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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP complaint, guideline study, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference Type:
study report
Report Date:

Materials and methods

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

Test material

Details on test material:
Test material name: Delta Damascone
Lot number: 2030337
Storage conditions: 2-10°C
Appearance: clear, colorless to pale yellow liquid
Purity: 97.6%
Expiry date: 18 January 2011
TNO dispende number: 100009

Test system

unchanged (no vehicle)
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.
Number of animals or in vitro replicates:
not applicable
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 cormeal 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 equilibaration 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 uotside 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 neuteral 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 sevreal years of experience with conduct of the ICE test.

Results and discussion

In vivo

Irritation parameter:
other: Irritation Index (= maximum mean corneal swelling (%) + maximum mean opacity (x 20) + mean fluorescein (x 20))
other: mean of three eyes
Time point:
other: 240 minutes
Max. score:
other: the examination of reversibility is not part of this study
Other effects:
In the isolated chicken eye (ICE) test, application of delta-damascone for 10 seconds caused very slight corneal swelling, slight corneal opacity and very slight fluorscein retention, which correspond to an Irritancy class of “Not irritating”.

Any other information on results incl. tables

Delta-damascone caused very slight corneal swelling, slight corneal opacity and very slight fluorscein retention. The calculated Irritancy categories were I, II, and I for swelling %, opacity, and fluorescein retention, respectively, which correspond to an Irritancy class of “Not irritating” As such, on the basis of the results obtained in the ICE test and according to its classification schemes, delta-damascone was considered to be not irritating to eyes.

Applicant's summary and conclusion