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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:
February 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
EC Number:
275-156-8
EC Name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
Cas Number:
71048-82-3
Molecular formula:
C13H20O
IUPAC Name:
[1α(E),2β]-1-(2,6,6-trimethylcyclohex-3-en-1-yl)but-2-en-1-one
Test material form:
liquid

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test substance: 30 μL
Negative control: 30 μL physiological saline 0.9%
Positive control: 30 μL benzalkonium chloride
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
At ca. 0, 30, 75, 120, 180 and 240 minutes after treatment
Fluorscein retention was only scored at ca 30 minutes after treatment.
Details on study design:
- Approximately 7 weeks old, male or female chickens (ROSS, spring chickens), bodyweight range approximately 1.5-2.5 kg, were used as eye-donors. Heads of these animals were obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, the Netherlands. Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they have reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium BP 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea were examined with a slit-lamp microscope to ensure that the cornea is not damaged. If undamaged, the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit cutting off the optical nerve to short. The enucleated eye was placed in a stainless steel lamp with the cornea positioned vertically and 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 isotonic saline from a bent, stainless steel tube, at a rate of ca. 0.10 - 0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32°C. After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they are not damaged. Corneal thickness was measured using the Depth Measuring Attachment No. I for the Haag-Streit slit-lamp microscope. Corneal thickness was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes that show opacity (score higher than 0.5), or are unacceptably stained with fluorescein indicating the cornea to be permeable, or eyes that show any other signs of damage, were rejected as test eyes and replaced. Three test eyes, one negative control eye and three positive control eyes were selected for testing. Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured again to determine the zero reference value for corneal swelling calculations. At time t=0, the following procedure was applied for each test eye: the clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards. Next, the eyes (corneas) were treated with the study substances. After rinsing, each eye in the holder was return to its chamber. After the final examination the test substance treated eyes and the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 μM and stained with PAS.
- In the ICE test, the eyes were examined at several time intervals after treatment to determine ocular effects using the parameters of corneal thickness (swelling), corneal opacity and fluorescein retention. Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV). In order to translate the eye irritancy scores from the ICE test to an EC/GHS-regulatory classification, it is necessary to reconcile four irritancy classes from the ICE study into three classes within the EC/GHS-regulatory classification scheme. This was achieved through application of the prediction model, which was based on scientific judgement and which is supported by several years of experience with conduct of the ICE test.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Irritation index
Remarks:
= maximum mean corneal swelling (%) + maximum mean opacity (x 20) + mean fluorescein (x 20))
Run / experiment:
240 minutes after treatment
Value:
32
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Maximum score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Irritancy category I
Irritation parameter:
percent corneal swelling
Run / experiment:
Maximum score
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Irritancy category II
Irritation parameter:
fluorescein retention score
Run / experiment:
Maximum score
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Irritancy category I
Irritation parameter:
cornea opacity score
Run / experiment:
30 minutes after treatment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
75 minutes after treatment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
120 minutes after treatment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
180 minutes after treatment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
240 minutes after treatment
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
30 minutes after treatment
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
75 minutes after treatment
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
120 minutes after treatment
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
180 minutes after treatment
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
240 minutes after treatment
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
30 minutes after treatment
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
In the isolated chicken eye (ICE) test, application of the substance for 10 seconds caused very slight corneal swelling (2%), slight corneal opacity and very slight fluorescein retention, which correspond to an Irritancy class of “Not irritating”.

Applicant's summary and conclusion

Interpretation of results:
other: Not irritating
Remarks:
in accordance with EU CLP (EC no 1272/2008 and its amendments)
Conclusions:
On the basis of the results obtained in the ICE test (OECD guideline 438) and according to the UN-GHS and the EU-CLP classification schemes, the substance was considered to be not irritating to eyes.
Executive summary:

In an Isolated Chicken Eye (ICE) test, performed according to OECD Guideline 438 and according to GLP, isolated chicken eyes were exposed to a single application of the test sample for 10 seconds using an application amount of 30 μL. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition an irritation index was given (Irritation Index = maximum mean corneal swelling (%) + maximum mean opacity (x 20) + mean fluorescein (x 20)). The maximum possible Irritation Index is circa 200. The negative control eye, treated with physiological saline, showed no corneal effects. The positive control, treated with a 5% aqueous solution of benzalkonium chloride, caused moderate swelling, severe opacity and severe fluorescein retention. The calculated Irritation Index was 145. The test substance caused very slight corneal swelling, slight corneal opacity and very slight fluorescein retention. The calculated Irritation Index was 32. In conclusion, based on the results obtained in the ICE test and according to its classification schemes, the test substance was considered to be not irritating to eyes.