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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:
18 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
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)
Version / remarks:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Benzaldehyde, hydroxy-, polymer with phenol
Cas Number:
106466-55-1
Molecular formula:
(C7H6O2.C6H6O)x
IUPAC Name:
Benzaldehyde, hydroxy-, polymer with phenol
Test material form:
solid: flakes
Details on test material:
- Appearance: Red – reddish brown solid flake
- Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH %)
Specific details on test material used for the study:
- The test material was applied as supplied, no formulation was required. Although the test material was further powdered.

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Characteristics of donor animals: approximately 7 weeks old
- Chicken heads were collected after slaughter in a commercial abattoir from chickens which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and heads transported to the testing laboratory at ambient temperature at the earliest convenience.
- After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at the testing laboratory and processed within approximately 2 hours of collection.

Test system

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

Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Selection: 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 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
- Preparation: 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 and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.


EQUILIBRATION AND BASELINE RECORDINGS
- The prepared 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 avoid too much pressure on the eye by the clamp. 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/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. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all 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 it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5 °C) during the acclimatisation and treatment periods.
- At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. Slight corneal thickness changes (0.0 – 1.6 %) were observed in the eyes, this is considered normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.
- After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test item was applied onto the centre of the cornea.

NUMBER OF REPLICATES
- Three test material treated eyes, three positive control treated eyes and one negative control eye were examined during the study.

NEGATIVE CONTROL USED
- 30 µL of physiological saline (Salsol solution, 0.9 % (w/v) NaCl)

POSITIVE CONTROL USED
- 30 mg of powdered Imidazole

APPLICATION DOSE AND EXPOSURE TIME
- 30 mg for 10 seconds

REMOVAL OF TEST SUBSTANCE
- After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed.

OBSERVATION PERIOD
- The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.

METHODS FOR MEASURED ENDPOINTS:
- Corneal thickness and corneal opacity were measured at all time points.
- Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.
- Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

SCORING SYSTEM:
- Corneal swelling was calculated according to the following formulae:

CS at time t = [(CT at time t – CT at time=0)/ CT at t=0] x 100

Mean CS at time t = (FECS(at time t) + SECS(at time t) + TECS(at time t)) / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point

- Small negative numbers for swelling (0 to -5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at time 0

Mean ΔCOmax = (FECOmax(30min to 240min) + SECOmax(30min to 240min) + TECOmax(30min to 240min)) / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

- Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t – FR at time=0

Mean ΔFR = (FEFR(30min) + SEFR(30min) + TEFR(30min)) / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

ICE CLASSIFICATION
- Corneal Thickness:
Mean Corneal Swelling 0 to 5 %: ICE Class I
Mean Corneal Swelling >5 to 12 %: ICE Class II
Mean Corneal Swelling >12 to 18 % ( >75 min after treatment ): ICE Class II
Mean Corneal Swelling >12 to 18 %( <75 min after treatment ): ICE Class III
Mean Corneal Swelling >18 to 26 %: ICE Class III
Mean Corneal Swelling >26 to 32 % ( >75 min after treatment): ICE Class III
Mean Corneal Swelling >26 to 32 % ( <75 min after treatment ): ICE Class IV
Mean Corneal Swelling >32 %: ICE Class IV

- Corneal Opacity:
Mean Maximum Opacity Score 0.0 - 0.5: ICE Class I
Mean Maximum Opacity Score 0.6 - 1.5: ICE Class II
Mean Maximum Opacity Score 1.6 - 2.5: ICE Class III
Mean Maximum Opacity Score 2.6 – 4.0: ICE Class IV

- Fluorescein Retention:
Mean Fluorescein Retention Score at 30 minutes post – treatment 0.0 - 0.5: ICE Class I
Mean Fluorescein Retention Score at 30 minutes post – treatment 0.6 - 1.5: ICE Class II
Mean Fluorescein Retention Score at 30 minutes post – treatment 1.6 - 2.5: ICE Class III
Mean Fluorescein Retention Score at 30 minutes post – treatment 2.6 – 3.0: ICE Class IV

CLASSIFICATION
- In the case where the result indicates Non-irritant or Corrosive/Severely Irritating, then the test material can be classified. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.
- No category = 3 ×I or 2×I, 1×II
- Category 1: 3×IV or 2×IV, 1×III or 2×IV, 1×II or 2×IV, 1×I, Corneal opacity ≥ 3 at 30 min (in at least 2 eyes), Corneal opacity = 4 at any time point (in at least 2 eyes), Severe loosening of epithelium (in at least 1 eye).
- No prediction can be made: Other combinations

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
75 minutes
Value:
14.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class III
Irritation parameter:
percent corneal swelling
Run / experiment:
240 minutes
Value:
16.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class IV
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Other effects / acceptance of results:
TEST MATERIAL
- Results can be seen in Table 1.
- The overall ICE class was 1xI; 1xII; 1xIV, based on these results the test material is classified as severely irritating. UN GHS Classification: Category 1.

POSITIVE CONTROL
- The positive control (Imidazole) was classified as severely irritating.

NEGATIVE CONTROL
- The negative control Physiological saline was non-irritating.

VALIDITY OF THE TEST
- The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Any other information on results incl. tables

Table 1: Summary of Results

Observation

Test Material

Positive Control

Negative Control

Value

ICE Class

Value

ICE Class

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

14.9 %

III

9.5 %

II

0.0 %

I

Mean maximum corneal swelling at up to 240 min

16.5 %

II

22.7 %

III

0.0 %

I

Mean maximum corneal opacity

4.00

IV

4.00

IV

0.00

I

Mean fluorescein retention

0.0

I

3.00

IV

0.00

I

Other Observations

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

None

Overall ICE Class

1xI; 1xII; 1xIV

1xIII; 2xIV

3xI

 

Applicant's summary and conclusion

Interpretation of results:
other: EU Criteria: Category 1. Causes severe eye damage
Conclusions:
Under the conditions of this study the test material is irritating to the eye.
Executive summary:

An in vitro eye irritation study of the test material was performed in isolated chicken’s eyes. The irritation effects of the test material were evaluated according to the standardised guidelines OECD 438 and EU Method B.48, under GLP conditions. 

After the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid.

Slight corneal swelling was observed during the four-hour observation period on test material treated eyes. Severe corneal opacity change (severity 4) was observed in all three eyes. No fluorescein retention change was noted on all three eyes.

The test material was fully stuck to the cornea and could not be washed off during the study. The assessment was based on the examination of the free corneal surfaces.

Under the conditions of this study the test material is irritating to the eye.

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