2,5-di-tert-butylhydroquinone

Endpoint summary

Description of key information

Skin Irritation / Corrosion

OECD 439 (EpiSkin) : Reconstructed human epidermis (RHE) test : non-conclusive

OECD 439 (EpiDerm) : Reconstructed human epidermis (RHE) test : non-irritant

OECD 431 (Reconstructed Human Epidermis (RHE) test method) : non-corrosive

Eye Damage

OECD 437 (BCOP) : not irritant / not damging to eyes

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

The test substance is identified as di-tert-butyl hydroquinone. The purity is 99.6%. The physical state/appearance of material is white solid. The expiry date of material is 01 March 2019. The substance can be stored at room temperature in the dark conditions.

Test system:

human skin model

Source species:

human

Cell type:

other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit- 24-well plate

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of the test item with 99.6% purity

Duration of treatment / exposure:

Tissues were treated with the test item for exposure periods of 3 and 60 minutes.

Number of replicates:

Duplicate

Details on study design:

Main Test

Pre-Incubation
0.9 mL of this assay medium was pipetted into the appropriate wells of two pre-labeled 6-well plates for both the 3-Minute and 60-Minute exposure periods.
EpiDerm™ tissues were transferred into the 6-well plates containing the assay medium. The 6-well plates containing the EpiDerm™ samples were pre-incubated (37 °C, 5% CO2) for approximately 1 hour before dosing.

Application of Test Item and Rinsing

Before pre-incubation was complete, a 24-well plate was prepared for use as a “holding plate” for both the 3-Minute and 60-Minute exposure periods. This plate was used to maintain the viability of the tissue inserts between rinsing following chemical exposure and MTT loading. Another 24-well plate was prepared for the MTT loading. 300 μL of either pre-warmed assay medium (holding plate) or MTT medium (MTT loading plate) was dispensed into each well. The two plates were placed into the incubator until required.After pre-incubation of the EpiDerm™ tissues, the medium was aspirated and replaced with 0.9 mL of fresh assay medium. The 6-well plate for the 3-Minute exposure period was returned to the incubator, while the other was being dosed for the 60-Minute exposure.For the 60-Minute exposure period, 50 μL of sterile distilled water (negative control) was added to the first two tissues. 25 mg of the test item and 50 μL of 8.0 N Potassium Hydroxide (positive control) were also applied to the corresponding tissues in turn. 25 μL of sterile water was added for wetting of the test item to increase tissue surface contact. The plate was returned to the incubator (37 °C, 5% CO2) for the 60-Minute exposure period.When dosing for the 60-Minute exposure period was complete, the same procedure was repeated for the 3-Minute exposure period. Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) to gently remove any residual test item. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT loading procedure was repeated.
After the 3-Hour MTT incubation was complete, the inserts were blotted and transferred to labeled 24-well plates for MTT extraction. 2 mL of MTT extractant (isopropanol) was used to completely immerse each insert and the plate was covered with plate sealer to prevent Isopropanol evaporation. The plates stood overnight at room temperature, to allow extraction to proceed.

Absorbency at 570nm (OD570) of each well was measured using the Labtech LT-4500 microplate reader.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

89.2

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute

Value:

94.9

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

The corrosivity potential of di-tert-butyl hydroquinone was evaluated using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues samples were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in isopropanol for MTT extraction. At the end of the formazan extraction period the optical density (OD) was measured at 570 nm. Percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) of test item for 3-minute exposure was 89.2% and after 60 minutes was 94.9%. Positive control showed the viability of 4.8 and 5.3% in 3 and 60 minutes exposure. The test item is not corrosive to skin and not classified for corrosivity in UN GHS or CLP regulations.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

yes

Remarks:

The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual % tissue viabilities of the three identically treated replicates is ≤18%.

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

yes

Remarks:

The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual % tissue viabilities of the three identically treated replicates is ≤18%.

GLP compliance:

yes

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: 01 March 2019
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Cell type:

non-transformed keratinocytes

Details on test system:

EpiDerm™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek

EpiDermTM Tissues : (0.63cm2)

lot number:25878
Assay Medium lot number:020818ALE

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

25 mg of test item was used in sterile water

Duration of treatment / exposure:

The test item is applied topically for a treatment period of 60 minutes.

Duration of post-treatment incubation (if applicable):

Post-exposure incubation period of 42 hours

Number of replicates:

Triplicate tissues

Details on study design:

Main Test

Pre-Incubation (Day 0)

mL of assay medium was pipetted into each well of sterile 6-well plates using one 6-well plate for pre-incubation of three inserts.The plates were incubated at 37 ºC, 5% CO2 in air for 60 ± 5 minutes. At the end of the first pre-incubation period, the inserts were gently blotted on absorbent paper to remove residual assay medium and the inserts transferred from upper wells into the lower wells of the 6-well plate. The plates were incubated at 37 ºC, 5% CO2 in air overnight for 18 ± 3 hours. Ensuring that there were no air bubbles present under the tissue inserts.

Application of Test Item and Rinsing (Day 1)
25 mg of the solid test item was then applied to the epidermis surface ensuring uniform covering of the tissues. The tissues were dosed at regular intervals, to allow for the time taken to rinse each tissue insert following exposure, and to ensure each tissue had an equal exposure period. Triplicate tissues, treated with 30 μL of DPBS, were used to serve as negative controls. Triplicate tissues, treated with 30 μL of SDS 5% (w/v), were used to serve as positive controls. After dosing the plates were incubated at 37 ºC, 5% CO2 in air for 35 ± 1 minutes. After 35 minutes the plates were removed from the incubator and kept in the biological safety cabinet at room temperature for the remainder of the 60-minute exposure period.At the end of the 60-minute exposure period, each tissue was removed from the well of the treatment plate using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (DPBS) without Ca++ and Mg++.

Post-exposure incubation (Day 2)
Using aseptic techniques 0.9 mL of assay medium was pipetted into the upper wells of new sterile 6-well plates using one 6-well plate per test item or control. These plates were used for further post-exposure incubation purposes. Each plate was labeled with details of the coded test item or control and study number.At the end of the 24 hour post-exposure incubation period the 6-well plates were removed from the incubator and the medium in the wells of the plates pipetted up and down three times (changing the tips between the test item and controls). The inserts were transferred into the upper wells of the new 6-well plates pre-filled with 0.9 mL assay medium per well. These plates were incubated at 37 ºC, 5% CO2 in air for 18 ± 2 hours longer (total post-exposure incubation period approximately 42 hours).

MTT Loading/Formazan Extraction (Day 3)

Using aseptic techniques 0.3 mL of 1.0 mg/mL MTT solution freshly prepared in assay medium was pipetted into the wells of a 24-well plate designated ‘MTT Loading Plate’. Following the 42-hour post-exposure incubation period, the 6-well plates containing the tissue inserts were removed from the incubator.After the final aspiration each tissue insert was placed on absorbent paper to dry before transfer to a new pre-labelled 24-well plate designated ‘MTT Extraction Plate’. The inserts were immersed by gently pipetting 2 mL of isopropanol into each insert. The 24-well MTT extraction plate was sealed with a suitable plate sealer to prevent isopropanol evaporation. The sealed plate was extracted overnight in the dark at room temperature.

Absorbance/Optical Density Measurements (Day 4)
Absorbency at 570 nm (OD570) of each well was measured using the Labtech LT 4500 microplate reader and LT-com analysis software.

Irritation / corrosion parameter:

% tissue viability

Value:

93.4

Negative controls validity:

valid

Remarks:

100% viability

Positive controls validity:

valid

Remarks:

7.7 % viability

Other effects / acceptance of results:

Positive Control
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤20% relative to the negative control treated tissues.

Negative Control
The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues was ≥1.0 and ≤2.5.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant.

Executive summary:

The test was conducted to evaluate the skin irritation potential following OECD 439, EpiDerm^TM Reconstructed Human Epidermal (RHE) model.The test item is applied topically for a treatment period of 60 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in RHE cultures following topical exposure to the test item by means of the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period may be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. The relative mean viability of the test item treated tissues was 93.4% after the 60-Minute exposure period. The test item was classified as non-irritant under GHS and CLP classification.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes

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: 01 March 2019
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell source:

other: A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum from an adult human

Details on test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit

Amount/concentration applied:

10 mg with 99.6% purity

Duration of treatment / exposure:

Tissues were treated with the test item for an exposure period of 15 minutes

Duration of post-treatment incubation (if applicable):

42-Hour post-exposure incubation period

Number of replicates:

two

Details on study design:

Pre-Test Procedure
To identify this possible interference, the test item is checked for the ability to directly reduce MTT.

Pre-incubation (Day 0: Tissue Arrival)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
Main test
Application of Test Item and Rinsing (Day 1)
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 10 μL (26.3 μL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
The optical density was measured at 570 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 min

Value:

64.7

Negative controls validity:

valid

Other effects / acceptance of results:

Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.

Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean OD570 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.

Test Item: The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item induced non-specific MTT reduction was 49.1% relative to the negative control. The test item was therefore considered incompatible with the test system and no conclusion of irritation potential can be made.

Executive summary:

The skin irritation potential of the di-tert-butyl hydroquinone was evaluated using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the post-exposure incubation period tissues were taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made for extraction of formazan crystals. At the end of the formazan extraction period the optical density was measured at 570 nm. The percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) was calculated. The relative mean viability of the test item treated tissues was 64.7% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The relative mean viability of negative control was 49.1% and for positive control was 14.7 %. The test item induced non-specific MTT reduction was 49.1% relative to the negative control. The test item was therefore considered incompatible with the test system and no conclusion of irritation potential can be made.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

The Bovine Corneal Opacity and Permeability (BCOP) Assay

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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Remarks:

The results of the test have been disregarded and not reported.

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

Remarks:

The results of the test have been disregarded and not reported.

GLP compliance:

yes

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:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Vehicle:

not specified

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Approximately 446mg of the solid test item was found to adequately cover the corneal surface. 0.75mL of each control item was applied to the appropriate corneas.

Duration of treatment / exposure:

240 minutes

Details on study design:

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM. A visual inspection of the corneas was performed prior to the pre-treatment opacity reading. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer . The average opacity for all corneas was calculated.Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.
Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 446mg of the solid test item was found to adequately cover the corneal surface. 0.75mL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (OD492) (without a reference filter) using the Labtech LT-4500 microplate reader and LT-com analysis software.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
No histopathology was required for this study.

Irritation parameter:

in vitro irritation score

Value:

0.4

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

20% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean during 2016 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 65.1 to 123.3.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing solids the negative control limit for opacity should be ≤2.4 and for permeability ≤0.072.

Interpretation of results:

GHS criteria not met

Conclusions:

The In Vitro irritancy score for test item is 0.4. Not requiring classification to UN GHS or EU CLP.

Executive summary:

A test was to conducted to identify test item that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The undiluted test item was applied for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The In Vitro irritancy scores of test item was 0.4 and negative control was 0.8 and positive control was 100.2. No category. Not requiring classification to UN GHS or EU CLP.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

SKIN IRRITATION / CORROSION

EpiSkin (OECD 439)

The skin irritation potential of the di-tert-butyl hydroquinone was evaluated using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the post-exposure incubation period tissues were taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made for extraction of formazan crystals. At the end of the formazan extraction period the optical density was measured at 570 nm. The percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) was calculated. The relative mean viability of the test item treated tissues was 64.7% after the 15-Minute exposure period and 42-Hours post-exposure incubation period. The relative mean viability of negative control was 49.1% and for positive control was 14.7 %. The test item induced non-specific MTT reduction was 49.1% relative to the negative control. The test item was therefore considered incompatible with the test system and no conclusion of irritation potential can be made.

EpiDerm (OECD 439)

The test was conducted to evaluate the skin irritation potential following OECD 439, EpiDerm^TM Reconstructed Human Epidermal (RHE) model. The test item is applied topically for a treatment period of 60 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in RHE cultures following topical exposure to the test item by means of the colorimetric MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period may be determined for test items which are found to be borderline non-irritant based upon the MTT reduction endpoint. The relative mean viability of the test item treated tissues was 93.4% after the 60-Minute exposure period.

RHE (OECD 431)

The corrosivity potential of di-tert-butyl hydroquinone was evaluated using the EpiDerm Human Skin Model after treatment periods of 3 and 60 minutes. Duplicate tissues samples were treated with the test item for exposure periods of 3 and 60 minutes. Negative and positive control groups were treated for each exposure period. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in isopropanol for MTT extraction. At the end of the formazan extraction period the optical density (OD) was measured at 570 nm. Percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues) of test item for 3-minute exposure was 89.2% and after 60 minutes was 94.9%. Positive control showed the viability of 4.8 and 5.3% in 3 and 60 minutes exposure.

EYE IRRITATION

OECD 437

An in vitro eye irritation study was conducted according to EU Method B.47 and OECD Guideline TG No. 437. This study is a key study and conducted with most modern validated method which was performed under GLP conditions. The undiluted test item was applied for 240 minutes. The In Vitro irritancy scores of test item was 0.4 and negative control was 0.8 and positive control was 100.2. The positive control and negative acceptance criterion results were satisfied.

Justification for classification or non-classification

Di-tert-butyl hydroquinone is not an irritant or a corrosive to skin or eyes and no classification is needed according to the CLP Regulation No. 1272/2008.