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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:
weight of evidence
Study period:
2014-08-14 to 2014-08-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals / tissue source

Species:
chicken
Strain:
other: COBB 500
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened by electric current). The heads were transported to TOXI-COOP ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (20.3 ºC to 21.4ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day. After collection, the heads were inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
- Time interval prior to initiating testing: 2 h
- indication of any existing defects or lesions in ocular tissue samples: none

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 μL/eye

Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
The control and test eyes were evaluated at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within ± 5 minutes were considered acceptable.
Number of animals or in vitro replicates:
The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye. The enucleated eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again, too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball 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 approximately 3 or 5 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity. The appropriate number of eyes was selected and after being placed in the superfusion apparatus, were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. Any eye with cornea thickness deviating more than 10 % from the mean value for the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5°C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a base line (t=0) for each individual eye. The cornea thickness of the eyes should not increase or reduce by more than ± 5-7 % between the acclimatisation time (-45 to -60 minutes) and the zero time. Slight thinning and changes in thickness (0% to 3%) were observed in the eyes. This is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Base line values were required to evaluate any potential test item related effects after treatment. The locations of any minor findings were marked on the record sheet as a drawing. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl 9 g/L solution

POSITIVE CONTROL USED: acetic acid 10 % (v/v) solution

APPLICATION DOSE AND EXPOSURE TIME: 30 µL for 10 sec

OBSERVATION PERIOD: 30, 75, 120, 180 and 240 minutes after the post-treatment

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire residual test item, if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to a minimum.

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

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximum corneal swelling at up to 75 min
Value:
5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean maximum corneal swelling at up to 240 min
Value:
9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
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

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Based on the overall ICE Class the negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: Based on the overall ICE Class the positive control Acetic acid 10% (v/v) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.
Positive and negative control values were within the corresponding historical control data ranges.

Any other information on results incl. tables

 Test item

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

5 %

I

Mean maximum corneal swelling at up to 240 min

9 %

II

Mean maximum corneal opacity

3.0

IV

Mean fluorescein retention

1.0

II

Other Observations

None

Overall ICE Class

2xII, 1xIV

 

 

Positive Control: Acetic acid 10% (v/v) solution

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

11 %

II

Mean maximum corneal swelling at up to 240 min

21 %

III

Mean maximum corneal opacity

4.0

IV

Mean fluorescein retention

1.0

II

Other Observations

Cornea opacity score 4 was observed in two eyes at 75 minutes after the post-treatment rinse

Overall ICE Class

1xII, 1xIII, 1xIV

 

 

Negative Control: NaCl (9g/L saline)

 

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

0 %

I

Mean maximum corneal swelling at up to 240 min

0 %

I

Mean maximum corneal opacity

0.0

I

Mean fluorescein retention

0.0

I

Other Observations

None

Overall ICE Class

3xI

 

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In an Isolated Chicken Eye model test the test item was determined to be not corrosive to eye.
Executive summary:

In the OECD 438 compliant in vitro Isolated Chicken Eye Test (ICET), the purpose was to evaluate the potential ocular corrosivity or severe irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose onto the cornea of isolated chicken eyes in order to potentially classify the test compound as ocular corrosive and/or severe irritant. The ICET does not fully replace the in vivo rabbit eye test (OECD 405); however, the ICET is used as part of a tiered testing strategy for regulatory purposes.

The test item, Acetic acid 10% (v/v) solution (positive control) and NaCl (9 g/L saline) (negative control) were applied in a volume of 30 μL/eye, in such a way that the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive or negative control. Three test item treated eyes and three positive control eyes and one negative control eye were used in this study.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.

Positive and negative controls showed the expected results. The experiments were considered to be valid.

In this in vitro eye corrosives and severe irritants study, using the Isolated Chicken Eye model with SIKA Hardener AI, no ocular corrosion or severe irritation potential was observed. According to the guideline OECD 438, the test item has been classified as “No prediction can be made”. However, to obtain a definitive classification in relation to the irritation potential, a further in vitro study (OECD 492) is required.