Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2020
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))
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Glycerol tribenzoate
EC Number:
210-379-6
EC Name:
Glycerol tribenzoate
Cas Number:
614-33-5
Molecular formula:
C24H20O6
IUPAC Name:
1,3-bis(benzoyloxy)propan-2-yl benzoate
Test material form:
solid: flakes
Details on test material:
room temperature: off-white coloured flakes or granulate; > 80 °C: colourless to yellowish liquid
Batch no. 20118
CAS no. 614-33-5
Production date 28. Jul. 2020
Expiry date 15. Aug. 2022
Specific details on test material used for the study:
Batch: 20118
Purity: 98.1%
Expiry: 15 Aug 2022

In chemico test system

Details of test system:
cysteine peptide, (Ac-RFAACAA-COOH)
lysine peptide (Ac-RFAAKAACOOH)
Details on the study design:
PERFORMANCE OF THE STUDY
Test solutions
Dilution buffers
• 2 mL Acetonitrile were mixed with 8 mL phosphate buffer, pH 7.50 (Peptide dilution buffer C)
• 2 mL Acetonitrile were mixed with 8 mL ammonium acetate buffer, pH 10.20 (Peptide dilution buffer K)
Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
• 0.667 mM Cys-Peptide solution was prepared by dissolving 10.0 mg of the peptide in 20 mL phosphate buffer, pH 7.50. (batch no. T20201102)
• 0.667 mM Lys-Peptide solution was prepared by dissolving 10.4 mg of the peptide in 20 mL ammonium acetate buffer, pH 10.20. (batch no. T20201103)
Peptide calibration standards
From each peptide stock solution, the following calibration standards were prepared in the appropriate dilution buffer (see chapter 7.1.1): 0.534 / 0.267 / 0.1335 / 0.0667 / 0.0334 / 0.0167 mM Peptide.
Calibration samples were analysed before the samples containing the test item. Blank dilution buffer was also measured.
Test item samples
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were prepared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item solution), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item solution) using the stock solutions described in chapters 6.1.3 and 7.1.2. A final volume of 1 mL per sample was prepared for each sample. Slight precipitation was observed immediately upon addition of the test item solution to the peptide solution.
Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22 h.
All three replicates for the Cys-peptide and Lys-peptide were turbid after incubation. They were centrifuged (10 min, 400 g) and only the clear supernatant was used for the measurement.
Vehicle / solvent:
other: Solvent controls For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide c
Positive control:
other: 6.6.1 Positive control Positive controls were treated identically as the test item. The following positive controls were used: • Cinnamaldehyde (CAS 104-55-2, food grade ≥95 %, batch no. MKCJ4653, expiration date: Feb. 2024) was used as 100 mM (± 10 %) so

Results and discussion

Positive control results:
The mean peptide depletion of the positive control cinnamaldehyde was within the range 60.8% - 100.0%, the peptide depletion of the positive control 2,3-Butanedione was within 10.0% - 45.0%. The standard deviation of the replicates of the positive control and test item was <14.5% in the Cys-peptide assay and <11.3% in the Lys-peptide assay respectively.

In vitro / in chemico

Results
Key result
Parameter:
cysteine depletion
Value:
0.3 %
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Precipitates were observed, immediately upon addition of the test item solution to the peptide solution of the Cys- and Lys-peptides. This may be caused due to low aqueous solubility of the test item.
After the incubation period, the precipitates were still present in the test solutions and they were centrifuged and only the clear supernatant was used to avoid clogging of the HPLC system.
and as a result. As consequence of the presence of precipitates, one cannot be sure, how much test item remained in solution to react with the peptides and peptide depletion may be underestimated.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
This study indicated no skin sensitisation potential though the test design was limited due to a lack of solubility and results are pending for other skin sensitisation studies