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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
Study period:
22-07-2019 to 22-07-2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal
Cas Number:
1226911-73-4
Molecular formula:
C14H18O
IUPAC Name:
(4E)-2,4-dimethyl-5-(4-methylphenyl)pent-4-enal
Test material form:
liquid
Details on test material:
- Physical state: Liquid
- Storage condition of test material: approximately 4ºC, in the dark and under nitrogen
- Other: colourless liquid

Test animals / tissue source

Species:
chicken
Strain:
other:
Remarks:
Spring Chickens (Gallus Gallus e.g. Ross 308 Broiler)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Recognised supplier (documented in the full study report)
- Number of animals: Not reported.
- Characteristics of donor animals (e.g. age, sex, weight): The chickens weighed approximately 3 kg and were approximately 56 days old prior to being humanely killed for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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.
- Time interval prior to initiating testing:
- Indication of any existing defects or lesions in ocular tissue samples: None.
- Indication of any antibiotics used: None reported.
- Selection and preparation of corneas: Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
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.
- Quality check of the isolated corneas: 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 approximately 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.03 ml of test material, undiluted
Duration of treatment / exposure:
The test item remained for 10 seconds in place and then was rinsed from the eye using 20 ml of isotonic saline.
Controls (negative and positive control items) were similarly applied to the cornea in the negative and positive control groups respectively.
Observation period (in vivo):
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 group consisted of two eyes.
Details on study design:
Selection and preparation of isolated eyes:
Full details are provided in the full study report. 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.
Eyes that had a high baseline fluorescein staining (>0.5) or corneal opacity score (>0.5) after the enucleation process were rejected.
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.
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 approximately 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.
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% v/v), 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.

Equilibration and baseline recordings: Taken at zero after 45 minutes incubation prior exposure.

Number of replicates: Triplicate for test item and positive control and duplicate for negative controls.

Application dose and exposure time: Application dose: 0.03 ml; Exposure time: 10 seconds.

Observation period: prior to treatment at 30, 75, 120, 180 and 240 minutes (±5 minutes) after the eyes had been decontaminated with the isotonic saline.

Removal of test substance: Test item remained for 10 seconds and then rinsed with 20ml of isotonic saline.

Methods for measured endpoints:
Endpoints used during the evaluation procedure were corneal opacity, swelling, fluorescein retention and morphological effects (e.g. pitting, sloughing or roughening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which is only determined at 30 minutes after test substance exposure) were determined at each of the above time points.
After the final examinations at the 240 minute time point, the eyes were preserved in neutral buffered formalin for possible histopathology. Eyes were retained until finalization of the final report.
Percentage corneal swelling was assessed from corneal thickness measurements.
Mean percentage of corneal swelling for all test eyes was calculated for all the time points. The overall category score was determined from the highest mean score for epithelial swelling as observed at any time point.
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.
The mean fluorescein retention scores for all test eyes are calculated at the 30 minute time interval only. These measurements are used for the overall classification for each test item.
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.
Once each endpoint had been established, ICE classes were determined based on a predetermined range in accordance with the criteria given in OECD TG 438. The overall in vitro irritancy classification of the test item was determined by using all the classification information and criteria given in OECD TG 438 and the UN GHS classification referenced in the guideline. Full details are provided in the full study report.
The test was considered acceptable as the concurrent negative or vehicle/solvent items and the concurrent positive controls were within the historic control data range and/or identified as GHS non classified and GHS category 1, respectively.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
mean (n=3)
Value:
1.55
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
mean (n=3)
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
mean (n=3)
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE Class II
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: None reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The laboratory previously demonstrated technical proficiency of the validated method with proficiency test items (information in the public domain). Additionally, concurrent positive and negative controls were within the historic control range (documented in the full study report), each meeting the validity criteria respectively.

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

Table 1.- Ocular reactions



























































Test Item
Maximal mean score for corneal opacity0,8ICE Class II
Mean score of Fluorescein Retention0,5ICE Class II
Maximal mean corneal swelling compared to time zero1,55%ICE Class II
Positive Control Item
Maximal mean score for corneal opacity3,3ICE Class IV
Mean score of Fluorescein Retention3ICE Class IV
Maximal mean corneal swelling compared to time zero39,06%ICE Class IV
Negative Control Item
Maximal mean score for corneal opacity0,3ICE Class I
Mean score of Fluorescein Retention0ICE Class I
Maximal mean corneal swelling compared to time zero1,63%ICE Class I

Corneal opacity scores


Maximum score was 1 in the test item group. Maximum score was 0.5 in the negative control. In the positive control group the Maximum score was 4.


No corneal effects were noted in one negative control treated eye during the study period.


Very faint opacity was noted in one negative control treated eye.


Severe corneal opacity was noted in two positive control treated eyes.


Complete corneal opacity was noted in one positive control treated eye. 


Very faint opacity was noted in one test item treated eye.


Scattered or diffused areas of opacity were noted in two test item treated eyes.


No morphological effects were noted in the test item, positive or negative control item treated eyes.


Fluorescein Retention scores


Maximum score was 0.5 in the test item group. Maximum score was 3 in the positive control group, and 0.0 in the negative control. 


No fluorescein retention was noted in the negative control treated eyes during the study period.


Confluent large areas of the cornea retaining fluorescein was noted in all positive control treated eyes. 


Very minor single cell staining was noted in the test item treated eyes.


 


Table 2.- Individual scoring - Test Item
































































































































































































End PointEye NumberTime
(after decontamination)
030 minutes75 minutes120 minutes180 minutes240 minutes
minutes
Corneal Opacity3A0.50.50.5111
6A0.50.51111
8A0000.50.50.5
Mean0.30.30.50.80.80.8
ICE ClassII
Fluorescein Retention3A 0.5    
6A 0.5    
8A 0.5    
Mean 0.5    
ICE ClassI
Corneal Thickness 3A0.660.640.680.660.640.65
6A0.630.610.660.640.620.64
8A0.650.610.630.640.590.59
Mean0.650.620.660.650.620.63
Mean Corneal Swelling (%) -4.121.550.00-4.64-3.09
ICE ClassI
Epithelium Condition3A NNNNN
6A NNNNN
8A NNNNN
ICE Classes Combined:2 x I, 1 x II
Classification:No Category 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following assessment of the data for all endpoints the Test Item was considered unlikely to have the potential to cause corrosivity/severe irritancy in vivo (UN GHS No Category and/or EU CLP No Category).
Executive summary:

The study was performed according to OECD TG 438 in accordance with GLP to evaluate possible corrosivity or severe irritancy potential of the Test Item as measured by its ability to induce toxicity in an enucleated chiken eye. 


Method 


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.


Results


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


























































Mean Corneal OpacityMean Fluorescein RetentionMean Corneal Thickness compared to time zero % (ICE class)Combination of the 3 Endpoints
(ICE class)(ICE class)3075120180240
  minsminsminsminsmins
0.80.5-4.121.550.00-4.64-3.09n/a
(II)(I)(I)(I)(I)(I)(I)
  (I)2 x I, 1 x II
Classification:No Category 

Conclusion


Following assessment of the data for all endpoints the test item was considered unlikely to have the potential to cause ocular corrosivity/severe irritancy in vivo (UN GHS No Category and/or EU CLP No Category).