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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Chicken heads for obtained from TARAVIS KFT, 9600 Sárvár, Rábasömjéni út 129, Hungary
- Age at study initiation: eyes for approx. 2 hours old (after head removal)
- Weight at study initiation: not relevant


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): 0.03 g (per eye)


CONTROLS
- Amount(s) applied (volume or weight with unit): 0.03g imidazole; 30 µL NaCl (in saline)
Duration of treatment / exposure:
After 10 seconds the cornea surface was rinsed thoroughly with approx. 20 mL saline solution at ambient temperature
Observation period (in vivo):
30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable)
Cornea thickness and opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.
Duration of post- treatment incubation (in vitro):
4 hours
Number of animals or in vitro replicates:
3 eyes (test substance and positive control)
1 eye (negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES:
With the eyes still in the chicken heads, eyelids were carefully cut away with scissors from the chicken eyes. One small drop of fluorescein solution 2% (w/v) was applied onto the cornea surface for a few seconds and then rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye, which may lead to corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain the appropriate humidity.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a stell clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), again avoiding too much pressure on the eye. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with saline solution dripping from a stainless steel tube, at a rate of approx. 3 to 5 drops/min. The door of the chamber was closed, except for manipulations and examination, to maintain temperature and humidity.
Eyes selected for testing were again examined with the slit lamp microscope. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl in saline (9 g/L) (n=1)

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED: Imidazole (n=3)

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g per eye for 10 seconds

OBSERVATION PERIOD: 4 hours

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Thorough rinsing with approx. 20 mL saline solution at ambient temperature
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity scores ranging from 0 (no opacity) to 4 (complete opacity; iris invisible) according to the test guideline were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention scores ranging from 0 - 3 according to the test guideline were measured at 0 and 30 min.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Strei BQ 900) with the slit-width set at 9.5, i.e. 0.095mm.
- Macroscopic morphological damage to the surface: not applicable
- Others (e.g, histopathology): not performed

SCORING SYSTEM:
- Mean corneal swelling (%): according to TG
- Mean maximum opacity score: according to TG
- Mean fluorescein retention score at 30 minutes post-treatment: according to TG

DECISION CRITERIA: Decision criteria as indicated in the TG were used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Any other information on results incl. tables

In an experimental study according to OECD test guideline 438 under GLP conditions, the substance scored into ICE class II for corneal opacity and fluorescein retention. It scored into ICE class I for corneal swelling.

With the overall ICE score of 1xI and 2xII, the substance could not be classified (no prediction can be made) according to the UN GHS classification and CLP.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Executive summary:

The substance was tested in an experimental study under GLP conditions according to OECD TG 438 (Isolated Chicken Eye). Eye (in heads) were obtained from a slaughterhouse and not older than 2 hours at study initiation. Eyes were carefully selected and prepared, especially avoiding pressure on the eye, which may result in opacity.

Three selected eye were treated for 10 seconds with the substance and then thoroughly rinsed.

The eyes were observed 30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable) for cornea thickness and opacity at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.

A negative control and a positive control (imidazole) were included, which were valid for all observations.

The substance scored into ICE class II for corneal opacity and fluorescein retention. It scored into ICE class I for corneal swelling.

With the overall ICE score of 1xI and 2xII, the substance could not be classified (no prediction can be made) according to the UN GHS classification and CLP.