[No public or meaningful name is available]

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 21 July 2020 Experimental completion date 21 July 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2020

Report date:

2020

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

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: • Guidance Document on ‘The Collection of Tissues for Histological Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160. Adopted July 6, 2018 Paris.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Molecular formula:

C20H36O2

IUPAC Name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Test material form:

liquid: viscous

Details on test material:

Chemical name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1‐yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers

Specific details on test material used for the study:

Identification: FRET 18-0091
Chemical Name: 1-[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan-1-yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers
Batch: RDEA571-42
Purity: 92.0%
Physical state/Appearance: Light yellow viscous liquid
Expiry Date: 31 December 2021
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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item or control items were applied to the appropriate corneas.

The negative control item, sodium chloride 0.9% w/v, was used as supplied.
The positive control item, ethanol, was used as supplied.

Duration of treatment / exposure:

The undiluted test item was applied for 10 minutes

Duration of post- treatment incubation (in vitro):

incubation period of 120 minutes.

Number of animals or in vitro replicates:

3

Details on study design:

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 75 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.

A pre treatment opacity reading was taken for each cornea using a calibrated opacitometer.

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 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 ºC 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 3 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.

The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final 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 (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

0.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The corneas treated with the test item were clear post treatment and post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Any other information on results incl. tables

In Vitro Irritancy Score

TheIn Vitro irritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	0.6
Negative Control	0.7
Positive Control	44.0

Criteria for an Acceptable Test

The positive controlIn VitroIrritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

According to UN GHS Classification, the test item FRET 18-0091 is given No Category classification under the conditions of the test.

Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short‑term maintenance of normal physiological and biochemical function of the bovine corneain vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS). Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

Method

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 formula to generate anIn VitroIrritancy Score (IVIS). 

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS	UN GHS
≤ 3	No Category
>3; ≤ 55	No prediction can be made
> 55	Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In VitroIrritancy Score
Test Item	0.6
Negative Control	0.7
Positive Control	44.0

 

Conclusion

According to UN GHS Classification, the test itemFRET 18-0091is given No Category classification under the conditions of the test.