4-tert-Butoxystyrene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

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:

study report

Title:

Unnamed

Year:

2007

Materials and methods

Principles of method if other than guideline:

The study procedures were based on the following documents:
- The Ocular Toxicity Working Group (OTWG) of the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Interagency Centre for the Evaluation of Alternative Toxicological Methods (NICEATM), Background Review Document (BRD): current status of in vitro test methods for identifying ocular corrosives and severe irritants: The Bovine Corneal Opacity and Permeability (BCOP) Test Method, March 2006.
- In Vitro Techniques in Toxicology Database (INVITTOX) protocol 124. Bovine Opacity and Permeability Assay - SOP of Microbiological Associates Ltd., 1999.
- Gautheron P., Dukic M., Alix D. and Sina J.F., Bovine corneal opacity and permeability test: An in vitro assay of ocular irritancy. Fundam Appl Toxicol 18:442-449, 1992.

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1-(tert-butoxy)-4-ethenylbenzene

EC Number:

619-127-3

Cas Number:

95418-58-9

Molecular formula:

C12H16O

IUPAC Name:

1-(tert-butoxy)-4-ethenylbenzene

Test material form:

liquid

Details on test material:

Clear and colourless

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, the Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
Eyes were collected and transported in physiological saline in a suitable container. The isolated corneas were stored at 32 °C in a petri dish with cMEM (Eagle’s Minimum Essential Medium) containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum.
- Indication of any existing defects or lesions in ocular tissue samples:
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl of undiluted test substance

POSITIVE CONTROL
- Amount: 750 µl
- Concentration: 10% (w/v) Benzalkonium Chloride solution prepared in physiological saline.

NEGATIVE CONTROL
- Amount: 750 µl
- Concentration: 0.9% (w/v) NaCl in milli-RO water

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

3 corneas for each treatment and control group.

Details on study design:

PREPARATION OF CORNEAS
The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 °C. The corneas were incubated for the minimum of 1 hour at 32 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against an air filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 3 were not used.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED
Physiological saline: 0.9% (w/v) NaCl in milli-RO water

POSITIVE CONTROL USED
The positive control was 10% (w/v) Benzalkonium Chloride solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750µl of the control or test substance was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position (to ensure uniform distribution of the control or the test substance over the entire cornea) for 10 minutes at 32 °C.

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three times with cMEM. The medium in the posterior compartment was also removed and both compartments were refilled with fresh cMEM.
- Post-exposure incubation: the corneas were incubated for 120 minutes at 32 °C. After the completion of the incubation period opacity determination was performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer OP-KIT, MC2
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490).

SCORING SYSTEM
In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value). Additionally the opacity and permeability values were evaluated independently to determine whether the test substance induced irritation through only one of the two endpoints.

DECISION CRITERIA:
In vitro score range In vitro classification
0-3 Non irritant
3.1-25 Mild irritant
25.1-55 Moderate irritant
55.1-80 Severe irritant
>80 Very severe irritant
Permeability values of >0.600 are considered severe irritant.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

other: permeability score

Value:

-0.006

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Value:

-0.1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The mean in vitro irritancy score (uncorrected) obtained with the negative control was less than 3.1 indicating that the negative controls did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control was within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

The ocular irritation properties of butoxystyrene were assessed on an isolated bovine cornea. The study procedures were based on recent BCOP documents. The possible ocular irritancy of butoxystyrene was tested through topical application for 10 minutes. The test substance was applied undiluted (750 µl) directly on top of the corneas. The in vitro irritancy scores of the corneas treated with butoxystyrene ranged from -0.1 to 0.0 with a mean of -0.1. Since all in vitro irritancy scores were less than 3.1 butoxystyrene was considered non irritant.

Finally, it is concluded that this test is valid and that butoxystyrene is non irritant in the Bovine Corneal Opacity and Permeability test under the described experimental conditions.