Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From February 01 to February 07, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
CAS No.: 23054-60-6 + 23054-61-7; Batch No.: 7723329-R802; Purity: 100 %; Appearance: colourless/pale straw clear liquid (white solid was confirmed by visual inspection upon arrival at test facility at room temperature. Test substance was clear liquid after incubation at 37 °C).

Test animals / tissue source

Species:
cattle
Strain:
other: Bos taurus
Details on test animals or tissues and environmental conditions:
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre_sur Dives, France.

Age: as French Authorities avoid the use of any organs from the head of bovines aged more than 12 months, bovine cattle were up to 12 months old (typically, 5 to 8 months old).

Reason for choice: bovine corneas are recommended by Regulatory Authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.

Transport from Supplier to Citoxlab France: the eyes were immerged in containers filled with cooled buffered Hanks medium and placed into a cooling-box with a sufficient amount of ice packs to ensure cooling until arrival at Citoxlab France. Containers with smooth internal surfaces were used for the transport to avoid damage to the corneas. Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].

Preparation of the corneas:
Upon arrival at Citoxlab France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using Hanks’ Balanced Salt Solution (HBSS) in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.

Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared. The corneas were used immediately.

Test system

Vehicle:
other: 0.9 % NaCl
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Test substance formulation:
Prior handling, a small portion of test substance was taken and placed at 37 °C until the liquid form was retrieved; i.e. in 10 minutes for the preliminary assay and 37 minutes for the main test. As the test substance was a surfactant, it was tested at the concentration of 10% (w/v) in the vehicle. The test substance formulation was a whitish emulsion. The test substance formulation was prepared within 4 hours of use and stored at room temperature until use.

Dosing:
A volume of 750 µL (± 8 µL) was applied on each cornea using the closed-chamber treatment method as follows: the test substance formulation was introduced into the anterior chamber of the corneal holder, through the dosing holes to cover the epithelial side of the cornea. The dosing holes were then sealed.
Duration of treatment / exposure:
10 minutes
Number of animals or in vitro replicates:
3
Details on study design:
Pre-incubation:
Both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea.
After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).

At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM without phenol red (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0.

Allocation of the corneas:
- The median value of the OPT0 values of all pre-incubated corneas (with OPT0 ≤ 7) was calculated,
- Three corneas with opacity values close to the median value were selected as vehicle control corneas,
- The remaining corneas were shared out between test substance and positive control-treated series using a manual distribution procedure.

Treatment of corneas:
The medium of the anterior chamber was removed and each substance was applied onto the epithelium of the cornea, test substance formulation, positive and vehicle controls application.
After application of the substance, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for the selected treatment time.

Rinsing of the corneas:
On completion of the treatment period, the test substance formulation was removed from the front opening of the anterior chamber (closed-chamber method) and the epithelium was rinsed as follows:
- the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red. Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. Care was taken to make sure that no air bubbles were present within the holders.
Following the 10-minute treatment and the rinsing step, the holders were incubated horizontally (corneas placed vertically) for 2 hours (± 10 minutes) in a water bath at +32°C (± 1°C). On completion of the 2-hour incubation period, the medium of both anterior and posterior chambers was renewed with pre-warmed cMEM (+32°C (± 1°C)), the second opacity measurement (OPT2) was then performed.

Opacity measurements:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.

Just before the first opacity measurement (i.e. OPT0), the opacitometer was calibrated using specific calibrators. Values obtained for each calibrator were as follows:
- calibrator No. 1: set to 75,
- calibrator No. 2: from 145 to 155,
- calibrator No. 3: from 218 to 232.

Just before the second opacity measurement (i.e. OPT2), the opacitometer was calibrated using the calibrator No. 1 set to 75.
Just after each opacity measurement (OPT0 and OPT2), the calibration of the opacitometer was checked by using the calibrator No. 1. The obtained value was between 73 and 77.
Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.

Permeability determination:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution.

As the test substance was a surfactant, the concentration of the fluorescein solution was 4 mg/mL. Before use, the fluorescein solution was validated.

For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32°C (± 1°C) for 90 minutes (± 5 minutes).

At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

Macroscopic examination:
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
3
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- No notable opaque spots or irregularities were observed on vehicle control corneas.
- Fluorescein fixation was observed on the corneas treated with the test substance.
- Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the study conditions, the obtained mean In Vitro Irritancy Score (IVIS) was 3, with individual IVIS of 5, 1 and 4 for each cornea. The mean IVIS indicates the test substance is not irritant to the eye. However, as two out of three corneas gave a prediction different from the mean of all three, results were considered borderline and no conclusion can be made.
Executive summary:

A study was conducted to determine the potential irritant and corrosive properties of the test substance to the eye according to OECD Guideline 437, in compliance with GLP. In this in vitro method, damage by the test substance is assessed by quantitative measurements of changes in corneal opacity and permeability. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at 32°C. A single experiment was performed using three corneas for each treated series (test substance, positive control and vehicle control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test substance was applied at the concentration of 10% (w/v) in the vehicle (0.9% NaCl), in a single experiment using a treatment time of 10 minutes and using the closed chamber treatment method. Vehicle and positive controls were treated concurrently. At the completion of the treatment period the epithelia were rinsed and the corneas were then incubated for 2 hours (± 10 minutes) at +32 °C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at 32 °C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Fluorescein fixation was observed on the corneas treated with the test substance. All acceptance criteria were fulfilled. The study was therefore considered as valid. Under the study conditions, the obtained mean In Vitro Irritancy Score (IVIS) was 3, with individual IVIS of 5, 1 and 4 for each cornea. The mean IVIS indicates the test substance is not irritant to the eye. However, as two out of three corneas gave a prediction different from the mean of all three, results were considered borderline and no conclusion can be made (Gerbeix, 2018).