2,5-di-tert-butylhydroquinone

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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.

Test guideline 2

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

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:

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

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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.

Applicant's summary and conclusion

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.