[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 17 February to 11 March 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:

2021

Report date:

2021

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 Direct peptide reactivity Assay (DPRA) is an in chemico test to determine the reactivity of a test substance towards peptides.
This assay has been validated for a broad range of low-molecular weight chemicals and it was found to detect reactive skin sensitizers from a broad range of so called applicability domains, i.e. chemicals reacting with proteins by different mechanisms. It was validated by ECVAM and proposed to be used as part of an integrated approachfor testing and assessment (IATA). It was recently also implemented in OECD test guideline 497 on Defined Approaches for skin sensitisation testing.

Test material

Test material information

Constituent 1

Reference substance name:

3,7-dimethylnon-6-en-1-ol

Cas Number:

41972-59-2

Molecular formula:

C11H22O

IUPAC Name:

3,7-dimethylnon-6-en-1-ol

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 Lys-peptide (Ac-RFAAKAA, MW 775.4) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity
of 95.6%. The Cys-peptide (Ac-RFAACAA, MW 750.1) was obtained from Genscript Inc. (Piscataway, NJ, USA). It has a purity of 95.6%. A reference sample of the Cys-peptide dimer (MW 1499.2) was obtained from Genscript Inc, it has a purity of 90.2%.
- 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:
(1) Lys-peptide: incubated at a final concentration of 0.5 mM in an ammonium acetate buffer at pH 10.5 in presence of a final level of 25% acetonitrile and in presence of a 50-fold excess of the test substance (25 mM) dissolved in acetonitrile.
(2) Cys-peptide: 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 a 10-fold excess of the test substance (5 mM) dissolved in acetonitrile.
- Precipitation noted: No precipitation noted

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Yes
- Verification of the suitability of the HPLC for test chemical and control substances: Yes

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 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 results are presented in Table 7.4.1/3 and 7.4.1/4 in the study report attached.
The positive control gave 71.2 % depletion of the Cys-peptide when measured with the LC-MS method.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

1.3 %

At concentration:

25 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

24.2 %

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:

OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Not applicable
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for reference controls A to C: Yes. Acceptance criteria: The standard deviation for Cys-peptide depletion should be < 14.9% and for Lys-peptide depletion < 11.6%. These criteria were fulfilled (0.9 % and 0.7 % SD, respectively).
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes. The co-elution controls indicated no co-elution with an UV-absorbing component.
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: as per the OECD TG 442C

Any other information on results incl. tables

When using HPLC-UV analysis only, the test substance led to 24.2% depletion of the Cys-peptide and 1.3% of the Lys-peptide and an average peptide depletion of 12.7%. It was therefore classified into the LOW reactivity class according to the classical DPRA prediction model.
When samples were analysed with high resolution LC-MS, the same amount of peptide depletion was observed. However, no direct adduct was formed in samples with the test chemical. At the same time the test chemical triggered significantly enhanced peptide dimerization, and the observed peptide depletion can be attributed to peptide oxidation / dimer formation rather than adduct formation / direct reactivity. No indication for a peptide adduct was found in this analysis. For the positive control, direct adduct formation could be verified indicating direct reactivity for the positive control. Thus, based on this more sophisticated and detailed analysis, the test chemical is not directly peptide reactive, but can only trigger peptide dimerization which is not considered itself as a molecular initiating event in skin sensitization.
Since this LC-MS based prediction model is not part of the validated DPRA protocol, this additional analysis should be used in a weight-of-evidence based analysis of the sensitization potential of a test chemical.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test substance was non-reactive and classified into the minimal reactivity class according to the prediction model. It is therefore considered a non-sensitizer according to the prediction model of DPRA.

Executive summary:

The Direct Peptide Reactivity Assay (DPRA) study was performed according to OECD TG 442C to assess the first key event in the skin sensitization adverse outcome pathway.

The test substance was dissolved in acetonitrile and mixed with a Cysteine- and a Lysine-containing peptide according to the standard operating procedure of the DPRA. One study with three replicates was conducted. After 24 h incubation time, peptide depletion induced by the test substancewas determined by HPLC-UV.

The test substance gave 24.2 % depletion of the Cys-peptide and 1.3 % depletion of the Lyspeptide. The substance is thus attributed to the “LOW” reactivity class according to the DPRA prediction model., rating it as a non-sensitizer according to the DPRA prediction model.