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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
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))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential.
To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC.
The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
a off-white powdery solid, without irritating odor

In vitro 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
Usage and calibration followed the corresponding SOP 114 00 526 in the current edition.
Column
An ACE Excel SuperC18 150x3 mm column with 3 μm particles and pre-column Phenom-enex SecurityGuard C18, 4x3 mm was used. This column was selected because it delivers substantially better peak shape for the peptides than the Agilent Zorbax SB-C18 column recommended in the OECD 442C guideline.
HPLC program
Eluent A H2O + 0.1 % TFA
Eluent B Acetonitrile + 0.085 % TFA
Flow rate 0.55 mL/min
Injection volume 7 μL
Column temperature 30 °C
Wavelength 1 220 nm
Wavelength 2 258 nm
Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
1 Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.:P170415-2-LR569638
Purity: 98.10 %
2 Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170415-2-LR569640
Purity: 98.85 %

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Parameter:
other: Mean peptide depletion [%]
Run / experiment:
1
Value:
ca. 4.67
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Parameter:
other: Mean peptide depletion [%]
Run / experiment:
3
Value:
ca. 4.55
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Parameter:
other: Mean peptide depletion [%]
Run / experiment:
4
Value:
ca. 1.61
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid

Any other information on results incl. tables

The mean peptide depletion in the Lys-peptide and Cys-peptide assay in experiment 1 was 4.67 %, therefore the test had to be repeated for verification of this result. Experiment 2 was not valid for the Lys-peptide and had to be repeated. The mean peptide depletion in the Lyspeptide and Cys-peptide in experiment 3 was 4.55 %. The result of the first experiment was considered verified. However due to an error in the first evaluation of experiment 1 experiment 3 was considered as the first valid experiment at this time and was repeated again because the mean peptide depletion fell within the range 3 - 10 %. In experiment 4 the mean peptide depletion was 1.61 % which confirmed again the results of the previous experiments as:

DPRA Prediction: Negative

Reactivity class: Minimal

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
All acceptance criteria were fulfilled for experiments 1, 3 and 4, therefore the test was con-sidered valid. In experiment 2 the standard deviation for the percent depletion of the test item was out of the acceptable range. The DPRA prediction for the test item 1-(9,9-Dibutyl-9H-fluoren-2-yl)-2-methyl-2-morpholin-4-yl-propan-1-one was negative with reactivity class minimal according to the Cysteine 1:10/Lysine 1:50 prediction model. The result was verified in a second and third experiment. The last verification experiment was unnecessary and was performed only because of a calculation error during initial assessment of run ac-ceptance criteria for experiments 1 and 2 but confirmed again the results of the previous experiments.
No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.