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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:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted 04 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08

In chemico test system

Details on study design:
The test substance was prepared at a 100 mM concentration in acetonitrile considering a purity/contents of 99.0% and its molecular weight. The C-containing peptide was incubated with
the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate prepared as a 50 mM solution in acetonitrile.
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Results and discussion

Positive control results:
67.6% (Cys-peptide) was depleted by the positive control substance ethylene glycol dimethacrylate using acetonitrile as solvent.
13.6% (Lys-peptide) was depleted by the positive control substance ethylene glycol dimethacrylate using acetonitrile as solvent.

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Peptide depletion [%] Cys
Run / experiment:
mean
Value:
0.67
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Peptide depletion [%] Lys
Run / experiment:
mean
Value:
0.62
Vehicle controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
other: inactive in the direct peptide binding assay
Executive summary:

The reactivity of the UVCB-substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the

test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was formulated at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The mean C-peptide depletion, caused by the test substance was determined to be 0.67%.

The mean K-peptide depletion, caused by the test substance was determined to be 0.62%.

The mean peptide depletion was calculated to be 0.64% and therefore the substance was determined to be inactive in the direct peptide binding assay.