[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:

September 18, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

IUPAC Name:

[(2-hydroxyacetyl)oxy](oxo) vanadium 2-hydroxyacetate]

Test material form:

solid

Test animals / tissue source

Species:

cattle

Strain:

other: Bos taurus

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: bovine eyes obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Characteristics of donor animals (e.g. age, sex, weight): bovine cattle up to 12 months old (typically, 5 to 8 months old)
- Storage, temperature and 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)].
- Time interval prior to initiating testing: upon arrival at Citoxlab France, the selection and preparation of corneas was performed as soon as possible.
- indication of any existing defects or lesions in ocular tissue samples: : 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 swivelled in order to observe the fringe areas and any scratches directly under the light.
- Indication of any antibiotics used: Hank’s medium contained an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Concentration (if solution): 20% (w/v) in the vehicle (i.e. NaCl 0.9%)

VEHICLE
- Amount(s) of formulation (test item + vehicle) applied (volume or weight with unit): 750 mg (± 75 mg)

Duration of treatment / exposure:

4 hours (± 5 minutes)

Number of animals or in vitro replicates:

three replicates for test item, vehicle control and positive control

Details on study design:

SELECTION AND PREPARATION OF 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.
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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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 swivelled in order to observe the fringe areas and any scratches directly under the light.

NUMBER OF REPLICATES
Three replicates for test item, for vehicle and positive controls

NEGATIVE CONTROL USED (VEHICLE CONTROL)
0.9% NaCl

POSITIVE CONTROL USED
20% imidazole solution in 0.9% NaCl (w/v)

APPLICATION DOSE AND EXPOSURE TIME
750 mg (± 75 mg) for 4-hour treatment

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least three time for the corneas treated with the test item formulations and the vehicle control; and four times for corneas treated with the positive control

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured by an opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Spectrophotometer UV/VIS Cary100 (OD490)
- Others (e.g, pertinent visual observations, histopathology): after permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

118

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

No notable opaque spots or irregularities were observed on vehicle control-treated corneas.
Opacity, fluorescein fixation and blue colouration of the corneas were observed all corneas treated with the test item formulation.
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:

other: Eye corrosive

Remarks:

Acoording to the criteria set up in the OECD guideline 437

Conclusions:

Eye corrosive

Executive summary:

Method

The objective of this study was to evaluate the potential irritant and corrosive properties of the test item, to the eye. The design of this study was based on the OECD Test Guideline 437.

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 vehicle control).

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

The test item, applied at the concentration of 20% (w/v) in the vehicle (i.e.NaCl 0.9%) and both vehicle and positive controls were tested using a treatment 4 hours(± 5 minutes)and 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. A second opacity measurement was then 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

Opacity, fluorescein fixation and blue colouration of the corneas were observed all corneas treated with the test item formulation.

In VitroIrritancy Score

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

The mean In Vitro Irritancy Score (IVIS) of the corneas treated with the test item formulation was 118.

 

Conclusion 

Under the experimental conditions of this study, the test item was identified asinducing serious eye damage.