2,5-di-tert-butylhydroquinone

Endpoint summary

Description of key information

OECD 442D (keratinosens): non-sensitizer

OECD 442C (DPRA method): sensitizer

OECD 429 (LLNA):sensitizer

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:

2017

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

Type of study:

activation of keratinocytes

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

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

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%

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.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

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

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

Sponsor’s identification: Eastman DTBHQ
Alternative name : 2,5-ditert-butylhydroquinone
CAS No: . 88-58-4
Batch No. :TS140610
Purity: 99.6%
Molecular weight :222.32 g/mol
Appearance: Tan to white crystals
Expiry/retest date: 03 November 2018
Storage conditions: Ambient temperature in the dark (15-25°C)

Details on the study design:

The solubility of Eastman DTBHQ in acetonitrile was assessed at a concentration of 100 mM.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution in acetonitrile of Eastman DTBHQ was prepared.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of Eastman DTBHQ and solutions of the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either Eastman DTBHQ or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of Eastman DTBHQ and solutions of the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either Eastman DTBHQ or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the Eastman DTBHQ and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of Eastman DTBHQ and the associated positive controls was quantified by HPLC using UV detection.

Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - Peptide peak area in replicate depletion samples (x 100)\
Mean Peptide peak area of reference control samples B

Positive control results:

Mean Depletion (%): 72.3

Parameter:

cysteine depletion

Value:

100 %

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Cinnamic Aldehyde

Parameter:

cysteine depletion

Value:

26.6 %

Vehicle controls validity:

not examined

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

Cinnamic Aldehyde

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

As per OECD442 guideline, a positive result with the DPRA may be used on its own to classify a chemical into UN GHS category 1.

Conclusions:

The overall result of 63.3% depletion places Eastman DTBHQ in the reactivity class of “high” and therefore it is predicted by DPRA to be a-skin sensitizer.

Executive summary:

In chemico Skin Sensitisation Direct Peptide Reactivity Assay (DPRA) following OECD TG 442C was conducted to assess the reactivity and sensitizing potential of Eastman 2,5-ditert-butylhydroquinone DTBHQ. Eastman (DTBHQ) and positive control samples vials were placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis. The concentration of both the Cysteine and Lysine peptides in the presence of Eastman DTBHQ and the associated positive controls was quantified by HPLC using UV detection. The positive control showed percentage depletion of 60.6 to 72.3 % which was within the acceptance criteria. The overall mean result of 63.3% depletion places Eastman DTBHQ in the reactivity class of “high” and hence it is predicted as a skin sensitizer by DPRA. The substance is classified as a chemical into UN GHS category 1.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

An in vivo LLNA study was conducted after a trio of in vitro studies were done and the results were ambiguous, requiring the conduct of an in vivo study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Identification: di-tert-butyl hydroquinone
CAS Number: 88-58-4
Batch: TS140610
Purity: 99.6%
Physical state / Appearance: white solid
Expiry Date: 03 November 2018
Storage Conditions: room temperature in the dark

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V., Inc., Horst, The Netherlands. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non-pregnant. After an acclimatization period of at least 5 days the animals were selected at random and given a number unique within the study by indelible ink marking on the tail and a number written on a cage card. At the start of the study the animals were in the weight range of 15 to 23 g, and were 8 to 12 weeks old.

The animals were housed in suspended solid floor polypropylene cages furnished with softwood wood flakes. Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study. The temperature and relative humidity were set to achieve limits of 19 to 25C and 30 to 70%, respectively. The rate of air exchange was at least fifteen changes per hour and the lighting was controlled by a time switch to give 12 hours continuous light and 12 hours darkness. The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

25, 10 or 5%

No. of animals per dose:

4

Details on study design:

Test Item Administration

Groups of four mice were treated with the test item at concentrations of 25%, 10% or 5% w/w in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of four mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL (250 μL) of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Terminal Procedures
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. The draining auricular lymph nodes from the four mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by -scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer".
The EC3 value was also calculated. The EC3 value is the concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation.

Positive control results:

A periodic positive control is run with α-Hexylcinnamaldehyde. The results were valid and the documentation is included in the study report.

Parameter:

SI

Value:

3.86

Test group / Remarks:

5%

Parameter:

SI

Value:

5.35

Test group / Remarks:

10%

Parameter:

SI

Value:

3.69

Test group / Remarks:

25%

 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index	Result
5	3.86	Positive
10	5.35	Positive
25	3.69	Positive

 

 

There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

Slight light brown residual test item on the ears was noted post-dose on Day 3 in animals treated with the test item at a concentration of 25% w/w in acetone/olive oil 4:1.

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3value) was calculated to be 3.35% (extrapolated).

Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 25% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% w/w. A further group of four animals was treated with acetone/olive oil 4:1 alone.

 

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (% w/w) in acetone/olive oil 4:1	Stimulation Index	Result
5	3.86	Positive
10	5.35	Positive
25	3.69	Positive

 

The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3value) was calculated to be 3.35% (extrapolated).

 

The test item was considered to be a sensitizer under the conditions of the test.

 

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

KERATINOSENS (in vitro)

Assessment of the Skin Sensitisation potential of 2,5-ditert-butylhydroquinone (DTBHQ) was intially conducted according to OECD Test Guideline 442D (the KeratinoSens test method) chemico study was assessed to evaluate the sensitization potential.  After 48h exposure of cells with 12 concentrations (0.195- 400 ug/ml) of di-tertbutyl hydroquinine, Luciferase measurements and MTT viability testing gave negative results in KeratinoSens method and Di-tertbutyl hydroquinine was concluded as non-sensitizer.

DPRA (in chemico)

Next OECD TG 442C Direct Peptide Reactivity Assay (DPRA) was assessed to evaluate the sensitization potential.  DTBHQ gave overall result of 63.3% depletion values which places yjer substance in the reactivity class of “high” and therefore DPRA predicted DTBHQ as a-skin sensitizer.

LLNA (in vivo)

Local Lymph Node Assay was conducted according to the OECD Test guideline 429 at concentration of 25% w/w. This concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. The EC3 value gave positive results in all three concentrations tested. The test item was considered to be a sensitizer under the conditions of the test.

Justification for classification or non-classification

Based on the WoE approach (in vitro, in chemico and in vivo studies) the substance need to be classified for Skin Sens 1B according to the CLP Regulation No. 1272/2008.