Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-03-2016 to 15-04-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
In accordance with GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
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)
Version / remarks:
adopted on 26 July 2013
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed

Test animals / tissue source

Species:
other: eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: approximately 1.5 - 2.5 kg
-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 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.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: Positive controls: Benzalkonium Chloride. Negative control: Physiological saline.
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
240 minutes
Number of animals or in vitro replicates:
3 eyes
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing: The eyes were rinsed with 20 mL saline
- Time after start of exposure: 10 seconds

SCORING SYSTEM: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes

TOOL USED TO ASSESS SCORE: All examinations were carried out with the slit-lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment.

CONTROLS: A negative control (30 µL physiological saline) and 3 positive controls (30 µL Benzalkonium Chloride 5%) were included.

Results and discussion

Results of ex vivo / in vitro studyopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Slit-lamp examination
Value:
6
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean values
Irritation parameter:
cornea opacity score
Run / experiment:
Slit-lamp examination
Value:
1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean values
Irritation parameter:
fluorescein retention score
Run / experiment:
Slit-lamp examination
Value:
1.3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Slit-lamp examination: The test substance caused corneal effects consisting of slight corneal swelling (mean of 6%), slight opacity (mean score of 1.0) and slight or moderate fluorescein retention (mean score of 1.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination: Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion, very slight or slight necrosis (two corneas) and moderate vacuolation of the epithelium (one cornea), and the epithelium partly detached from the basement membrane (one cornea). Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).

Applicant's summary and conclusion

Interpretation of results:
other: Category 2 (irritating to eyes)
Remarks:
in accordance with EU CLP criteria (1272/2008) and its amendments.
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is considered to be an eye irritant. Therefore, the substance should be classified Category 2 irritating to eyes in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC). In accordance with GHS criteria, the substance should be classified as Category 2B (mildly irritating to eyes).
Executive summary:

In accordance to OECD guideline 438 and GLP the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of slight corneal swelling (mean of 6%), slight opacity (mean score of 1.0) and slight or moderate fluorescein retention (mean score of 1.3). The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion, very slight or slight necrosis (two corneas) and moderate vacuolation of the epithelium (one cornea), and the epithelium partly detached from the basement membrane (one cornea). Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the test substance is considered to be eye irritating.