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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-05-25 to 2020-07-07
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
adopted 2019-06-18
Deviations:
yes
Remarks:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate peptide buffer and acetonitrile (parallel dilution) instead of conducting a serial dilution as stated in the OECD 442C Guideline.
Principles of method if other than guideline:
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate peptide buffer and acetonitrile (parallel dilution) instead of conducting a serial dilution as stated in the OECD 442C Guideline. This procedure was selected, since this preparation is similar to the preparation of the test item samples and controls. Furthermore, the DPRA proficiency study was conducted under these conditions.
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
1,2-Heptanediol
Cas Number:
3710-31-4
Molecular formula:
C7H16O2
IUPAC Name:
1,2-Heptanediol

In chemico test system

Details on the study design:
PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in approximately 20 mL aliquots of the appropriate buffer solution (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
- Preparation of the test chemical solutions: The test item was weighed into volumetric flask and dissolved immediately before testing in acetonitrile to prepare a 100 mM stock solution.
- Preparation of the positive controls, reference controls and co-elution controls:
Positive controls: The positive control chemical (Cinnamaldehyde) was prepared at a concentration of 100 mM in acetonitrile.
Reference controls: The reference control A, B and C1 samples of both peptides were prepared at a concentration of 500 μM in acetonitrile.

Reference control A= For the verification of the HPLC system suitability (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=1 with 3 fold injections.
Reference control B= For the stability of the reference controls over time (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=6
Reference control C1= Peptide stability control for the solvent used to dissolve the test item and the positive control (samples containing 0.5 mM peptide dissolved in the appropriate peptide buffer and acetonitrile). n=3

Co-elution controls: Sample prepared of the respective peptide buffer and the test item or the positive control without peptide. n=1, each

INCUBATION
- Incubation conditions: 500 μM cysteine and lysine peptide solutions were incubated in glass autosampler vials with 5 mM or 25 mM of the test item, respectively. The reaction solutions were incubated in the dark at 22.5 - 30ºC for 24 ± 2 hours prior to initiation of the analysis run.
- Precipitation noted: No, the solubility of the test item in acetonitrile at a nominal concentration of 100 mM was achieved.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys: Calibration standards of both peptides were prepared in a solution of 20% acetonitrile buffer using phosphate buffer (pH 7.5) for the cysteine peptide and ammonium acetate buffer (pH 10.2) for the lysine peptide.The following calibration solutions were prepared from the peptide stock solution of each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A blank of the dilution buffer was also included in the standard calibration curve for both peptides. The blank is 25% acteonitrile:buffer solution with phosphate buffer pH 7.5 for the cysteine peptide and with ammonium acetate buffer pH 10.2 for the lysine peptide without peptide.
- Verification of the suitability of the HPLC for test chemical and control substances: Analysis was performed according to a proficiency study conducted at the testing facility.

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Results and discussion

Positive control results:
Cinnamic aldehyde showed 70.6 % ± 0.403 % mean cysteine peptide depletion and 43.3 % ± 2.92 % mean lysine peptide depletion.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
mean
Parameter:
other: mean lysine depletion (migrated information)
Value:
0.743
Vehicle controls validity:
valid
Remarks:
Reference Control C1
Negative controls validity:
valid
Remarks:
Co-elution Control
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
mean
Parameter:
other: mean cystein depletion (migrated information)
Value:
2.74
Vehicle controls validity:
valid
Remarks:
Reference Control C1
Negative controls validity:
valid
Remarks:
Co-elution Control
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY: Analysis was performed according to a proficiency study conducted at the testing facility.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for reference controls A to C: Yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): Yes

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
The DPRA can be used as part of a testing battery (including e.g. h-CLAT (human cell line activation test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
Conclusions:
Under the conditions of this assay, the test item was not predicted to be a skin sensitizer by DPRA.
Executive summary:

The purpose of this GLP compliant study (based on the based on the OECD Guidelines for the Testing of Chemicals: Test Guideline No. 442C: In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA)) was to assess the reactivity and sensitizing potential of the test item. This direct peptide reactivity assay can be used as part of a testing battery (including e.g. h-CLAT (human Cell Line Activation Test), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals. The test item was dissolved in acetonitrile when incubated for 24 ± 2 hours in the range between 22.5 and 30 °C.


There were no co-elution peaks in either the cysteine or lysine assays. Solutions of the test item were analyzed by the DPRA method in both the cysteine and lysine containing synthetic peptides. Based on this assay, with an overall depletion value of 1.74%, the test item was placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer.