Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 May 2019 - 15 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
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)
Version / remarks:
09 October 2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
BISPHENOL A POLYETHYLENE GLYCOL DIETHER DIMETHACRYLATE
EC Number:
609-946-4
Cas Number:
41637-38-1
Molecular formula:
C23H24O4 (C2H4O)n
IUPAC Name:
BISPHENOL A POLYETHYLENE GLYCOL DIETHER DIMETHACRYLATE
Test material form:
liquid

Test animals / tissue source

Species:
other: bovine cattle (Bos taurus)
Strain:
other: not applicable
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)].

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 HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled 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:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL per cornea
- Concentration: undiluted
Duration of treatment / exposure:
Exposure period of 10 minutes (± 30 seconds), followed by rinsing
Observation period (in vivo):
Not applicable.
Duration of post- treatment incubation (in vitro):
2 hours (± 10 minutes)
Number of animals or in vitro replicates:
Triplicate corneas for each tested substance (test item, negative control, positive control).
Details on study design:
EYES SELECTION
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any eyes with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number. After pre-incubation, corneas that showed any macroscopic defect or an opacity value over 7 were discarded.

TREATMENT
The corneas from the same series were always processed in the same order at each step. The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea for 10 minutes in a water bath at +32°C (± 1°C).

RINSING OF THE CORNEAS
The purpose of rinsing was to eliminate as much item as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item and both negative and positive controls were removed from the front opening of the anterior chamber (open-chamber method) and the corneas were rinsed with pre-warmed cMEM containing phenol red. Rinsing of the test item-treated corneas was performed as follows:
. residual amounts of test item, adhering to the walls of the anterior chamber, were removed using a cotton bud and a pipette of heated cMEM (32°C),
. the corneas were rinsed six times with pre-warmed cMEM containing phenol red (i.e. until the test item had been completely removed from the chamber or until the phenol red was not discoloured).

Then, all corneas were finally rinsed with pre-warmed cMEM without phenol red.


SCORING SYSTEM
- Opacity:
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea.

The average change in opacity for the corneas treated with the negative control was calculated and this value was subtracted from the change in opacity for each cornea treated with the test item or the positive control to obtain a corrected opacity value (cOPT). The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.

- Permeability:
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution (4mg/mL) 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 corrected OD490 nm (cOD490 nm) value (i.e. permeability) for each cornea treated with the test item or the positive control was calculated by subtracting the average negative control cornea OD490 nm value from the original OD490 nm value of each cornea. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.

- Scoring:
In vitro irritancy score (IVIS) = cOPT + (15 x cOD490 nm)

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS (macroscopic examination):
Negative control and test item-treated corneas: None
Positive control-treated corneas: Opacity, fluorescein fixation and thickening of the corneas

ACCEPTANCE CRITERIA:
For the validation of an experiment, the following criteria had to be fulfilled:
. the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean,
. the mean opacity and mean OD490 nm of the negative control corneas should be less than the established upper limit of historical mean.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Not eye irritating
Conclusions:
Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item to the eye. The Bovine Corneal Opacity and Permeability (BCOP) test method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage.

The design of this study was based on the OECD Test Guideline No. 437 and the study was performed in compliance with Citoxlab France standard operating procedures and with the OECD Principles of Good Laboratory Practice.

 

Method

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 item, positive control and negative control).

Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer.

The test item was applied undiluted, using a treatment time of 10 minutes (± 30 seconds) and the open-chamber treatment method. Negative and positive controls were also applied using the same treatment time but thanks to the closed-chamber treatment method.

At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. 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.

 

Results

Macroscopic examination

No notable opaque spots or irregularities were observed on the three test item-treated corneas.

In Vitro Irritancy Score

All acceptance criteria were fulfilled. The study was therefore considered as valid.

 

The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.

As the mean IVIS was = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

 

Conclusion

Under the experimental conditions of this study, the test item was identified as not requiring classification for eye irritation or serious eye damage (UN GHS No Category).