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The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 December 2016 - 2 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Yttrium chloride hexahydrate
Cas Number:
10025-94-2
Molecular formula:
YCl3.6H2O
IUPAC Name:
Yttrium chloride hexahydrate
Test material form:
solid: particulate/powder
Details on test material:
- Name of the test material (as cited in the report): yttrium trichloride hexahydrate
- Physical state: solid
- Appearance: white to yellowish crystalline powder
- Further information on test material confidential.
Specific details on test material used for the study:
- Treatment of test material prior to testing: The test item was tested neat (in its original form).
- Correction factor: No correction factor was applied for this type of study.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes were 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 were 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): Eyes were transported at ambient temperature in a cool box, immerged in cooled buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for transport to avoid damage to the corneas. The corneas were selected on the day of arrival in the laboratory.
- Time interval prior to initiating testing: Maximum 24 hours, stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C.
- 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, neovascularisation, 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.
- Indication of any antibiotics used: Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final).

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 mg (± 75 mg)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL (± 8 µL)
- Concentration (if solution): 0.9% (w/v)

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µL (± 8 µL)
- Concentration (if solution): 20% (w/v) in 0.9% NaCl (w/v)

Duration of treatment / exposure:
4 hours (± 5 minutes)
Duration of post- treatment incubation (in vitro):
90 minutes ± 5 minutes
Number of animals or in vitro replicates:
3 per group (test item, positive control, negative control)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- Macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, neovascularisation, 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.
- 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 Hank’s Balanced Salts Solution (HBSS) until all corneas had been prepared.
- The corneas were washed 3 times for 15 min in HBSS plus penicillin/streptomycin (100 units/100 μg/mL final) at room temperature. The corneas were used within a maximum of 24 hours. Each cornea was 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.
- 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. The corneas were then mounted in the corneal holders (one cornea per holder) with the endothelial side against the O-ring of the posterior chamber. The anterior half of the holder was then positioned on top of the cornea and tightened with screws.
- 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 (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.

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

NUMBER OF REPLICATES: 3

TREATMENT METHOD
- Closed chamber method for negative and positive controls.
- Open chamber method for test item treatment.
- Application of test item: The window-locking ring and glass window from the anterior chamber were removed. The test item was gently applied onto the epithelium of the cornea, as uniformly as possible in order to ensure that it covers the whole epithelial surface. The glass window of the anterior chamber was then replaced (without the window-locking ring).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 4 times with pre-warmed cMEM containing phenol red, then the corneas were finally rinsed with pre-warmed cMEM without phenol red.

POST-EXPOSURE INCUBATION: Yes, 90 min in 5 mg/mL fluorescein stain at +32°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: 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 permeability: Passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

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 of the negative control corneas should be < 4.4;
- the mean OD490 nm of the negative control corneas should be < 0.043.

DECISION CRITERIA:
The IVIS cut-off values for identifying the test item as inducing serious eye damage (UN GHS Category 1) or as not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are the following:
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1

A single experiment composed of at least three corneas is sufficient for a test item when the resulting classification is unequivocal.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean of 3
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Remarks:
range: 88 to 97
Other effects / acceptance of results:
FURTHER DETAILS ON RESULTS
Mean opacity scores (range):
- negative control: 1.3 (0 to 3)
- positive control (corrected corneal opacity): 118.7 (107-127)
- test item (corrected corneal opacity): 87.7 (79-93)

Mean permeability scores (range):
- negative control: 0.036 (0.017-0.051)
- positive control (corrected optical density): 2.912 (2.456-3.272)
- test item (corrected optical density): 0.386 (0.224-0.648)

Mean in vitro irritancy score (range):
- positive control: 162 (156-168)
- test item: 93 (88-97)

No notable opaque spots or irregularities were observed on negative control corneas.
Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the test item and with the positive control.

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 93.

As the mean IVIS was > 55, the test item was considered as inducing serious eye damage (UN GHS Category 1).

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the experimental conditions of this study, the test item was identified as a test item inducing serious eye damage (UN GHS Category 1).