Glycerol tribenzoate

Skin sensitisation

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

Test material information

Constituent 1

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:

The DPRA prediction with measured values is “negative” according to the Cysteine
1:10/Lysine 1:50 prediction model based on solubilised test item. Thus, under the
experimental conditions reported, the test item Glycerol tribenzoate (GTB) shows no
or minimal reactivity towards the two model synthetic peptides at the level of dissolved test item in the peptide test solution. This result should, however, be treated
with caution.
Due to precipitation of the test item in the test solution with the Cys- and Lys-peptides, a negative conclusion should be treated with caution. The conclusion is therefore reported as being “inconclusive”.