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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:
07.2017
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:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes
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
- Freshly prepared immediately prior to use
- Stock solution: 100 mM
- Vehicle: no solvent compatible with the DPRA was found to dissolve the test item in the required concentration (100 mM)

CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: Ac-RFAACAA
-- Lysine- (K-) containing peptide: Ac-RFAAKAA
- 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

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis starting from 22 to 26 hours after sample preparation and the analysis time will be set up in order to keep the HPLC analysis time less than 30 hours.
- Calibration samples: samples of a known peptide concentration measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples will be incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours and visually investigated for any precipitate that may occur during the exposure period.
- Reference controls, co-elution controls as well as the positive control will be set up in parallel (see table below)

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: Agilent, 1200 Series, with Chemstation, Rev. B.04.01
- Wavelength: 220 nm and 258 nm
- Detector: UV detector

DATA EVALUATION
- The percent peptide depletion (PPD) will be calculated according to the following formula:
PPD = [1 – (Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference Control C)] * 100

ACCEPTANCE CRITERIA
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.
The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

EVALUATION RESULTS
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
- In case the mean peptide depletion cannot be determined due to invalid K-peptide depletion the evaluation is performed as follows:
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

Results and discussion

Positive control results:
not available

In vitro / in chemico

Results
Parameter:
other: mean depletion % of peptide
Run / experiment:
0
Vehicle controls valid:
not applicable
Negative controls valid:
not applicable
Positive controls valid:
not applicable
Remarks on result:
not determinable because of methodological limitations
Other effects / acceptance of results:
not available

Any other information on results incl. tables

No solvent compatible with the DPRA was found to dissolve the test item in the required concentration (100 mM).

The test item solubility was tested in acetonitrile, water, isopropanol, methanol, ethanol, 1,4 -butandiol and dimethylformamide in a concentration of 100 mM. In none of the solvents the substance showed visible partial solubility so that lower concentrations were not tested.

Therefore the study could not be performed due to technical limitations.

Applicant's summary and conclusion

Interpretation of results:
other: The study could not be performed
Conclusions:
Because no solvent compatible with the DPRA was found to dissolve the test item in the required concentration (100 mM) the study could not be performed.
Executive summary:

In the planned study the skin sensitisation effect of the test item should be assessed in an in chemico assay according to OECD 442C and in compliance to GLP.

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

No solvent compatible with the DPRA was found to dissolve the test item in the required concentration (100 mM).

The test item solubility was tested in acetonitrile, water, isopropanol, methanol, ethanol, 1,4 -butandiol and dimethylformamide in a concentration of 100 mM. In none of the solvents the substance showed visible partial solubility so that lower concentrations were not tested.

Therefore the study could not be performed.