Pentaerythritol tetrabenzoate

Endpoint summary

Description of key information

In vitro skin sensitization screenings following OECD 442C and OECD 442D were performed on the test item pentaerythritol tetrabenzoate were performed. There were no indications of a skin sensitiation potential from the test item according to the valid in vitro tests. The test item pentaerythritol tetrabenzoate can be condsidered to be non-sensitizing.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24.09. - 26.10.2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The direct peptide reactivity assay (DPRA) is an in chemico assay to quantify the reactivity of the test item towards cysteine and lysine containing peptides. This reactivity is related to the skin sensitisation potential. To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item is measured using HPLC. The assay is used for supporting the discrimination between skin sensitizers (i.e. UN GHS Category 1) and non-sensitizers in accordance with the UN GHS. A categorization in the sub-categories 1 A and 1 B is not possible.

Specific details on test material used for the study:

Name: Pentaerythritol tetrabenzoate
Appearance: off-white solid
Composition: Pentaerythritol tetrabenzoate >96%
CAS No.: 4196-86-5
EINECS-No.: 224-079-8
Molecular formula: C33H28O8
Molecular weight: 552.576 g/mol
Purity: >96%
Homogeneity: homogeneous
Expiry date: Nov. 2018
Storage: Room Temperature (20 ± 5°C)

Details on the study design:

The study was performed in order to evaluate the reactivity of the test item Pentaerythritol tetrabenzoate towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, respectively. The peptide concentration after the incubation was measured using HPLC-UV.
Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

Peptides with ≥95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.: P151223-TL472063
Purity: 98.12%
Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170415-2-LR569640
Purity: 98.85%

Key result

Run / experiment:

other: Mean value

Parameter:

other: Cys-peptide depletions

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Key result

Run / experiment:

mean

Parameter:

other: Lys-peptide depletion

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

not applicable

Conclusions:

Under the experimental conditions of this OECD 442C study reported, the test item Pentaerythritol tetrabenzoate does not possess skin sensitisation potential.

Executive summary:

The study was performed in order to evaluate the reactivity of the test item Pentaerythritol tetrabenzoate towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 22 h and 5 min for the Cys-peptide and 22 h for the Lys-peptide at 25 °C, respectively. The peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.The mean peptied depletion was 0.00%.

In conclusion, the DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item Pentaerythritol tetrabenzoate does not possess skin sensitisation potential.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03.04.2018 - 13.04.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

This in vitro study evaluates the potential of the test item Pentaerythritol tetrabenzoate to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

Specific details on test material used for the study:

Name: Pentaerythritol tetrabenzoate
Appearance: off-white solid
Composition: Pentaerythritol tetrabenzoate >96%
CAS No.: 4196-86-5
EINECS-No.: 224-079-8
Molecular formula: C33H28O8
Molecular weight: 552.576 g/mol
Purity: >96%
Homogeneity: homogeneous
Expiry date: Nov. 2018
Storage: Room Temperature (20 ± 5°C)

Details on the study design:

This in vitro study was performed to assess the potential of the test item Pentaerythritol tetrabenzoate to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” (Bauch et al. 2012). It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010). In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments are performed.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: luciferase induction

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions of this study following OECD 442D, the test item, Pentaerythritol tetrabenzoate, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Executive summary:

This in vitro study following OECD 442D was performed to investigate the potential of Pentaerythritol tetrabenzoate to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line. The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II: 244 μM, 292 μM, 351 μM, 421 μM, 505 μM, 606 μM, 727 μM, 873 μM, 1047 μM, 1257 μM, 1508 μM, 1810 μM.

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. DL-lactic acid (5000 μM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control. The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold. No cytotoxic effect was observed in any of the tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.

Finally the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction in experiment I and II: 244 μM, 292 μM, 351 μM, 421 μM, 505 μM, 606 μM, 727 μM, 873 μM, 1047 μM, 1257 μM, 1508 μM, 1810 μM. In all tested concentrations of the test item no increase ≥ 1.5 fold in luciferase induction was measured. In addition no dose-dependent effect was observed. Therefore, the result of both experiments is negative.

Since the highest concentration is lower than 2000 μM (prescription of Draft OECD Guideline 442D in case of no cytotoxicity up to the highest test item concentration), one acceptance criterion is not met in both experiments. In this case according to the Draft OECD Guideline 442D a negative result should normally be considered as inconclusive. However, in this study, the result was declared as valid and negative, since the deviation was considered as uncritical because of the following circumstances: Due to the low solubility of the test item, the highest possible concentration was 1810 μM. This value is higher than the second highest concentration which would have been used (1666 μM), if the test item would have been soluble up to the maximum concentration as required by the guideline (2000 μM). Therefore, only one more test concentration would have been necessary to comply with the requirements of the Guideline. In addition, even if the next higher test item concentration (e.g. 2000 μM) would have shown a statistically significant increased induction of ≥ 1.5 fold compared to the solvent control, the conclusion would still be negative, since this effect has to be detected in at least in two consecutive test item concentrations for a positive result. Furthermore, there was no evidence of an increase in luciferase induction in any of the concentrations tested. All values were lower or equal to those of the growth control and the negative control. Based on these reasons and the fact that the final result is not affected by the deviation, the negative result obtained in this study is concluded to be valid.

Under the experimental conditions of this study, the test item, Pentaerythritol tetrabenzoate, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test item pentaerythritol tetrabenzoate can be condsidered to be non-sensitizing and does not

require classification and labelling for skin sensitiyation according to CLP Regulation (EC) No. 1272/2008.