles, desmosomes, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns characteristic of in vivo epidermis. Each lot of tissues was Quality Assured by MatTek, Inc. according to specific QC standards including: histology (cell layers), tissue viability (MTT mean optical density) and reproducibility (SD).

Vehicle:

unchanged (no vehicle)

Details on test system:

The normal human-derived keratinocytes were cultured at the air-liquid interface in a chemically defined medium on a permeable polycarbonate insert (surface 0.5 cm2). They were cultured in chemically defined serum free medium to form a multi-layered epithelium similar to that found in native epidermis.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTEK EpiDerm 200-SIT
- Tissue batch number(s): 27116 Kit S
- Shipping Date: 11 Sep 2017
- Delivery date: 12 Sep 2017
- Date of initiation of testing: 13 Sep 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ~37°C and ~5% CO2
- Temperature of post-treatment incubation (if applicable): ~37°C and ~5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed with PBS and blotted to remove test substance

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: uQuant Plate Reader, Bio-Tek Instruments
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was assessed with an MTT QC assay. Application of tissue culture water to the tissues for four hours produced an MTT OD value of 1.86 for Lot No. 27116 Kit S. This value met the criteria of >1.0 but < 3.0 indicating adequate viability of tissues. The historical tissue viability values from the 1996 EpiDerm Database (N=184) for water are mean= 1.47; standard deviation = 0.13; Range ± 2 standar deviatons = 1.21 - 1.73.
- Barrier function: Tissues were exposed to 1% Triton X-100 for 4, 6, 8 and 10 hours. The time of exposure required to reduce the tissue viability (ET-50) using the MTT viability assay is determined (See MatTek EpiDerm MTT ET-50 Protocol) for each lot of tissue. ET-50’s must fall within the range of the 1996 EpiDerm database of 4.77 – 8.72 hours. The ET50 for Lot No. 27116 Kit S was 7.26 hours.
- Morphology: Tissue viability and barrier function tests are wtihin the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a function stratum corneum, a viable basal cell layer and intermediate spinous and granular layers.
- Contamination: The cells used to produced EpiDerm tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants as follows: HIV (oligonucleotide-directed amplification), Hepatitis-B (oligonucleotide-directed amplification), Hepatitis-C (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture)
- Reproducibility: Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness. Tissues were used consistently on the same day, i.e. following overnight storage at 4°C (Wednesday morning) to maintain reproducibility.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
- The test substance is considered to be irritating to skin if the viability after 60 minutes exposure is less than 50%
-The test substance is considered to be non-irritating to skin if the viability after 60 minutes exposure is greater than or equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 microliters was applied to the top of the tissue
- Concentration (if solution): not applicable

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliters was applied to the top of the tissue
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 microliters was applied to the top of the tissue
- Concentration (if solution): 5%

Duration of treatment / exposure:

Tissues were exposed to controls and test substance for approximately 35 ± 1 minutes in a humidified incubator at ~37°C and ~5% CO2, and at room temperature for the remainder of the 60 minute total exposure period.

Duration of post-treatment incubation (if applicable):

Rinsed EpiDerm tissues were returned to the humidified incubator at ~37°C and ~5% CO2 for 42 ± 2 hours.

Number of replicates:

n=3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes

Value:

100.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: not observed
- Colour interference with MTT: not observed

ACCEPTANCE OF RESULTS:
The mean OD540 of the negative control tissues was 2.464, and the mean viability of positive control tissues was 2.2%. The standard deviation (SD) calculated from individual percent tissue viabilities of the three identically-treated replicates was 0.53% for the negative control and 0.39% for the positive control. All controls passed the acceptance criteria. All calibrations of the uQuant microplate reader passed the acceptance criteria during the course of the study.

Interpretation of results:

GHS criteria not met

Conclusions:

In the in-vitro EpiDerm SIT Skin Model, the relative mean tissue viability obtained after a 1-hour treatment with Tetrakis(2-ethylbutyl) Orthosilicate compared to the negative control tissues was 100.4%. This finding does not warrant classification of Tetrakis(2-ethylbutyl) Orthosilicate as a Category 2 Skin Irritant under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Executive summary:

The MatTek EpiDerm™ SIT skin model was used to assess the potential dermal irritation of Tetrakis(2 -ethylbutyl) Orthosilicate by determining the viability of the tissues following exposure to the test substance via MTT. The objective of this study was to assess the dermal irritation of neat (undiluted) Tetrakis(2 -ethylbutyl) Orthosilicate. Tissues were exposed to test substance and controls for one hour. Pre-testing for coloration and MTT auto-reduction were negative, therefore no data corrections were necessary. The MTT data show the positive control, 5% sodicum dodecyl sulfate (SDS), reduced tissue viability to 2.2% of negative control after 1 hour exposure. The MTT data show that the test substance, Tetrakis(2 -ethylbutyl) Orthosilicate produce tissue viability of 100.4% of negative control after 1 hour exposure. This finding does not warrant classification of Tetrakis(2 -ethylbutyl) Orthosilicate as a Category 2 Skin Irritant under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 Aug 2017 - 31 Aug 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:

26 July 2013

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Tetrakis(2-ethylbutyl) Orthosilicate CAS 78-13-7 Lot# 8E-33-134

Species:

other: Isolated Bovine Cornea

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

The bovine eyes, in a Hanks’ Balance Salt Solution (HBSS) with penicillin-streptomycin, were received from Spear Products on 31 Aug 2017 and transported to MB Research in a refrigerated container.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 ml of the test article

Duration of treatment / exposure:

10 (±1) minutes at 32 (±1)°C

Observation period (in vivo):

N/A

Duration of post- treatment incubation (in vitro):

All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution.

Number of animals or in vitro replicates:

3 bovine corneas per group

Details on study design:

Application/Treatment of test article:
Following the pretest observations, the MEM solution was removed from the anterior chamber. A volume of 0.75 ml of test article, ethanol or MEM solution was applied to the epithelium of each of the three test article corneas, three positive control corneas and three negative control corneas and three vehicle control corneas in a manner which ensured that the entire cornea was covered. All corneas were dosed via the closed-chamber method. All holders and corneas were placed in a horizontal position (anterior side up) in the 32 (±1)°C incubator. After 10 (±1) minutes), the test article, ethanol or MEM was removed from the epithelium of the cornea and anterior chamber of the holder by washing with MEM solution containing phenol red. A final rinse was made with MEM without phenol red. The anterior and posterior chambers of the holders were then refilled with fresh MEM solution. Opacity measurements were made following the 10-minute exposure and MEM solution refill. All corneas were incubated at 32 (±1)°C for an additional two hours at which time the MEM solution in the anterior and posterior chambers was removed and the holders refilled with fresh MEM solution.

Opacity Measurement:
A measurement of opacity was taken with each treated cornea compared to the blank supplied with the OP-KIT. This reading was used in the final IVIS calculations. Immediately following the two-hour opacity measurement, the MEM solution was removed from the anterior chamber and replaced with 1.0 ml of 0.4% sodium fluorescein solution in Dulbecco's phosphatebuffered saline (PBS). Each holder was then returned to the 32 (±1)°C incubator in a horizontal position (anterior side up) ensuring contact of the fluorescein with the cornea. After 90 (±5) minutes, the fluid from the posterior chamber was removed and the amount of dye that passed through the cornea (permeability) was measured as the optical density at 490 nm by a spectrophotometer. A 1:1000 dilution of the fluorescein was prepared and measured in the spectrophotometer as a measure of consistency.

Irritation parameter:

cornea opacity score

Remarks:

2-hour corrected

Value:

-1.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

In vitro irritation score (IVIS) calculated from opacity score

Value:

-1.24

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The ethanol positive control IVIS was 29.95, which fell within the acceptance range of 16.96 – 37.00 (± 2 standard deviations of the historical mean).

Interpretation of results:

GHS criteria not met

Conclusions:

The mean corneal opacity, and in-vitro irritation score for Tetrakis(2-ethylbutyl) Orthosilicate were -1.33 and -1.24, respectively. These findings do not warrant classification of Tetrakis(2-ethylbutyl) Orthosilicate as a substance inducing serious eye damage (Category 1) under the Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).

Executive summary:

Tetrakis(2-ethylbutyl) Orthosilicate (neat) was applied to isolated bovine cornea to assess for the potential to induce serious eye damage. Ocular damage was assessed at 4 hours post-instillation and scored through measurement of corneal opacity and permeability. The mean corneal opacity, and in-vitro irritation score for Tetrakis(2-ethylbutyl) Orthosilicate were -1.33 and -1.24, respectively. These findings do not warrant classification of Tetrakis(2-ethylbutyl) Orthosilicate as a substance inducing serious eye damage (Category 1) under the Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 August 2017 - 1 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Source: Gelest, Inc. (Morrisville, PA)
Lot #8E-33-134
Expiration date: May 2020

Species:

human

Details on test animals or tissues and environmental conditions:

EpiOcular™ tissues, Lot No. 23495 Kit E, were received from MatTek on 29 Aug 2017 and refrigerated at 2-8°C. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium for an additional overnight equilibration of 16 to 24 hours. After the overnight incubation, the tissues were moistened with 20 μl of
phosphate-buffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

RECONSTRUCTED HUMAN CORNEAL-LIKE EPITHELIUM (RHCE) TISSUE
- Model used: MatTEK EpiOcular OCL-200
- Tissue batch number(s): 23495 Kit E
- Shipping date: 28 Aug 2017
- Delivery date: 29 Aug 2017
- Date of initiation of testing: 30 Aug 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: (37±1°C, 5±1% CO2
- Temperature of post-treatment incubation (if applicable): 37±1°C, 5±1% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Tissues were rinsed with PBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: uQuant Plate Reader, Bio-Tek Instruments
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Tissue viability was assessed with an MTT QC assay. Application of tissue culture water to the tissues for four hours produced an MTT OD value of 1.64 for Lot No. 23495 Kit E. This value met the criteria of >1.10 based on the MatTek 1996 QC EpiOcular Database (N=184)indicating adequate viability of tissues.
- Barrier function: Tissues were exposed to 0.3% Triton X-100 for 5, 20, and 60 minutes. The time of exposure required to reduce the tissue viability (ET-50) using the MTT viability assay is determined (See MatTek EpiOcular MTT ET-50 Protocol) for each lot of tissue. ET-50’s must fall within the range of 12.2 – 37.5 hours based on the 1996 QC EpiOcular database (average = 24.9 ± 6.3). The ET50 for Lot No. 23495 Kit E was 7.26 hours.
- Morphology: Tissue viability and barrier function tests are within the acceptable ranges and indicate appropriate formation of the epithelium. Appropriate morphology has also been established for the Validated Reference Method EpiOcular EIT and therefore detailed histological examination does not need to be demonstrated by a test method user for each tissue batch.
- Contamination: The cells used to produced EpiOcular tissues are screened for potential biological contaminants by the manufacturer. Tests were performed by MatTek for each of the potential biological contaminants as follows: HIV (oligonucleotide-directed amplification), Hepatitis-B (oligonucleotide-directed amplification), Hepatitis-C (oligonucleotide-directed amplification), and Bacteria, yeast and other fungi (long-term antibiotic, antimycotic free culture)
- Reproducibility: Each lot of tissues was Quality Assured by MatTek according to specific QC standards including: histology, tissue viability (MTT mean optical density), reproducibility (SD) and tissue thickness. Tissues were used consistently on the same day, i.e. following overnight storage at 4°C (Wednesday morning) to maintain reproducibility.

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

DECISION CRITERIA
- The test substance is considered to be an eye irritant or substance causing serious eye damage (GHS Category 1 or 2) if the viability following treatment is less than or equal to 60%
- The test substance is considered to be non-irritating (GHS No Category) to eye if the viability following treatment is greater than 60%

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50μl of the test article

Duration of treatment / exposure:

The tissues were then incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

The tissues were rinsed with phosphate-buffered saline (PBS) and soaked in 5 ml of room-temperature assay medium in a 12-well plate for 12 minutes. The soaked tissues were then incubated in fresh assay medium at 37±1°C, 5±1% CO2 for 120 minutes.

Number of animals or in vitro replicates:

2 replicate tissues

Details on study design:

Assessment of Colored Substance:
Since test article Tetrakis(2-ethylbutyl) Orthosilicate CAS 78-13-7 was non-colored, it was assessed to determine if it would become colored in water or extractant. 50 μl of the test article were incubated in a six-well plate with 1 ml of tissue culture water for at least one hour in a humidified 37±1°C, 5±1% CO2 incubator. An additional 50 μl of the test article were added to 2 ml of extractant (isopropanol) and incubated for 2 to 3 hours in a six-well plate, at room temperature with shaking. Two aliquots of the test article plus TCH2O or test article plus extractant from each well were measured at 570 nm using the plate reader. Nocolor developed in the water or the extractant, resulting in an OD570 no more than 0.08 after subtraction of blank (undosed tissue culture water or isopropanol, respectively), so no colorant controls were added to the assay for this test article.

Application/Treatment of test article:
50 μl of the test article were applied to duplicate EpiOcular™ tissues. A negative control (50 μl of TCH2O) and a positive control (50 μl of methyl acetate) were each tested concurrently. Each treatment with test article or control was conducted in duplicate. The tissues were then incubated at 37±1°C, 5±1% CO2 for 30±2 minutes. After dosing and incubation, the tissues were rinsed with phosphate-buffered saline (PBS) and soaked in 5 ml of room-temperature assay medium in a 12-well plate for 12 minutes. The soaked tissues were then incubated in fresh assay medium at 37±1°C, 5±1% CO2 for 120 minutes.

Cell Viability Measurement:
At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in Dulbecco's Modified Eagle's Medium). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiOcular™ tissue was rinsed with PBS and then treated with 2.0 ml of extractant solution (isopropanol) per well overnight at room temperature in the dark. Two aliquots of the extracted MTT formazan from each well were measured at 570 nm using a plate reader (μQuant Plate Reader, Bio-Tek Instruments, Winooski, VT).

Irritation parameter:

other: Mean Tissue Viability (%)

Value:

98.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The R-squared value calculated for the plate reader linearity check was 0.9999, which met the acceptance criterion of greater than 0.999. All other quality controls passed the acceptance criteria. The mean OD570 of the negative control tissues was 2.137, and the mean relative viability of the positive control tissues was 19.8%. The differences in viability between identically treated tissues were 0.04 - 2.95%. All controls passed the acceptance criteria.

Interpretation of results:

GHS criteria not met

Conclusions:

Eye irritation was assessed with EpiOcular EIT (OCL-200), a 3D model of reconstructed human cornea-like epithelium test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Duplicate tissues were treated with Tetrakis (2-ethylbutyl) Orthosilicate (50 uL) for a period of 30±2 minutes. The relative mean tissue viability of reconstructed cornea-like epithelium exposed to Tetrakis (2-ethylbutyl) Orthosilicate was 98.2%, which is above the threshold of 60% for requiring classification as a chemical causing serious eye damage or irritation.

Executive summary:

The eye irritation potential of Tetrakis (2 -ethylbutyl) Orthosilicate was evaluated using the Reconstructed Human Cornea-like Epithelium (RHCE; OECD TG 492), an alternative to the Draize Rabbit Eye Test. Duplicate tissues were treated with Tetrakis (2 -ethylbutyl) Orthosilicate for an exposure period of 30±2 minutes, followed by determination of the cytotoxicity expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan from MTT. Serious eye damage or irritation is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is ≤ 60% of the mean viability of the negative controls. In this case, the mean relative viability following exposure to Tetrakis (2 -ethylbutyl) Orthosilicate was >60%, therefore this substance is identified as a chemical not requiring classification for serious eye damage or irritation under the Regulation (EC) 1272/2008 on classification, labelling and packaging of substances and mixtures (CLP).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Since under REACH in-vitro studies are sufficient for compliance with Annex VII, no key in-vivo study has been conducted.

Corrosivity has been evaluated in-vitro using the human EpiDerm SCT Skin Model. The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. EpiDerm tissues were exposed to Tetrakis(2-ethylbutyl) Orthosilicate, skin corrosion was evaluated on the basis of cell viability. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with Tetrakis(2-ethylbutyl) Orthosilicate compared to the negative control tissues was 91.7% and 104.7%, respectively. Tetrakis(2-ethylbutyl) Orthosilicate is considered to be not corrosive; however more information is required on the irritating potential of Tetrakis(2-ethylbutyl) Orthosilicate within the top-down in-vitro testing approach.

EpiDerm SIT (EPI-200), the 3D-model of human epidermal keratinocytes seeded onto a dermal collagen matrix substitute, has been used to evaluate the in vitro irritation of Tetrakis(2-ethylbutyl) Orthosilicate. Triplicate human epidermal tissues were treated with Tetrakis(2-ethylbutyl) Orthosilicate (30 uL) for a period of 60 minutes and evaluated after 42 hours. The relative mean tissue viability of epidermal keratinocytes exposed to Tetrakis(2-ethylbutyl) Orthosilicate was 100.4%, which is above the 50% threshold for skin irritancy. Tetrakis(2-ethylbutyl) Orthosilicate is not considered to be a skin irritant.

The in vitro bovine corneal opacity and permeability (BCOP) test generated an in vitro irritation score (IVIS) of -1.24 for Tetrakis(2-ethylbutyl) Orthosilicate which is below the threshold of IVIS > 55 for requiring classification as a substance inducing serious eye damage. Eye irritation was assessed with the EpiOcular EIT (OCL-200), a 3D model of reconstructed human cornea-like epithelium test method for identifying chemicals not requiring classification and labelling for eye irritation or serious eye damage. Duplicate tissues were treated with Tetrakis(2-ethylbutyl) Orthosilicate (50 uL) for a period of 60 minutes and evaluated after 18 hours. The relative mean tissue viability of reconstructed cornea-like epithelium exposed to Tetrakis(2-ethylbutyl) Orthosilicate was 98.2%, which is above the threshold of 60% indicating no classification is required.

Justification for classification or non-classification

Tetrakis(2-ethylbutyl) Orthosilicate did not produce any evidence of skin irritation or corrosion in two in-vitro assays. These findings do not warrant classification of Tetrakis(2-ethylbutyl) Orthosilicate as a Category 1 Skin Corrosive or Category 2 Skin Irritant under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).

Tetrakis(2-ethylbutyl) Orthosilicate did not produce evidence of serious eye damage in an in-vitro BCOP assay or eye irritation in an RhCE in-vitro assay and does not warrant classification as Eye Damage Category 1 or Category 2 Eye Irritant under the Regulation (EC) 1272/2008 on classification, labeling and packaging of substances and mixtures (CLP).