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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jan 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: The Rabbit Enucleated Eye Test (REET) was used. The study was well documented, meets generally accepted scientific principles, was similar to the ICCVAM-recommended Test Method Protocol for Isolated Rabbit Eye Test Method (IRE), acceptable for assessment
Remarks:
This study incorporates a step-wise procedure which is in accordance with OECD Test Guideline 405, UK Home Office regulations and HRL, Shardlow, UK Ethical Testing Strategy.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Principles of method if other than guideline:
- Principle of test: The rabbit enucleated eye test (REET) study assesses the irritancy potential of the test substance following application onto the cornea of enucleated rabbit eyes.
- Short description of test conditions: The eyes were isolated from sacrificed rabbits and processed within 2 h postmortem. Each enucleated eye was positioned in a perspex clamp with the cornea positioned vertically, and transferred to a superfusion apparatus for examination of damage. Once approved, a saline drip was positioned to irrigate each eye, and the eyes equilibrated for 0.5 h in the superfusion apparatus. Baseline measurements were then recorded, and surface damage was assessed, before each eye was positioned horizontally and the test substance applied. At 10 seconds after treatment, the corneas were rinsed with saline solution and returned to the superfusion chamber for analysis of toxic effects. Measurements of changes in corneal opacity, fluorescein retention, corneal thickness (swelling) and morphologic changes to the corneal surface were recorded and scored.
- Parameters analysed / observed: Assessment of corneal opacity, swelling of the cornea (thickness), condition of the cornea (morphologic changes), and fluorescein retention by the corneal epithelium were measured at pre-enucleation, post equilibration and at approximately 60, 120, 180, and 240 minutes after exposure treatment.
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, London, England

Test material

Constituent 1
Chemical structure
Reference substance name:
Pyridine-2-carboxylic acid
EC Number:
202-719-7
EC Name:
Pyridine-2-carboxylic acid
Cas Number:
98-98-6
Molecular formula:
C6H5NO2
IUPAC Name:
pyridine-2-carboxylic acid
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Remarks:
Hsdlf:NZW
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Harlan Laboratories UK Ltd., Leicestershire, UK
- Number of animals: not specified
- Characteristics of donor animals: male rabbits, 12 to 20 weeks old
- Storage, temperature and transport conditions of ocular tissue: immediately after enucleation, the eyes were positioned in a perspex clamp and placed within the chamber of the superfusion apparatus, with a saline drip to irrigate the cornea.
- Time interval prior to initiating testing: the eyes were allowed to equilibrate for 30 min in the superfusion apparatus, and were then tested for corneal thickness, damage (fluorescein), and opacity.
- Indication of any existing defects or lesions in ocular tissue samples: eyes with greater than 10% corneal swelling relative to pre-enucleation measurement, or stained with fluorescein, were rejected from testing.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.1 mL
- Concentration: 10% w/w aqueous preparation
Duration of treatment / exposure:
10 sec
Number of animals or in vitro replicates:
3 treated eyes
2 control eyes
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- To select eyes used in the study, assessments of corneal opacity, swelling of the cornea, condition of the cornea, and fluorescein retention by the corneal epithelium were done at pre-enucleation.

EQUILIBRATION AND BASELINE RECORDINGS
- All eyes equilibrated in a perspex clamp within the superfusion apparatus for 30 min, and then were again screened by assessment of corneal opacity, swelling of the cornea, condition of the cornea, and fluorescein retention by the corneal epithelium.

NUMBER OF REPLICATES
- Three treated eyes

APPLICATION DOSE AND EXPOSURE TIME
- A volume of 0.1 mL of the test substance as a 10% w/w aqueous preparation, was applied to each treated eye, for 10 seconds, then washed off with saline solution.

OBSERVATION PERIOD
Measurements were recorded at approximately 60, 120, 180, and 240 minutes after exposure treatment.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: a minimum of 20 mL of saline solution was used.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp biomicroscope examination using the McDonald-Shadduck Score System (five grades ranging from 0 [normal cornea] to 4 [involvement of the entire stromal thickness])
- Damage to epithelium based on fluorescein retention: Slit-lamp biomicroscope examination (five grades ranging from 0 [absence of staining] to 4 [extreme staining])
- Swelling: measured with untrasonic pachymeter on a slit-lamp biomicroscope; slit-width setting: not specified
- Macroscopic morphological damage to the surface: documented if observed.

SCORING SYSTEM:
- Mean corneal swelling (%): less than 10% difference from pre-enucleation to post-equilibration
- Mean maximum opacity score: free of opacities up to 60 min after treatment
- Mean fluorescein retention score at 60 minutes post-treatment: free of fluorescein retention

DECISION CRITERIA:
The REET test has not been accepted as a validated test by the European Authorized Agencies for the assessment of eye irritation, however the ICCVAM has approved the REET as a legitimate alternative to in vivo testing. This step-wise procedure is in accordance with OECD TG 405, UK Home Office regulations, and HRL, Shardlow, UK Ethical Testing Strategy. The results of this study are believed to be of value in predicting the ocular irritation potential of the test substance in man.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean of 3 eyes at 240 min
Value:
>= 4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation parameter:
fluorescein retention score
Run / experiment:
mean of 3 eyes at 240 min
Value:
>= 4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation parameter:
percent corneal swelling
Run / experiment:
mean of 3 eyes at 60 min
Value:
12
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation parameter:
percent corneal swelling
Run / experiment:
mean of 3 eyes at 120 min
Value:
15.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Irritation parameter:
percent corneal swelling
Run / experiment:
mean of 3 eyes at 240 min
Value:
14
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
not examined
Other effects / acceptance of results:
OTHER EFFECTS:
No further morphological effects were observed after an observation period of 240 minutes.

Applicant's summary and conclusion

Interpretation of results:
other: Cat 1 H318 according to Regulation (EC) No. 1272/2008 is required.
Conclusions:
The test substance was considered to have the potential to cause severe ocular irritancy ex vivo. It is reasonable to assume that the test substance requires classification as Category 1 (irreversible effects on the eye) according to CLP Regulation (EC) No. 1272/2008. CLP: Category 1, H 318: causes serious eye damage.

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