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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
eye corrosion in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
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:
yes
Remarks:
without impact on the study conclusion
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
yes
Remarks:
without impact on the study conclusion
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
chicken
Details on test animals or tissues and environmental conditions:
The eyes collected from chickens obtained from a slaughterhouse 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. Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline. The eyes were enucleated afterwards.
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 firmly 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 of the 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 superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.1°C and 32.1°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 had been examined and approved (see table in appendix 4), the eyes were incubated between 45 and 64 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 endpoint.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 mg
Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
3 enucleated chicken eyes
Details on study design:
Immediately following the zero reference measurements, three eyes (in their holder) were removed from the superfusion apparatus, placed in a horizontal position, and 30 mg of the test item was applied, after being reduced in fine powder, during 10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item. Then the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature. The eye (in its holder) was subsequently returned to the superfusion apparatus in the original upright position. Concurrent negative control (physiological saline – Dutscher Batch No. 3012808) and positive control (sodium hydroxide – Fisher Scientific, Batch No. 1550248) were included in this experiment. One eye was treated with the negative control and three eyes were treated with the positive control.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
ca. 1.7
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
ca. 1.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
ca. 5
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item 1,4-DIBROMONAPHTHALENE is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification