Phenyl 4-hydroxybenzoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

publication

Title:

Assessment of the sensitizing potency of preservatives with chance of skin contact by the loose-fit coculture-based sensitisation assay (LSCA)

Author:

Sonnenburg A. et al.

Year:

2015

Bibliographic source:

Arch Toxicol 89, 2339-2344

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In the publication the results after conducting the loose-fit coculture-based sensitisation assay (LSCA) were provided. The coculture of primary human keratinocytes and allogenic dendritic cell-related cells (DC-rc) were used to emulate the in vivo situation of human skin.

GLP compliance:

not specified

Remarks:

Study is published and it is not further specified, if the GLP did apply

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Phenyl 4-hydroxybenzoate

EC Number:

241-698-9

EC Name:

Phenyl 4-hydroxybenzoate

Cas Number:

17696-62-7

Molecular formula:

C13H10O3

IUPAC Name:

phenyl 4-hydroxybenzoate

Specific details on test material used for the study:

- Source: TCI Deutschland GmbH, Eschborn, Germany

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Keratinocytes were isolated from skin which was received as residual material from plastic surgery. PBMCs were enriched from buffy coats by density centrifugation. Cells were cocultured in serum-free KGM-2 (PromoCell, Heidelberg, Germany) on 12-well cell culture plates. Increasing concentrations of test substances were tested until significant cytotoxicity or decreased solubility occurred. Viability of cells was measured by 7-AAD staining. Each substance was tested with three different DC-rc/KC-donor pairs to account for interindividual variability. TNBS at a concentration of 100 µmol/L served as positive control.

FACS ANALYSIS:
FACS analysis was performed on a FACS Calibur flow cytometer (BD Biosciences). Cells were gated to a distinct population of DC-rc according to their scatter properties. Of three cell populations to be seen in a scatter dot plot, one is CD14+, one CD4+CD8+ and one CD86+. The latter is considered to be the DC-rc population and is therefore gated. The experiments were assumed to be reliable, when the addition of TNBS led to an at least 1.4-fold increase in CD86 expression compared to zero controls. Relative upregulation of CD86-expression was calculated by the following equation: MFI (CD86 of treated cells)/MFI (CD86 of vehicle control). 7-AAD staining was used to determine and exclude dead cells from the calculation. Dose-effect relationships for relative CD86-expression and viability as well as EC50 values were determined using GraphPadPrism software.

Categorisation:
For categorisation of sensitising potency, the substance concentration which led to a half-maximal increase in CD86-expression (EC50 sens) was assessed. Substances were categorized as follows: half-maximal increase in CD86-expression <12.5 µM: extreme; <50 µM: strong; <100 µM: moderate and >100 µM: weak. Substances failing to elicit significant rise in CD86-expression up to the maximum test concentration were deemed non-sensitisers.
Likewise, categorisation of irritative potency is based on the concentration that resulted in cell death of 50% (EC50vit) of DC-rc as compared to vehicle controls. Substances eliciting 50% cell death at concentrations below 50 µM were considered to be irritative. Those with an EC50vit value from 50 to 1000 µM were considered to be weakly irritative. Substances that did not reach the 50% threshold or with EC50(vit) values above 1000 µM were rated as non-irritative.

Results and discussion

In vitro / in chemico

Results

Key result

Run / experiment:

other: mean of triplicates

Parameter:

other: EC50sens (µmol/L)

Value:

69.23

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Based on a EC50sens value of 69.23 µmol/L the test substance can be considered as moderate skin sensitising. The EC50vit was 416.17 µmol/L, indicating that the substance is weakly irritative.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results from an in vitro skin sensitisation assay, namely the loose-fit coculture-based sensitisation assay (LCSA), the test item phenylparaben must be considered as moderate skin-sensitiser.

Executive summary:

In a loose-fit coculture-based sensitisation assay (LCSA), keratinocytes were isolated from skin which was received as residual material from plastic surgery. PBMCs were enriched from buffy coats by density centrifugation. Cells were cocultured in serum-free KGM-2 (PromoCell, Heidelberg, Germany) on 12-well cell culture plates. Each substance was tested with three different DC-rc/KC-donor pairs to account for interindividual variability. TNBS at a concentration of 100 µmol/L served as positive control. Dose-effect relationships for relative CD86-expression and viability as well as EC50 values were determined using GraphPadPrism software. Based on a EC50sens value of 69.23 µmol/L, can the test substance be considered as moderate skin sensitising. The EC50vit was 416.17 µmol/L, indicating that the substance is weakly irritative.