Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The Isolated Chicken Eye (ICE) test is an in vitro method that can be used to identify chemicals inducing serious eye damage (Category 1 according to the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008 on the Classification, Labelling and Packaging of Substances and Mixtures) as well as chemicals that do not require classification for eye irritation or serious eye damage as defined by UN GHS and Regulation (EC) No 1272/2008. The ICE test was evaluated by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM), in conjunction with the European Centre for the Validation of Alternative Methods (ECVAM) and the Japanese Centre for the Validation of Alternative Methods (JaCVAM) (2006 and 2010).
The purpose of this method is to describe the procedures involved to evaluate the eye hazard potential of a test chemical as measured by its ability to induce toxicity in an enucleated chicken eye. Toxic effects to the cornea are measured by (i) a qualitative assessment of opacity (ii) a qualitative assessment of damage to epithelium based on application of fluorescein to the eye (fluorescein retention) (iii) a quantitative measurement of increased thickness (swelling) and (iv) a qualitative evaluation of macroscopic morphological damage to the surface.
The ICE test can also be used to identify chemicals not requiring classification according to the UN GHS Classification System.
Preferred species of choice since the test was developed using this species and allows for comparison with in vivo eye irritation data and they are specified in the appropriate test guidelines.

Data source

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

Materials and methods

Test guideline
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
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): o-sec-butylphenol
- Substance type: Mono-alkylphenol
- Analytical purity: 99.15%
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: A00000035
- Expiration date of the lot/batch: 2017-01-28
- Purity test date: 2016-02-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: yes
- Solubility and stability of the test substance in the solvent/vehicle: yes
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

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

FORM AS APPLIED IN THE TEST (if different from that of starting material)
- liquid

Test animals / tissue source

Species:
other: enucleated chicken eye
Strain:
other: Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)
Details on test animals or tissues and environmental conditions:
Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK. The chickens weighed 3kg and were approximately 56 days old prior to being humanely killed for human consumption.
Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
For the purpose of this study the test item was used as supplied.
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 mL


Duration of treatment / exposure:
The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline.
Duration of post- treatment incubation (in vitro):
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.
Number of animals or in vitro replicates:
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Multiple chicken heads (Spring chickens (Gallus Gallus e.g. Ross 308 Broiler)), were supplied by Baileys Turkeys Ltd., Cheshire, UK. The chickens weighed 3kg and were approximately 56 days old prior to being humanely killed for human consumption.
Heads were removed immediately after the chickens had been humanely killed at the source, for use on the same day. The time interval between collection of chicken heads and placing the eyes in the superfusion chamber following enucleation was minimized although all eyes had to fall within the acceptance criteria identified in the test guideline.
Following slaughter, the intact chicken heads were placed into individual plastic compartments within a plastic box in order to minimize any damage to the eyes. The base of each compartment was lined with a paper towel moistened with isotonic saline. The heads were transported to the test facility at ambient temperature.
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.
Eyelids were carefully excised whilst taking care not to damage the cornea. The integrity of the cornea was measured with a drop of 2% (w/v) sodium fluorescein to the surface of the cornea and then rinsed with isotonic saline after a few seconds. The treated eyes were examined with the use of the Haag-Streit BQ 900 (Switzerland) microscope, to examine for damage to the cornea. An acceptable eye for the ICE test was one where the fluorescein retention and corneal opacity scores are ≤ 0.5.
Acceptable eyes were dissected from the skull and pulled from the orbit by holding the nictitating membrane firmly with surgical forceps. The tissue behind the eye was carefully removed with bent, blunt-tipped scissors. Once the eye was removed from the orbit a portion of the optic nerve remained. Other connective tissue was removed from the eye on an absorbent tray liner.
Enucleated eyes were transferred to an appropriate clamp keeping the cornea vertical. They were then transferred to chambers within the superfusion apparatus ensuring the corneas received sufficient isotonic saline from the saline drip. The temperature of the chambers was at 32 ±1.5 °C.

EQUILIBRATION AND BASELINE RECORDINGS
Once all eyes were placed in the superfusion apparatus, the eyes were examined again with the Haag-Streit BQ 900 to ensure the eyes had not been damaged by the dissection procedure.
Corneal thickness measurements are taken with a depth measuring device no. 1 on the Haag- Streit BQ 900 slit-lamp microscope at the center of each cornea.
Eyes were replaced when: (i) the fluorescein score was > 0.5; (ii) the corneal opacity score was > 0.5; or (iii) there was any additional signs of damage, (iv) the corneal thickness measurements for individual eyes deviated more than 10% from the mean value for all eyes.
After the approval process the eyes were incubated for 45 minutes for equilibrium purposes.
Time zero measurements for corneal thickness and opacity were taken to serve as a baseline.
The baseline for the fluorescein measurements were taken at dissection.

NUMBER OF REPLICATES
The test item and positive control item groups consisted of three eyes and the negative control item consisted of two eyes.

NEGATIVE CONTROL USED
Identification: Sodium chloride 0.9% w/v
Batch: 3011424
Purity: 0.9%
Physical state / Appearance: clear colorless liquid
Expiry Date: 01 January 2017
Storage Conditions: room temperature

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
Information as provided by the supplier.
Identification: Benzalkonium chloride
Batch: A0324149
Purity: unknown
Physical state / Appearance: clear colorless viscous liquid
Expiry Date: 29 July 2016
Storage Conditions: room temperature over silica gel

APPLICATION DOSE AND EXPOSURE TIME
Immediately following the zero reference measurements, each test eye (including clamp) was removed from the superfusion apparatus and placed horizontally (cornea facing upwards) into a petri dish. 0.03 mL of test item was applied to the cornea. The entire surface of the cornea was evenly covered. The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline. The treated eye (including clamp) was subsequently returned to the superfusion apparatus in the original upright position.
0.03 mL of the positive control item, Benzalkonium chloride (5%), was similarly applied to the cornea of each positive control eye and 0.03 mL of the negative control item was applied to the cornea of each negative control eye.

OBSERVATION PERIOD
Treated corneas were evaluated prior to treatment and at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The test item remained in place for 10 seconds and was then rinsed from the eye using 20 mL of isotonic saline.
- Indicate any deviation from test procedure in the Guideline: No deviations reported.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes, at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline
- Damage to epithelium based on fluorescein retention: yes, only determined at 30 minutes after test substance exposure
- Swelling: assessed from corneal thickness measurements: yes, at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline
- Macroscopic morphological damage to the surface: yes, at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline

SCORING SYSTEM:
- Mean corneal swelling (%): The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
- Mean maximum opacity score: Corneal opacity was calculated with the most densely opacified areas for scoring. The mean value for all test eyes was calculated for all time points. The highest mean score, as observed at any time point was given an overall category for each test item
- Mean fluorescein retention score at 30 minutes post-treatment
-Pitting, sloughing, roughening of the corneal surface, and adhering of test item are all morphological effects that can be noted on the cornea. The classification of these findings was subject to interpretation.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: yes.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Remarks:
Maximal mean score for corneal opacity
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.5 ICE Class I
Positive controls validity:
valid
Remarks:
4.0: ICE Class IV
Remarks on result:
other: ICE Class IV
Irritation parameter:
fluorescein retention score
Remarks:
Mean score of Fluorescein Retention
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.5 ICE Class I
Positive controls validity:
valid
Remarks:
3.0 ICE Class IV
Remarks on result:
other: ICE Class IV
Irritation parameter:
percent corneal swelling
Value:
48.57
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
5.19% ICE Class II
Positive controls validity:
valid
Remarks:
39.81% ICE Class IV
Remarks on result:
other: ICE Class IV
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: complete corneal opacity

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

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

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Following assessment of the data for all endpoints the test item was considered to have the potential to cause ocular corrosivity/severe irritancy in vivo.
Executive summary:

The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item as measured by its ability to induce toxicity in an enucleated chicken eye.

0.03 mL of the test item was applied onto the cornea of each of three enucleated eyes. A further three enucleated eyes were treated with positive control item. A further two enucleated eyes were treated with negative control item.

Maximal ocular irritation observations recorded for the test item treated eyes were as follows:

Mean Corneal

Opacity

(ICE class)

Mean

Fluorescein

Retention

(ICE class)

% Mean Corneal Thickness

(ICE class)

Combination of the

3 Endpoints

30

mins

75

mins

120 mins

180 mins

240 mins

4.0

(IV)

3.0

(IV)

18.10

31.43

44.76

48.57

46.67

 

(IV)

3 x IV

Classification

Category 1

Mins= Minutes following treatment

Following assessment of the data for all endpoints the test item was considered to have the potential to cause ocular corrosivity/severe irritancy in vivo.