Reaction products of alkenyl dihydrofuran-2,5-dione and polyisoalkenyl dihydro-2,5-furandione with alkylene glycol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 to 29 April 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin and Streptomycin, and transported to the laboratory on ice packs. The corneas were refrigerated on arrival and used within 24 hours of receipt.

A pH measurement of the test item at a concentration of 10% W/w in sterile water.
pH measurement:
Initial reading = 3.02
After 10 minutes = 3.04

For the purpose of this study the test item was used as supplied.
The negative control item, 0.9% w/v sodium chloride solution, was used as supplied.
The positive control item, Ethanol, was used as supplied.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative and positive controls

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 ml/eye

Duration of treatment / exposure:

10 minutes

Observation period (in vivo):

The holders were incubated, anterior chamber facing forward, at 32 +/- 1 °C for 120 minutes.

Number of animals or in vitro replicates:

3 tested eyes, 3 negative controls and 3 positive controls

Details on study design:

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 (epithelial side uppermost) 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(MEM) and plugged. The holders were incubated at 32+/- 1 °C for 60 minutes. At the end of the incubation period each comea was examined for defects. Only corneas free of damage were used.
The medium from both chambers of each holder was replaced with fresh complete MEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were 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 °C for 10 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 MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each comea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 +/- 1 °C for 120 minutes.
After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed.
Following the opacity measurement the permeability of the comeas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 +/- 1 °C for 90 minutes.
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each comea was applied to a designated well on a 96-well plate and the optical density at 492 mn (OD492) was measured using the Anthos 2001 microplate reader.
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.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

3 duplicates

Value:

ca. 7

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

In vivo

Irritant / corrosive response data:

Individual and mean comeal opacity measurements and individual and mean corneal permeability measurements are given in Table 1 of the attached document.
The condition of each comea post treatment and at the final opacity measurement is given in Table 2 of the attached document.
The in vitro irritancy scores are summarized in the table hereunder.
The comeas treated with the test item were clear post treatment and slightly cloudy post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The comeas treated with the positive control item were cloudy post treatment and post incubation.

Any other information on results incl. tables

Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived fonnula to generate an In Vitro Irritancy Score.

The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control comeas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

The following formula was used to determine the In Vitro Irritancy Score:

In Vitro Irritancy Score = mean opacity value + (15 x mean OD492 value)

Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

The condition of the cornea was visually assessed immediately after rinsing and at the final opacity measurement.

In Vitro Irritancy Scores  Treatment  In vitro Irritancy Score  Test Item 7.0   Negative Control  0.9 Positive Control   32.3

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The eye irritancy potential of the test material was assessed in accordance with OECD Guideline 437. The test item was considered not to be an ocular corrosive or severe irritant. The result of this study has identified the test item as not causing serious eye damage. As such, the test material does not meet the GHS criteria for classification.

Executive summary:

Introduction

A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea.

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 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 fonnula to generate an In Vitro Irritancy Score (IVIS).

Method

The test item is classified according to the prediction model below:

IVIS CLASSIFICATION

<3 No category. Not requiring classification to UN GHS or EU CLP

> 3 and < 55 No prediction of eye irritation can be made

> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

The In Vitro irritancy scores are summarized as follows:

Test Item: 7.0

Negative Control: 0.9

Positive Control: 32.3

Conclusions

The test item was considered not to be an ocular corrosive or severe irritant.

The result of this study has identified the test item as not causing serious eye damage.