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Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(2-ethylbutyl) orthosilicate
EC Number:
201-085-9
EC Name:
Tetrakis(2-ethylbutyl) orthosilicate
Cas Number:
78-13-7
Molecular formula:
C24H52O4Si
IUPAC Name:
tetrakis(2-ethylbutyl) orthosilicate
Test material form:
liquid
Details on test material:
Tetrakis(2-ethylbutyl) Orthosilicate
CAS 78-13-7
Expiration: May 2020
Clear colorless liquid
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

In chemico test system

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

Results and discussion

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.

In vitro / in chemico

Resultsopen allclose all
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.

Applicant's summary and conclusion

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).

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