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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-07-20 to 2020-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: EU method B.59: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)
Version / remarks:
2017-02-14
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2020-06-26
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(allyloxy)ethanol
EC Number:
203-871-7
EC Name:
2-(allyloxy)ethanol
Cas Number:
111-45-5
Molecular formula:
C5H10O2
IUPAC Name:
2-(allyloxy)ethanol
Test material form:
liquid

In chemico test system

Details on the study design:
TEST CHEMICALS
Synthetic peptides:
- Peptides with 95.8 % (cysteine) and 93.84 % (lysine) purity
- Sequence cysteine peptides: Ac-RFAACAA-OH (MW = 750.9 g/mol)
- Sequence lysine-Peptide: Ac-RFAAKAA-OH (MW = 775.9 g/mol)

Buffers:
- Phosphate buffer pH 7.5 ± 0.05
- Ammonium acetate buffer pH 10.2 ± 0.05

Peptide Stock Solutions:
- Cysteine peptide stock solution: 0.667 mM, 0.501 mg/mL
- Lysine peptide stock solution: 0.667 mM, 0.518 mg/mL

Control samples:
- Reference control A (system suitability): Peptide stock solutions combined with acetonitrile, 3 replicates
- Reference control B (stability of the peptides): Peptide stock solutions combined with acetonitrile and measured before and after the reaction, 3 replicates
- Reference control C (solvent control): Peptide stock solutions combined with the respective solvent of the test item (and acetonitrile in case of cysteine peptide), 3 replicates
- Positive control (cinnamaldehyde): Peptide stock solutions combined with positive control stock solution (100 mM) (and acetonitrile in case of cysteine peptide), 3 replicates
- Co-elution controls (test item and peptide coelution): Test item stock solution (and acetonitrile in case of cysteine peptide) combined with the respective buffer solutions in each run

Calibration solutions:
Six calibration standard points were prepared by serial dilution of the peptide stock solutions with the following nominal molarities: STD 1 = 0.534 mM, STD 2 = 0.267 mM, STD 3 = 0.1335 mM, STD 4 = 0.0667 mM, STD 5 = 0.0334 mM and STD 6 = 0.0167 mM. As dilution buffer a 20% acetonitrile buffer solution (phosphate or ammonium acetate) was used. For the zero standard point (STD 7 = 0 mM) dilution buffer was used.

TREATMENT
Peptide stock solutions (750 µL) were combined with test item/positive control/solvent stock solutions (50 µL for cysteine peptides or 250 µL for lysine peptides) were combined and placed to the HPLC autosampler for 24 ± 2 h incubation at 22.5 - 30 °C in the dark. HPLC analysis of the batch of reaction samples started 24 ± 2 h hours after the test chemical was added to the peptide solution. The batches consisted of 2 parts: one part with the A reference controls, the calibration standards and the co-elution controls. These samples could be run before the 24 ± 2 h incubation time ends and right before the other part started or right after the other part. The other part contained the B and C reference controls, the positive controls and the reaction samples and these samples were run right after the 24 ± 2 h incubation time ended. The total HPLC analysis time was less than 21 hours. 3 replicates of reaction samples were measured.

TECHNICAL EQUIPMENT
- HPLC: Shimadzu LC-2030i Prominence (serial number L21445402951AE)
- Detector: D2 lamp (220 nm)
- Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
- Column temperature: 30°C
- Sample temperature: 25°C
- Injection volume: 7 µL
- System equilibration: running mobile phase A and mobile phase B in a ratio of 1:1 for 2 hours at 30°C column temperature and running the gradient twice before injecting the first sample
- Run time: 20 min
- Mobile Phase A: 0.100 % (v/v) trifluoroacetic acid in ultrapure water
- Mobile Phase B: 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Results and discussion

Positive control results:
The acceptance criteria were met for the positive control with a cysteine peptide depletion value of 65.49 % ± 0.31 % and a mean lysine peptide depletion value of 52.71 % ± 0.87 %.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: 1
Parameter:
other: mean peptide depletion (%) for cysteine
Value:
9.4
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: mean peptide depletion (%) for lysine
Value:
1.27
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: 1
Parameter:
other: mean peptide depletion of both peptides (%)
Value:
5.34
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Any other information on results incl. tables

Co-elution: The test chemical did not absorb at 220 nm significantly (since the peak of co-elution control did not reach 10 % compared to the respective reference control) when tested with the cysteine and lysine peptides. Therefore, no co-elution was observed with either of the peptides. The range of retention time for cysteine peptide was between 8.229 and 8.458 and the range of retention time for lysine peptide was between 6.126 and 6.283.


 


System suitability: Reference control A replicates were included in the HPLC run sequence to verify the HPLC system suitability prior to analysis. The mean peptide concentration of reference control A sample replicates was 0.50 mM for the cysteine and 0.49 mM for the lysine peptide.


 


Table 2: Cysteine peptide depletion values for the positive control and the test item















































Sample



Peptide conc. Calculated (mM)



Mean peptide conc. (mM)



Peptide depletion (%)



Standard deviation (SD)


for peptide depletion (%)



CINNAMALDEHYDE,


rep I



0.16



 


 


0.16



65.72 %



 


 


0.31 %



CINNAMALDEHYDE,


rep II



0.16



65.14 %



CINNAMALDEHYDE,


rep III



0.16



65.62 %



Test item, rep I



0.46



 


0.44



4.82 %



 


7.27 %



Test item, rep II



0.40



17.78 %



Test item, rep III



0.46



5.59 %



 


Table 3: Lysine peptide depletion values for the positive control and the test item















































Sample



Peptide conc. Calculated (mM)



Mean peptide conc. (mM)



Peptide depletion (%)



Standard deviation (SD)


for peptide depletion (%)



CINNAMALDEHYDE,


rep I



0.23



 


 


0.23



53.68 %



 


 


0.87 %



CINNAMALDEHYDE,


rep II



0.24



51.99 %



CINNAMALDEHYDE,


rep III



0.23



52.46 %



Test item, rep I



0.48



 


0.49



2.09 %



 


0.71 %



Test item, rep II



0.49



0.82 %



Test item, rep III



0.49



0.91 %



 

Applicant's summary and conclusion

Interpretation of results:
other: No peptide reactivity
Conclusions:
Based on these results and the cysteine 1:10 / lysine 1:50 prediction model, the test item showed no or minimal reactivity towards the synthetic peptides; thus is not a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.
Executive summary:

In the course of this study, the skin sensitisation potential of the test item was studied using the Direct Peptide Reactivity Assay method (DPRA Method). For the test chemical and positive control substance, in order to derive a prediction, two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item. Peptide depletion which resulted from the positive control cinnamaldehyde was 65.49 % ± 0.31 % for cysteine peptides and 52.71 % ± 0.87 % for lysine peptides, fulfilling all acceptance criteria for the positive control. The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for cysteine (0.48 – 0.50 mM) and lysine peptides (0.49 mM) and the CV % for the nine reference controls B and C in acetonitrile were 1.9 % and 0.4 % percentages for cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay. The percent cysteine peptide depletion value of the test item was 9.40 % ± 7.27 % while the percent lysine peptide depletion was 1.27 % ± 0.71 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore, the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 5.34 %, which did not exceed the 6.38 % threshold of the applicable prediction model and fell into the no or minimal reactivity class.