[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 22 September 2021 to 23 September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study performed according to OECD TG 442C without deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Version / remarks:

2021

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ECVAM. (2014), DB-ALM protocol 154: Direct peptide reactivity assay (DPRA) for skin sensitisation testing

Version / remarks:

21 October 2021

Deviations:

not specified

GLP compliance:

no

Remarks:

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

Test material information

Constituent 1

Reference substance name:

(E)-1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene

Cas Number:

35856-81-6

Molecular formula:

C16H16O

IUPAC Name:

(E)-1-methoxy-4-(3-phenylprop-1-en-1-yl)benzene

Test material form:

liquid

In chemico test system

Details of test system:

other:

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- 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
- 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:

acetonitrile

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

Results

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: log kmax

Value:

-3.5

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

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

Conclusions:

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 k_max. 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 k_max = -3.5 for negatives and classified as a GHS 1B/NC substance according to the kDPRA prediction model.