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Skin sensitisation

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skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 22 September 2021 to 23 September 2021
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Study performed according to OECD TG 442C without deviation.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)
Version / remarks:
not specified
according to guideline
other: ECVAM. (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitisation testing
Version / remarks:
21 October 2021
not specified
GLP compliance:
Internal study performed with the GLP spirit
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
The kinetic Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the speed of the reaction of a test substance towards peptides to give a quantitative estimate of the Molecular Initiating Event in skin sensitization as an indication of sensitizer potency.
This assay has been validated for a broad range of low-molecular weight chemicals. It was found that the reaction constant derived from the assay correlates to the potency of reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated in a multi-laboratory trial and peer-reviewed by an international expert panel. It was recently implemented in OECD test guideline 442c on the Key event 1 of skin sensitization.
The kDPRA can be used for sub-classification of chemicals into GHS categories 1A and 1B/NC. In combination with evidence from the KeratinoSens and/or h-CLAT/GARD assay, it can be further used to determine a Point of Departure for risk assessment.

Test material

Constituent 1
Chemical structure
Reference substance name:
Cas Number:
Molecular formula:
Test material form:

In chemico test system

Details of test system:
Details on the study design:
- Preparation of the peptide/derivative stock solutions: The Cys-peptide (Ac-RFAACAA, MW 750.9) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity of 98.4%.
- Preparation of the test chemical solutions: The test substance was dissolved in Acetonitrile
- Preparation of the positive controls, reference controls and co-elution controls: Not reported

- Incubation conditions:
The Cys-peptide Ac-RFAACAA is incubated at a final concentration of 0.5 mM in phosphate buffer at pH 7.5 in presence of a final level of 25% acetonitrile and in presence of 0.32 – 5 mM of the test chemical (dissolved in the acetonitrile fraction) in a final volume of 120 µL.

At 10, 30, 90, 150, 210 and 1440 min after the start of the incubation, the reaction is stopped by the addition of a 3 mM solution of monobromobimane (40 µL added to 120 µL incubation solution). The remaining peptide is thus fluorescently labelled after an incubation time of 5 min and can then be quantified by measuring the fluorescence using an excitation filter of 390 nm and an emission filter of 480 nm.
Vehicle / solvent:
Positive control:
cinnamic aldehyde

Results and discussion

Positive control results:
Cinnamic aldehyde fulfilled the acceptability criteria for the positive control. The log k (in M-1s-1) of the positive control cinnamic aldehyde at 90 min should be within the following range: -1.75 to -1.40. The measured logarithmic reaction rate at 90 min was -1.57. This value is within the acceptance range.

In vitro / in chemico

Key result
test chemical
Run / experiment:
other: log kmax
At concentration:
5 mM
Vehicle controls validity:
Negative controls validity:
not examined
Positive controls validity:
Remarks on result:
no indication of skin sensitisation
Outcome of the prediction model:
no or minimal reactivity [in chemico]
Other effects / acceptance of results:
Acceptance criteria for the vehicle control: The coefficient of variation of the 12 vehicle control values of a plate should be <12.5% for 5 of the 6 time points. These criteria were fulfilled (1.79 % to 4.48 % CV at the six time points)

Chemicals which are non-reactive or which have a log kmax < -2.0 are not categorised as UN GHS subcategory 1A sensitizers by the kDPRA. The measured rate constant log kmax is -3.5 (default value for non-reactive substance), therefore the test substance is not a UN GHS subcategory 1A sensitizer according to the kDPRA prediction model.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
The test substance was non-reactive in the kDPRA. It is therefore classified into the GHS 1B/NC class, i.e. not as a strong 1A sensitizer, according to the prediction model of the kDPRA.
Executive summary:

The kinetic Direct Peptide Reactivity Assay (DPRA) study was performed according to OECD TG 442C to assess the skin sensitizing properties of the test substance.

The test substance was dissolved in acetonitrile and mixed with the Cysteine- containing peptide in five different ratios according to the standard operating procedure of the kDPRA. The Peptide depletion was monitored at six different time points by fluorescent derivatization of the parent peptide. One study with three replicates was conducted. The resulting depletion matrix vs. incubation time and test concentration was used to calculate the maximal reaction rate with the test peptide, expressed as Log kmax. The reaction rate was then used for GHS sub-classification.

The test substance was non-reactive in the kDPRA assay and thus rated with the default log kmax = -3.5 for negatives and classified as a GHS 1B/NC substance according to the kDPRA prediction model.