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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-03-23 to 2018-04-11
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guideline
according to
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:
adopted 09 October 2017
GLP compliance:
yes (incl. certificate)

Test material

Specific details on test material used for the study:
- CAS No.: 39464-70-5
- Description: Clear yellow to amber viscous liquid
- Storage condition: At room temperature
- Purity: 100% (UVCB)

As the test item was a surfactant containing mixture, it was tested undiluted (i.e. in its original form). Thus, no vehicle was used.

Test animals / tissue source

not specified
Details on test animals or tissues and environmental conditions:
- Source: from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Age of donor animals: up to 12 months old (typically, 5 to 8 months old).
- Transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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)]

Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): undiluted
Duration of treatment / exposure:
10 minutes (± 30 seconds)
Duration of post- treatment incubation (in vitro):
2 hours (± 10 minutes)
Number of animals or in vitro replicates:
Details on study design:
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 then used immediately.
Storage of the corneas: as the corneas were not used immediately, they were stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4 °C, for a maximum of 24 hours before use. On the day of use, the corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded.

A single experiment was performed. The treatment time was dependent upon the nature of the test item.
Treatment time:
As the test item was a surfactant containing mixture, a treatment time of 10 minutes (± 30 seconds) was used in the study.
Assembly of the corneas and the holders:
The corneas were 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.
For 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. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.
Allocation of the corneas:
The test item, the negative and positive controls were tested on three corneas each.

The corneas were distributed as follows:
- 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 negative control corneas,
- the remaining corneas were shared out between test item and positive control-treated series using a manual distribution procedure.

To rigorously respect the treatment and any other incubation times, the three corneas from the same series were always processed in the same order at each step.
Treatment of corneas:
The medium of the anterior chamber was removed and each item was applied onto the epithelium of the cornea, as specified in the Test item, positive and negative controls application.

The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series. After application of the items, 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:
The purpose of rinsing was to eliminate as much items as possible, while taking care not to damage the cornea. On completion of the treatment period, the test item was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test item adhering to the walls of the anterior chamber was removed using a pipette of heated cMEM (32 °C),
- the corneas were rinsed four 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, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

The rinsing efficiency was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red). No difficulties were encountered during the rinsing. 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 (by ensuring that each chamber was filled to overflowing with pre-warmed cMEM). 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. 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. For opacity measurement, care was taken to make sure that no air bubbles were present within the holders containing corneas (by ensuring that each compartment was filled to overflowing with heated cMEM) and each holder was wiped dry.
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 item was a mixture containing surfactants, the concentration of the fluorescein solution was 4 mg/mL. Before use, the fluorescein solution was validated. For this purpose, the solution of fluorescein was diluted in cMEM in order to obtain a 5 µg/mL solution and the Optical Density at a wavelength of 490 nm (OD490 nm) of this dilution was measured. As the value obtained was between 0.850 and 0.940 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. Then, the corneas were discarded.

Results and discussion

In vitro

Irritation parameter:
in vitro irritation score
Run / experiment:
mean of triplicates
Vehicle controls validity:
not examined
Negative controls validity:
Positive controls validity:
Remarks on result:
not determinable
no prediction can be made
Other effects / acceptance of results:
Macroscopic examination:
Fluorescein fixation was observed on one cornea treated with the negative control. Opacity, fluorescein fixation and thickening of the corneas were observed on the corneas treated with the test item and positive control.

In vitro Irritancy Score:
The individual and mean opacity and permeability values for the test item, positive control and negative control were measured and 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 46. As the mean IVIS was > 3 and ≤ 55, the eye hazard potential of the test item could not be predicted. The test item could not be identified as inducing serious eye damage (UN GHS Category 1) or as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). For detailed results please refer to Table 1 in box "Any other information on results incl. tables".

Any other information on results incl. tables

Table 1: Results for the test item






Neg correction



Neg correction



Test item




















































Applicant's summary and conclusion

Interpretation of results:
other: no prediction can be made
In conclusion, based on the mean in vitro irritation score of 46 obtained in the bovine corneal opacity and permeability assay (OECD 437), no prediction can be made regarding the classification of the test substance.
Executive summary:

The eye irritation potential of Ethoxylated phenol phosphate (100% purity) was investigated in the bovine corneal opacity and permeability assay (OECD 437). A mean in vitro irritation score of 46 was determined. The positive control induced the appropriate responses, indicating the validity of the assay. According to the UN GHS criteria, this mean in vitro irritation score does not allow to make any prediction regarding classification of the test item.