Glycerol tribenzoate

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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)

Specific details on test material used for the study:

Batch: 20118
Purity: 98.1%
Expiry: 15 Aug 2022

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

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.

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.

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”.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase LuSens test method

Specific details on test material used for the study:

Batch no. 20118
Expiry date 15. Aug. 2022
Purity 98.1%

Details of test system:

Lusens transgenic cell line [442D]

Details on the study design:

The LuSens cell line was specially designed for this test system by the BASF SE (Ludwigshafen, Germany). It employs the use of a luciferase reporter gene placed under the control
of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor
activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany)
carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene
(Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

The LuSens cell line was obtained from the BASF SE (Ludwigshafen, Germany). For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen in the
cell bank of LAUS GmbH to allow a continuous stock of cells (mycoplasma contamination
free), which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 14 were used. For the repetitions,
cells of passage 6 and 7 were used. After thawing the cells were cultivated in DMEM (9 %
FBS (Fetal bovine serum)) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with
5.0 ± 0.5 % CO2.

Vehicle / solvent control:

DMSO

Negative control:

DL-Lactic acid

Positive control:

EGDMA (120 M) [442D]

Positive control results:

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a distinct
increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: Luciferase induction

Remarks:

[fold]

Value:

2.4

At concentration:

250 other: uM

Cell viability:

104.6%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: Luciferase induction

Remarks:

[fold]

Value:

2.5

At concentration:

250 other: uM

Cell viability:

107.5%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In order to come to a conclusion, a minimum of two valid and independent repetitions need
to indicate a positive or negative result according to the above-described criteria. If the first
two repetitions come to the same result (i.e. either being negative or being positive) no further testing is required. In case that the first two repetitions give discordant results (i.e. one
is negative and the other is positive), a third independent repetition needs to be conducted
to complete the study. The potential to activate the Nrf2 transcription factor of a test substance is determined by the result of the majority of the repetitions of an experiment. If two
of two or two of three repetitions are negative/positive, the substance is considered as negative/positive.
The luciferase induction was ≥ 1.5 fold and statistically significant compared to the solvent
control in 2 (or more) consecutive non-cytotoxic tested concentrations in repetition I and II.
Therefore, the test item Glycerol tribenzoate (GTB) is considered to have the potential to
activate the Nrf2 transcription factor under the conditions of the LuSens test.

In conclusion, it can be stated that under the experimental conditions of this study, the test
item, Glycerol tribenzoate (GTB), was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor.