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Toxicological information

Eye irritation

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Administrative data

eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - October 2013
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
7 September 2009
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder

Test animals / tissue source


Test system

unchanged (no vehicle)
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg of test item was applied to an eyeball
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes +/- 5 minutes
Number of animals or in vitro replicates:
3 eyeballs for test item and postive control
1 eyeball for negative control
Details on study design:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay. The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg). Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 31 July 2013 at 9.00 a.m. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in polystyrene boxes humidified with towels moistened with isotonic saline. The eyes were enucleated at Phycher on 31 July 2013 between 10:30 am and 10:50 am.

The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane fumly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion ofthe optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away. The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the super:fusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the isotonic saline drip. The chambers of the superfusion apparatus was temperature controlled at 32°C ± 1.5°C. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected. Once all eyes have been examined and approved, the eyes were incubated approximately 45 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that end point.

Test item
The test item was applied for 10 seconds and then rinsed from the eye with 20 mL of isotonic saline at ambient temperature, except for the eyes No.9, 10 and 11 which were rinsed with 25 mL of isotonic saline. The eye (in its holder) was subsequently returned to the super:fusion apparatus in the original upright position.
Concurrent negative control (physiological saline - Cooper, Batch No. 19FC12GA) and positive control (Sodium Hydroxide Batch No.MKBF9973V) were included in this experiment.

Treated corneas are evaluated pretreatment and starting at 30, 75, 120, 180 and 240 minutes (+/- 5 minutes) after the post-treatment rinse.
The endpoints evaluated were corneal opacity, swelling, fluorescein retention and morphologicla effects. All of the endpoints, with the esception of fluorescein retention (which was determined only at pretreatment and 30 minutes after test item exposure) were determined at each of the above time points.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
maximal mean score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: ICE class I
Irritation parameter:
fluorescein retention score
Run / experiment:
mean score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: ICE class II
Irritation parameter:
percent corneal swelling
Run / experiment:
maximal mean score
Vehicle controls validity:
not applicable
Negative controls validity:
Positive controls validity:
Remarks on result:
other: ICE class II
Other effects / acceptance of results:
Result for positive control: 3 x IV
Result for negative control: 3x I

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In accordance with the Regulation (EC) No. 1272/2008, the test item must not be classified in category 1 "irreversible effects on the eye".
Executive summary:

In accordance with the Regulation (EC) No. 1272/2008, the test item must not be classified in category 1 "irreversible effects on the eye".