2,5-di-tert-butylhydroquinone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

2,5-di-tert-butylhydroquinone

EC Number:

201-841-8

EC Name:

2,5-di-tert-butylhydroquinone

Cas Number:

88-58-4

Molecular formula:

C14H22O2

IUPAC Name:

2,5-di-tert-butylhydroquinone

Specific details on test material used for the study:

Purity:100% (from MSDS)
Expiry Date: 01 MAR 2019 (from test item bottle)
Physical state: White to tan crystalline solid
Storage Conditions: Room Temperature

In vitro test system

Details on the study design:

Description of the test system:
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be upregulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Method of administration of test item:
single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

Method of administration of reference items:
single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%.

single application of culture medium with 1% DMSO was applied as the negative control.
Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.

Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT. The validity of each repetition was assessed following acceptance criteria described in section 12.1.

Study Design

Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT).
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 370C, 5% C02, 2 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MIT testing (2 plates)

Data Analysis
XCellR8 Forms F0056 A and B: Data Analysis for KeratinoSensTM (version 03) were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP 10057. The spreadsheets have been validated in-house (July 2017).

The following parameters were calculated in the KeratinoSensTM test method:

-the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item and positive control;
-the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity);
-For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
-The percentage of viability as compared to the Negative control

Results and discussion

In vitro / in chemico

Results

Parameter:

other: Luciferase measurements and M TT viability testing were performed

Remarks:

Luciferase measurements and M TT viability testing were performed

Value:

0

Negative controls validity:

valid

Remarks:

DMSO

Remarks on result:

no indication of skin sensitisation

Remarks:

No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold

Other effects / acceptance of results:

Test results are acceptable if:
- The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
-The Imax and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
- average induction in the three replicates for cinnamic aldehyde at 32 11M is between the XCellR8 historical range (currently 1.6 and 3)
- EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 and 39 VIM).
Note: At least one of these criteria must be met, otherwise the run is discarded. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
-CV% of blank values < 20%

Applicant's summary and conclusion

Interpretation of results:

other: No category. Not requiring classification to UN GHS.

Remarks:

No category. Not requiring classification to UN GHS.

Conclusions:

The human skin sensitisation potential of di-tertbutyl hydroquinine was assessed using the validated in vitro method, the KeratinoSensTM assay, adapted to fully animal-free by XCellR8, and validated in-house to determine keratinocyte activation.After 48h exposure of cells with 12 concentrations of di-tertbutyl hydroquinine, Luciferase measurements and MTT viability testing were performed.Di-tertbutyl hydroquinine was classified as non sensitiser.

Executive summary:

In Vitro Assessment of the Skin Sensitisation Potential of di-tertbutyl hydroquinine was conducted according to OECD Test Guideline 442D (the KeratinoSens^TMtest method). After 48h exposure of cells with 12 concentrations of di-tertbutyl hydroquinine, luciferase measurements and MTT viability testing were performed. A single application of culture medium with 1% DMSO was applied as the negative control. Three repetitions (runs) were performed. Solubility results of di-tertbutyl hydroquinine indicated the item was soluble in DMSO at 20 mg/ml. Testing on this study were done with subsequent dilution in cell culture medium 1% DMSO giving a top concentration of 400 ug/ml. No EC1.5 was determined as as no concentration induced the luciferase activity above the 1.5 threshold for lowest and highest tested concentrations. Di-tertbutyl hydroquinine caused luciferase induction 21.5 in repetition 2, at 3.125 ug/ml, however there was no dose-response induction and di-tertbutyl hydroquinine was classified as a non-sensitiser.