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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:
13. April - 28. April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

In chemico test system

Details on study design:
HPLC system
Designation: HPLC_4
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981

Test solutions
Dilution buffers
• 2 mL Acetonitrile were mixed with 8 mL phosphate buffer, pH 7.5 (Peptide dilution buffer C)
• 2 mL Acetonitrile were mixed with 8 mL ammonium acetate buffer, pH 10.2 (Peptide dilution buffer K)
Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
• 0.667 mM Cys-Peptide solution was prepared by dissolving 22.5 mg of the peptide in 45.0 mL phosphate buffer, pH 7.5. (batch no. 20180425)
• 0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45.0 mL ammonium acetate buffer, pH 10.2. (batch no. 20180426)
Peptide calibration standards
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer (see chapter 7.2.1): 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM Peptide.
Calibration samples were analysed before the samples containing the test item. Blank dilution buffer was also measured.
Test item samples
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were pre-pared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions described in chapters 7.2.2 and 7.1. A final volume of 1 mL per sample was prepared for each sample.

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22.08 h for Cys-peptide and 24 h for Lys-peptide.
All three replicates of test item incubated with Cys-peptide and all three replicates of Posi-tive Control Cinnamaldehyde were turbid after incubation.

Results and discussion

Positive control results:
a) The mean peptide depletion and standard deviation of the three replicates of the positive control Cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Depletion %
Run / experiment:
Lys-Peptide, mean
Value:
20.35
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Depletion %
Run / experiment:
Cys-Peptide, mean
Value:
99.44
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The DPRA predicition is “positive” with high reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.
Executive summary:

The study was performed in order to evaluate the reactivity of the test item2,2'-Azobis(2,4-dimethylvaleronitrile)towards Cysteine (Cys-) and Lysine (Lys-) containing peptides. A test item solution in Acetonitrile was incubated 24 ± 2 h at 25 °C together with Cysteine and Lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.

The peptide depletion values after incubation are shown in Table

Results

 

Cys-peptide
depletion [%]

Lys-Peptide
depletion [%]

Mean peptide
depletion [%]

Experiment

99.44

20.35

59.89