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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:
key study
Study period:
15.08.2016 - 19.08.2016
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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Department of Health of the Government of the United Kingdom
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent

In chemico test system

Details on study design:
TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: acetonitrile

CONTROLS
- Positive control: Cinnamic Aldehyde (100 nM solution in acetonitrile)

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA-OH (MW = 751.5 g/mol)
-- Lysine- (K-) containing peptide: Ac-RFAAKAA-OH (MW = 776 g/mol).
- Stock solution:
-- C-containing peptide: 0.667 mM of peptide in pH 7.5 phosphate buffer
-- K-containing peptide: 0.667 mM of peptide in pH 10.2 ammonium acetate buffer
- Ratios
- C-containing peptide: 0.5 mM peptide, 5 mM test substance
- K-containing peptide: 0.5 mM peptide, 25 mM test substance

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Incubation: at 25°C for minimum 22 hours incubation

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Column: Agilent Zorbax SB C18, 3.5 µm, 100 x 2.1 mm
- Wavelength: 220 nm (UV)

DATA EVALUATION
- % peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of reference control samples B)

EVALUATION RESULTS
- Mean cysteine and lysine % depletion: =<0 - =<6.38; Class 'No or minimal reactivity'; DPRA prediction: Negative
- Mean cysteine and lysine % depletion: =<6.38 - =<22.62; Class 'Low reactivity'; DPRA prediction: Positive
- Mean cysteine and lysine % depletion: =<22.62 - =<42.47; Class 'Moderate reactivity'; DPRA prediction: Positive
- Mean cysteine and lysine % depletion: =<42.47 - =<100; Class 'High reactivity'; DPRA prediction: Positive

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Peptide depletion
Run / experiment:
C-containing peptide (mM)
Value:
5.46
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Peptide depletion
Run / experiment:
K-containing peptide (mM)
Value:
0
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: Mean peptide depletion
Value:
2.73
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
No co-elution peaks were observed in either the Cysteine or Lysine assays. Small micro droplets were observed in the incubated Lysine peptide sample indicative of potential phase separation. As the test item is present in the Lysine incubation samples in excess relative to the amount of peptide (the concentration of the test item estimated to be present was 15.7 mM) then enough test item is considered to have remained in solution so as to ensure the result is valid and hence there is considered to have been no impact on the data reported.

Applicant's summary and conclusion

Interpretation of results:
other: DPRA prediction: negative (no or minimal reactivity) according to OECD 442C
Conclusions:
The test substance showed no peptide reactivity in the DPRA test under the current conditions.
Executive summary:

In the current study the skin sensitisation effect of the test item was assessed in the Direct Peptide Reactivity Assay (DPRA) in chemico assay according to OECD 442C and GLP.

This method quantifies the remaining concentrations of cysteine- or lysine-containing peptides after 24 hours incubation with the test item at 25 +/-2.5 °C. The percentage of depletion of cysteine- and lysine-peptides is calculated and used in a prediction model to assign the test chemical to one of four reactivity classes and in this way discriminate between sensitisers and non-sensitisers.

The test item was dissolved at a 100 mM concentration in acetonitrile (ACN) and 3 samples of the test item were incubated with a typical concentration of 0.667 mM of cysteine in a pH 7.5 phosphate buffer or 0.667 mM of lysine in a pH 10.2 ammonium acetate buffer. Incubation was done for 22 hours. The remaining non-depleted concentrations were determined by HPLC with gradient elution and UV-detector at 220 nm.

The mean C-peptide depletion, caused by the test substance was determined to be 5.46%.

The mean K-peptide depletion, caused by the test substance was determined to be -0.045%.

Negative depletions are considered to be "zero" for the calculation of the mean peptide depletion. Based on the results and the prediction model, the mean depletion was 2.73% and following the depletion model the reactivity class is 'no to minimal reactivity' and the DPRA prediction is negative.