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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Jun - 24 Aug 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted Feb 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on study design:
TEST METHOD
The DPRA is an in chemico method which quantifies the remaining non-depleted concentration of cysteine- or lysine-containing peptides following 24 h incubation with the test substance at 25 ± 2.5 °C. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by HPLC with gradient elution and UV detection at 220 nm. Cysteine- and lysine peptide percent depletion values are then calculated and used in a prediction model which allows assigning the test substance to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

TEST SYSTEM
- Supplier: GenScript, Piscataway, US and RS Synthesis, Louisville, US
- Synthetic cysteine- (C-) containing peptide:
Alternative name: Ac-RFAACAA-COOH
Molecular weight: 751.9 g/mol
- Synthetic lysine- (K-) containing peptide:
Alternative name: Ac-RFAAKAA-COOH
Molecular weight: 776.2 g/mol

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: ethylene glycol dimethacrylate
The positive control was prepared as a 50 mM preparation in acetonitrile.

CO-ELUTION CONTROL
- Sample prepared of the respective peptide buffer and test substance but without peptide.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM preparation in acetonitrile. The test substance was soluble in the vehicle.

PEPTIDE STOCK SOLUTION PREPARATION
C-containing peptide:
- Solvent: phosphate buffer (pH 7.5)
- Concentration: 0.667 mM
K-containing peptide:
- Solvent: ammonium acetate buffer (pH 10.2)
- Concentration: 0.667 mM
The peptide stock solution was used for preparing the calibration samples and the test-substance and control samples.

INCUBATION CONDITIONS
- Peptide to test substance ratios: C-containing peptide: 1:10 (0.5 mM peptide, 5 mM test substance); K-containing peptide: 1:50 (0.5 mM peptide, 25 mM test substance)
- Temperature: 25 ± 2.5 °C
- Duration: 24 ± 2 h
- Light conditions: in the dark

NUMBER OF REPLICATES
for each peptide in triplicates for treatment and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Agilent HP 1100 with DAD (Software: Dionex Chromeleon)
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column „Security Guard“ C18, 4 mm x 2 mm
- Analytical balance: Accuracy 0.1 mg
- HPLC mobile phase:
A: 0.1% (v/v) trifluoracetic acid (99+%) in de-ionized water, HPLC grade
B: 0.085% (v/v) trifluoracetic acid (99+%) in acetonitrile, HPLC grade
- Flow: 0.35 mL/min
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 25
% B: 10, 25, 90, 90, 10, 10
- Wavelength: 220 and 258 nm
- Injection volume: 2 µL

OTHER
Prior to HPLC analysis the samples were visually investigated for any precipitate that may have formed during the exposure period.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: % mean depletion of cystein-peptide
Value:
-0.29
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: % mean depletion of lysine-peptide
Value:
-0.69
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Parameter:
other: % overall mean depletion
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Any other information on results incl. tables

Table 5. Mean peptide depletions of cysteine, lysine and both peptides.

 

Cysteine-Peptide

Lysine-Peptide

Mean of both depletions (%)

Mean depletion (%)

SD

Mean depletion (%)

SD

Test substance

-0.29

0.13

-0.69

0.99

0.00

Positive control

55.00

4.60

6.11

0.44

30.55

The mean C-peptide depletion, caused by the test substance was determined to be -0.29%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.69%.

Negative depletions were considered to be 0 for calculation of the mean peptide depletion, which was thus calculated to be 0.0%.

No co-elution of test substance and peptides was noticed.

The acceptance criteria were met.

TEST-SPECIFIC CONFOUNDING FACTORS

- Precipitation: The test substance was soluble in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after 24 -hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.

- Co-elution: No co-elution of the test substance and peptides occurred as demonstrated by the consistent values of the area ratios 220 nm/ 258 nm.

 

COMPARISON WITH HISTORICAL CONTROL DATA

The positive control caused depletion of the cysteine-peptide comparable to historic data. The lysine-peptide caused a mean peptide depletion of 6.11 which is amongst the lowest values observed for this reaction in the lab. However, the lysine reaction is known to be minimal, only, and the validity of the test is not considered to be affected.

Applicant's summary and conclusion

Interpretation of results:
other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
Conclusions:
Based on the observed results it was concluded that the test substance revealed a minimal or no reactivity in the direct peptide reactivity assay under the test conditions chosen.