Tetrakis(2-ethylbutyl) orthosilicate

Endpoint summary

Description of key information

In-Vitro Skin Sensitization: Direct Peptide Reactivity Assay- Inconclusive (OECD 442C)

In-Vitro Skin Sensitization: Human Cell Line Activation Assay- Inconclusive (OECD442E, Vehicle-100% Ethanol)

In-Vivo Skin Sensitization: LLNA: BrdU-ELISA- Sensitizing (OECD TG 442B)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 Aug 2017 - 30 Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Gelest, Inc. OS-140 Lot# 8E-33-134
- Expiration date of the lot/batch: May 2020
- Purity test date: 07 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a dessicator at room temperature
- Stability under test conditions: stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: not applicable, test material used as received

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

Details on the study design:

TEST SYSTEM OVERVIEW:
DPRA is an in chemico method which quantifies the remaining concentration of cysteine or lysine-containing peptide following 24 ± 2 hours of incubation with the test chemical at a temperature of 25 ± 2.5ºC. Relative peptide concentration was measured by high performance liquid chromatography (HPLC) with gradient elution and ultraviolet (UV) detection at 220 nm. Cysteine and lysine peptide percent depletion values were calculated and used in a prediction model which allowed the assigning of the test chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. One or more calibration curves were generated from analyses of standard solutions of cysteine and lysine peptides analyzed with the test samples.

SOURCE/STORAGE OF PEPTIDES:
Cysteine peptide: RS Synthesis, Louisville, KY USA and stored under frozen conditions (approximately -25°C) in darkness.
Product Name: 021317-1
Product Description: Custom Synthetic Peptide
Sequence: Ac-RFAACAA-COOH
Lot Number: 170221
Purity: 98.77%
Molecular Weight: 751.5 g/mole
Appearance: White, Powder
Expiration date: Not Given

Lysine peptide: RS Synthesis, Louisville, KY USA and stored under frozen conditions (approximately -25°C) in darkness.
Product Name: 021317-2
Product Description: Custom Synthetic Peptide
Sequence: Ac-RFAAKAA-COOH
Lot Number: 170217
Purity: 96.46%
Molecular Weight: 776.5 g/mole
Appearance: White, Powder
Expiration date: Not Given

POSITIVE CONTROL:
Cinnamic aldehyde (CAS #104-55-2): Sigma-Aldrich stored under ambient conditions in darkness. The reference substance was used as a positive control for the assay.
Product Name: Cinnamaldehyde – natural, ≥95%,FG
Product Number: W228613
CAS Number 104-55-2
Batch Number: MKBS2662V
Purity: 99%
Molecular Weight: 132.16 g/mole
Appearance: Yellow Liquid
Expiration date: 06/30/2019

SUBSTANCE PREPARATION:
Cysteine Peptide: 0.668 mM stock solution in pH 7.5 phosphate buffer (0.1M); calibration standards prepared in 20:80 ACN:pH 7.5 phosphate buffer
Lysine Peptide: 0.666 mM stock solution in pH 10.2 ammonium acetate buffer (100 mM); calibration standards prepared in
20:80 ACN:pH 10.2 ammonium acetate buffer
Positive Control: Cinnamic aldehyde 100 ± 10 mM in Acetonitrile
Test Substance: Tetrakis (2-ethylbutyl) Orthosilicate 100 ± 10 mM in Acetone

REFERENCE SAMPLE PREPARATION:
For each peptide assay, three sets of reference control samples were prepared:
Reference Control A (three replicates): 750 μL of the appropriate cysteine or lysine peptide stock solution; 250 μL of ACN
Reference Control B (six replicates): 750 μL of the appropriate cysteine or lysine peptide stock solution; 250 μL of ACN
Reference Control C (three replicates per solvent): 750 μL of the appropriate cysteine or lysine peptide stock solution; 250 μL of ACN or appropriate solvent (Lysine) or 200 μL of ACN and 50 μL of solvent (Cysteine) used to dissolve test substance

Co-elution Control: prepared for each peptide assay without the appropriate peptide stock solution; the appropriate buffer solution was used instead. The
co-elution control sample was used to verify whether the test substance absorbed at 220 nm and eluted at a similar retention time as the appropriate peptide.

NUMBER OF REPLICATES: Three replicate solutions of tetrakis(2-ethylbutyl) orthosilicate and cysteine or lysine containing peptides at a molar ratio of 1:10 and 1:50, respectively, were incubated for 24 ± 2 hours.

QUANTITATION:
Photometric Analysis: Agilent Series 1100 HPLC equipped with an Agilent Series 1100 variable wavelength detector, Agilent Series 1100 autosampler and Agilent Series 1100 autosampler thermostat.
Chromatographic separations: Agilent Zorbax SB-C18 analytical column (100 mm x 2.1 mm, 3.5 um particle size) with a Phenomenex Security Guard C18 cartridge (4 mm x 2.0 mm).

Quantitation of the respective peptide was determined by comparison of test samples to concurrent external standards containing the respective peptide in buffer. Peptide depletion was calculated by the comparison of mean peak area of solvent matched control samples to the test substance samples.

PREDICTION MODEL:
Mean of Cysteine and Lysine % Depletion Reactivity Class DPRA Prediction
0% ≤ mean % depletion ≤ 6.38%; No or minimal reactivity; Negative
6.38% < mean % depletion ≤ 22.62%; Low reactivity; Negative
22.62% < mean % depletion ≤ 42.47%; Moderate reactivity; Positive
42.47% < mean % depletion ≤ 100%; High reactivity; Positive

Positive control results:

The assay is considered valid if: the mean percent depletion values of the three positive control replicates should be between 60.8% and 100% with a standard deviation (SD) as of < 14.9% for the cysteine peptide and between 40.2% and 69.0% with an SD of < 11.6% for the lysine peptide.

The mean depletion of cysteine and lysine for the positive control substance cinnamic aldehyde was 70.7 ± 0.34 (N=3, CV= 0.48%) and 64.9 ± 0.99 (N=3, CV=1.5%) respectively.

Key result

Run / experiment:

other: Lysine

Parameter:

other: % peptide depletion

Remarks:

mean

Value:

0.251

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No or Minimal Reactivity

Remarks:

Negative DPRA prediction

Key result

Run / experiment:

other: Cysteine

Parameter:

other: % peptide depletion

Remarks:

mean

Value:

1.12

Vehicle controls validity:

not applicable

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No or Minimal Reactivity

Remarks:

Negative DPRA prediction

Other effects / acceptance of results:

The HPLC/UV system suitability assay is considered to be valid if the following conditions are met: a.) the calibration curve should have an r2 >0.99; b.) the mean peptide concentration of reference controls (Set A) should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls in ACN should be < 15.0%; c.) the mean percent depletion values of the three positive control replicates should be between 60.8% and 100% with a standard deviation (SD) as of < 14.9% for the cysteine peptide and between 40.2% and 69.0% with an SD of < 11.6% for the lysine peptide.

The test chemical data should be considered to be valid if the following criteria are met: a.) the mean peptide concentration of the reference controls (Set C) for the appropriate solvent used should be 0.50 ± 0.05 mM; b.) the SD for the percent depletion values of the three test substance replicates should be < 14.9% for the cysteine peptide and < 11.6% for the lysine peptide.

The system suitability for the reference cysteine peptide assay sequence passed all guideline acceptance criteria. The system suitability for the reference lysine peptide assay sequence passed all guideline acceptance criteria.

Interpretation of results:

study cannot be used for classification

Conclusions:

The in-chemico Direct Peptide Reactivity Assay was used to assess the potential of Tetrakis(2-ethylbutyl) Orthosilicate for its potential to interact with cysteine and lysine peptides showed no or minimal reactivity. However, as a precipitate was observed immediately upon addition of the test chemical solution to the peptide solution, due to low aqueous solubility of the test chemical, in this case it cannot be confirmed how much test chemical remained in the solution to react with the peptide. Therefore a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result.

Executive summary:

The mean depletion of cysteine and lysine for Tetrakis(2-ethylbutyl) Orthosilicate was 1.12 ± 1.0 (N=3, CV=91.7%) and 0.251 ± 0.43(N=3; CV =173%), respectively. Based on the cysteine 1:10/lysine 1:50 model, Tetrakis(2-ehtlybutyl) Orthosilicate would be predicted as having no or minimal reactivity and a negative DPRA prediction. However, as a precipitate was observed immediately upon addition of the test chemical solution to the peptide solution, due to low aqueous solubility of the test chemical, in this case it cannot be confirmed how much test chemical remained in the solution to react with the peptide. As per OECD TG 442C, in this case a positive result could still be used to support the identification of the test chemical as a skin sensitizer but no firm conclusion on the lack of reactivity should be drawn from a negative result. Therefore the results of this assay are considered inconclusive and cannot be used as a line of evidence in the in-vitro skin sensitization adverse outcome pathway for concluding on the skin sensitization potential of Tetrakis (2-ethylbutyl) Orthosilicate under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Remarks:

Human Cell Line Activation Test

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

other: OECD 442E In-Vitro Skin Sensitization: Human Cell Line Activation Test (hCLAT)

Deviations:

yes

Remarks:

Vehicle used: 100% Ethanol

Principles of method if other than guideline:

Principle of test: The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Gelest, Inc. OS-140 Lot# 8E-33-134
- Expiration date of the lot/batch: May 2020
- Purity test date: 07 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a dessicator at room temperature
- Stability under test conditions: stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: soluble in vehicle based on initial solubility screen

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

Details on the study design:

The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers

THP-1 cells (ATCC No. TIB-202; Lot# 60731979) are cultured, at 37°C under 5% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 μg/mL streptomycin. The positive controls for this assay are 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity) and the negative control is lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity).

A dose finding assay is performed to determine the CV75, being the test chemical concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value is used to determine the concentration of test chemicals for the main assay involving CD86/CD54 expression measurement. The test substance working solutions are mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well or 96-well flat-bottom plate. The treated plates are then incubated for 24±0.5 hours at 37°C under 5% CO2. After 24±0.5 hours of exposure, CD54/86 expression is measured by FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies at 4°C for 30 min. Cell viability was measured by Propidium Iodine staining.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Cell viabilities of medium and solvent/vehicle controls should be > 90%. The RFI values of boths CD86 and CD54 should not exceed positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). The MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
-Acceptance criteria for positive control: RFI values of both CD86 and CD54 should meet the positive criteria CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) and cell viability should be more than 50%.
-Acceptance criteria for test chemicals: Cell viability should be more than 50% in at least four tested concentrations. Negative results are acceptable only for test chemicals exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5000 ug/mL is used as the maximal test concentration of a test chemical than a negative result is acceptable even if the cell viability is above 90%.

Key result

Run / experiment:

other: CD86 Main Assay #1

Parameter:

other: EC150 (ug/mL)

Remarks:

concentration showing the Relative Fluorescence Intensity values in CD86

Value:

5 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD54 Main Assay #1

Parameter:

other: EC200 (ug/mL)

Remarks:

concentration showing the Relative Fluorescence Intensity values in CD54

Value:

5 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD54 Main Assay #2

Parameter:

other: EC200 (ug/mL)

Remarks:

concentration showing the Relative Fluorescence Intensity values in CD54

Value:

5 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: CD86 Main Assay #2

Parameter:

other: EC150 (ug/mL)

Remarks:

concentration showing the Relative Fluorescence Intensity values in CD86

Value:

5 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: 100% Ethanol (1% final dilution) was shown not to be a senstizer in THP-1 cells. Both positive controls (DNCB and Nickel Sulfate) in 100% Ethanol as vehicle produced expected results. Two additional proficiency controls (Isopropanol- non sensitizer; 2-Mercapobenzothiazole- moderate sensitizer) produced expected results.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 100% Ethanol (1% final dilution) met the criteria for the solvent control with vaibility > 90% and RFI values of boths CD86 and CD54 did not exceed the positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in either replicate. The negative control utilized, isopropanol did not exceed the positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in either replicate.

-Acceptance criteria for positive control: For DNCB and 2-Mercaptobenzothiazole, RFI values of both CD86 and CD54 met the positive criteria CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in both replicates.

Interpretation of results:

study cannot be used for classification

Conclusions:

The in-vitro Human Cell Line Activation Assay (hCLAT) was used to assess the potential of Tetrakis(2-ethylbutyl) Orthosilicate for its potential to activate dendritic cells through measurement of markers CD86 and CD54. Since Tetrakis (2-ethylbutyl) Orthosilicate was shown to be insoluble both in saline and DMSO, the test substance was tested in 100% Ethanol. In this modified hCLAT assay, Tetrakis (2-ethylbutyl) Orthosilicate was negative for CD54 or CD86 markers of dendritic cell activation. It should be noted that the experimentally measured Log Kow is >6.91. Test chemicals with a Log Kow greater than 3.5 tend to produce false negative results. Therefore, a conclusion on the lack of activity cannot be drawn with sufficient confidence in case of a negative result.

Executive summary:

In a modified hCLAT assay, the EC150 and EC200 for Tetrakis (2-ethylbutyl) Orthosilicate was >5000 uL/mL (highest dose tested). Based on these results, Tetrakis (2-ethylbutyl) Orthosilicate would be predicted as having no dendritic activation and a negative hCLAT prediction. However, it should be noted that the experimentally measured Log Kow is >6.91. As per OECD TG 442E, in this case a positive result could still be used to support the identification of the test chemical as a skin sensitizer but no firm conclusion on the lack of activation should be drawn from a negative result. Therefore the results of this assay are considered inconclusive and cannot be used as a line of evidence in the in-vitro skin sensitization adverse outcome pathway for concluding on the skin sensitization potential of Tetrakis (2-ethylbutyl) Orthosilicate under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 2017 - Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Gelest, Inc. OS-140 Lot# 8E-33-134
- Expiration date of the lot/batch: May 2020
- Purity test date: 07 June 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored in a dessicator at room temperature
- Stability under test conditions: stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: soluble in vehicle based on initial solubility screen

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

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories (Bar Harbor, ME)
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Pathogen and opportunist-free as evaluated by JAX® Mice, Clinical & Research Services (JMCRS)
- Age at study initiation: 8 weeks
- Weight at study initiation: Irritation Screen: 19.2 - 20.9g; Main Test: 18.4 - 22.9g
- Housing: During acclimation, mcie were housed two per cage in suspended wire-bottom cages; during the study, mice were housed one per cage.
- Diet (e.g. ad libitum): PMI Rodent Chow (Diet No. 5001)
- Water (e.g. ad libitum): Tap water, ad-libitum
- Acclimation period: 5 days
- Indication of any skin lesions: The animals were observed prior to the study start to ensure that no skin lesions were present on the ears.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 67.5 - 72.8°F
- Humidity (%): 5.7 to 49.6%
- Photoperiod (hrs dark / hrs light): 12 hrs dark 12 hrs light
- IN-LIFE DATES: From: 22 Nov 2017 To: 13 Dec 2017

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10%, 25%, 50%

No. of animals per dose:

5 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Test substance soluble in Acetone: Olive Oil (4:1) and formed a clear light yellow liquid up to 50%
- Irritation: Irritation screen was conducted at 25%, 50% and 100%. Test substance produced ear swelling of 52.9% over control at 100%. Ear swelling of 27.9% was oboserved at 50%, however the standard deviation of the two animals used in the screen overlapped the 25% cutoff specified in the OECD TG. Therefore 50% was chosen as the high dose for the main assay.
- Systemic toxicity: All animals gained weight during the irritation screen, no systemic toxicity was evident.
- Ear thickness measurements: Mean% ear swelling values for 25%, 50%, and 100% were 5.6%, 5.6%, and 17.6% over control for Day 3-1 respectively and 11.1%, 27.8%, and 52.9% over control for Day 6-1 respectively.
- Erythema scores: Mean erythema scores on Day 6 for 25%, 50% and 100% were 0, 0 (left, right), 0.5, 0 (left, right), and 0.5, 0.5 (left, right) respectively. Flaking skin was observed at 100%.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Mousel Local Lymph Node Assay (LLNA) BrdU:ELISA (OECD TG 442B)
- Criteria used to consider a positive response: The ratio of the mean proliferation in each treated group to that in the concurrent VC group, termed the SI, is determined, and should be ≥1.6 before further evaluation of the test substance as a potential skin sensitizer is warranted

TREATMENT PREPARATION AND ADMINISTRATION:
Preliminary Dermal Irritation Screen: Three groups of CBA/J mice (two animals per group) were treated with increasing concentrations of the test article (25% and 50% (v/v) in AOO, and 100%). Treatment was via topical application of the test article concentrations to the dorsum of each ear once daily for three
consecutive days. The test article was spread over the entire dorsal surface of the ear using a micropipette to deliver 25 μl/ear.

Main Test: The test article concentrations used in the main LLNA study were chosen such that the maximum concentration tested was the highest achievable solution of the test article in the vehicle, while avoiding both overt systemic toxicity and excessive local dermal irritation. Five groups of CBA/J mice (five animals per group) were treated by topical application of the test article concentrations, vehicle control or positive control in the same manner as in the screen.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For each test group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Students’ t-Test was run to statistically compare each test article dose group to the vehicle control group. Although specified in the test guidelines, these calculations and results were not incorporated into the interpretation of the data. An SI value of 1.6 or more is the sole determinant for a positive sensitization response.

Positive control results:

The positive control, hexyl-cinnamic aldehyde (25% v/v) produced an SI of 5.4, indicating a positive LLNA: BrdU:ELISA assay response at the exposure level indicated in the OECD 442B test guideline.

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10% (v/v) Tetrakis(2-ethylbutyl) Orthosilicate

Key result

Parameter:

SI

Value:

2.5

Test group / Remarks:

25 % (v/v) Tetrakis(2-ethylbutyl) Orthosilicate

Key result

Parameter:

SI

Value:

4.9

Test group / Remarks:

50% (v/v) Tetrakis(2-ethylbutyl) Orthosilicate

Key result

Parameter:

other: EC1.6 (%)

Value:

15.5

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION:
Stimulation Indices were calculated by dividing the mean optical density value (a representation of the total amount of LNC proliferation) for each animal by themean optical density value for the vehicle control group. The mean SI and S.D. for each treatment group was then calculated. A result is regarded as a positive response in the LLNA if at least one concentration results in a 1.6-fold or greater increase in LNC proliferation relative to that of vehicle control animals, as indicated by an SI of 1.6 or more.

EC1.6 CALCULATION:
The EC1.6 was calculated to be 15.4% (v/v), according to the following:

EC1.6 = c + [(1.6-d) /(b-d)] x (a-c)
= 10% + [(1.6-1.1)/(2.5-1.1)] x (25%-10%)
= 10% + (0.357 x 15%)
= 10% + 5.4%
=15.4%

Where:
a = test article concentration at SI value “b”
b = SI value nearest to but greater than 1.6
c = test article concentration at SI value “d”
d = SI value nearest to but less than 1.6

CLINICAL OBSERVATIONS: Abnormal physical signs including localized hair loss on head were observed (1 animal at 10% v/v and 25% v/v, and 3 animals at 50% v/v test article concentrations).

BODY WEIGHTS: All animals gained weight by study termination

Test Article Dosing- No difficulties were experienced with the application of the test article to the ears or with the retention of

test article by the ear surface.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The in-vivo mouse LLNA Brdu: ELISA Assay to assess Tetrakis(2-ethylbutyl) Orthosilicate for its skin sensitization potential produced positive indication of stimulated proliferation in the mouse lymph node. This finding warrants classification of Tetrakis (2-ethylbutyl) Orthosilicate as a Category 1 skin sensitizer under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Executive summary:

Tetrakis (2-ethylbutyl) Othosilicate was applied to the ears of CBA:JN mice at 10%, 25% and 50%. The stimulation index (SI) for Tetrakis(2-ethylbutyl) Orthosilicate was 1.1, 2.5 and 4.9 at 10%, 25%, and 50% respectively. Under OECD TG 429B, an SI of 1.6 is considered a positive indication of skin sensitization. The calculated EC1.6 for Tetrakis (2-ethylbutyl) Orthosilicate was determined to be 15.5%. This data indicates a positive prediction for skin sensitization. While GHS criteria does not yet exist for sub-categorization into Category 1A high potency or 1B low/moderate potency based on an SI of 1.6, the magnitude of the dose response is suggestive of low to moderate potency. In addition, published literature supports that application of the current LLNA sub-categorization cutoff criteria of 2% (see GHS Rev. 7 §3.4.2.2.3.3) to the EC1.6 produces comparable performance in sub-categorization for the LLNA: BrdU-ELISA (Ha et al. 2017 Reg. Tox. Pharm. 85:124-131). The EC1.6 for Tetrakis (2-ethylbutyl) Orthosilicate was calculated as 15.5%, supporting a Category 1B classification.Therefore, the available test data warrants classification of Tetrakis (2-ethylbutyl) Orthosilicate as a Category 1B skin sensitizer under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A weight of evidence approach was applied to determine the skin sensitization potential of Tetrakis (2-ethylbutyl) Orthosilicate using both in-vitro and in-vivo data.

The three in-vitro assays used in the “2 out of 3” defined approach target different key events of the skin sensitization adverse outcome pathway: key event #1 is the assessment of protein binding in chemico (OECD 442 C: DPRA); key event #2, the trigger of an antioxidant/inflammatory response in keratinocytes (OECD 442D: KeratinoSens) and; key event #3, the activation of dendritic cells (OECD 442E: h-CLAT). In the DPRA (key event #1), no or minimal peptide binding was observed with Tetrakis (2 -ethylbutyl) Orthosilicate. However, it was noted that due to the limited solubility of the test substance (precipation observed immediately and at 24 hours) the substance/peptide solutions were considered emulsions and therefore the results could be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case. In the human cell line activate test (h-CLAT, key event #3), no activation of CD86 or CD54 was observed in dendritic THP-1 cells. However it should be noted that the Log Kow for Tetrakis (2-ethylbutyl) Orthosilicate was measured to be > 6.91. According to OECD TG 442E, test chemicals with a Log Kow >3.5 tend to produce false negative results. Therefore, negative results from test chemicals with a Log Kow >3.5 should not be considered.

Consequently, two inconclusive results were obtained in the DPRA and h-CLAT. Given the high Log Kow (>6.91) and extremely low water solubility (<LOD) which dictated the inconclusive results in the first two in-vitro assays, similar issues would be expected for other in-vitro assays as well, and would not likely contribute definitive information. Following the "2 out of 3" in-vitro defined approach, a conclusion on the skin sensitization potential could not be determined for Tetrakis (2-ethylbutyl) Orthosilicate.

Therefore, to produce definitive data to allow a conclusion on the skin sensitization potential of Tetrakis (2-ethylbutyl) Orthosilicate, an in-vivo Local Lymph Node Assay in the mouse was conducted.

Tetrakis (2-ethylbutyl) Othosilicate was applied to the ears of CBA:J mice at 10%, 25% and 50% according to OECD TG 442B LLNA: Brdu ELISA. The stimulation index (SI) for Tetrakis(2-ethylbutyl) Orthosilicate was 1.1, 2.5 and 4.9 at 10%, 25%, and 50% respectively. Under OECD TG 442B, an SI of 1.6 is considered a positive indication of skin sensitization. The calculated EC1.6 for Tetrakis (2-ethylbutyl) Orthosilicate was determined to be 15.5%. This data indicates a positive prediction for skin sensitization. While GHS criteria does not yet exist for sub-categorization into Category 1A high potency or 1B low/moderate potency based on an SI of 1.6, the magnitude of the dose response is suggestive of low to moderate potency. In addition, published literature supports that application of the current LLNA sub-categorization cutoff criteria of 2% (see GHS Rev. 7 §3.4.2.2.3.3) to the EC1.6 produces comparable performance in sub-categorization for the LLNA: BrdU-ELISA (Ha et al. 2017 Reg. Tox. Pharm. 85:124-131). The EC1.6 for Tetrakis (2-ethylbutyl) Orthosilicate was calculated as 15.5%, supporting a Category 1B classification.

Therefore, the available weight of evidence warrants classification of Tetrakis (2-ethylbutyl) Orthosilicate as a Category 1B skin sensitizer under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).