Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 March 2016 - 4 April 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 Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Number of animals: No data
- Characteristics of donor animals: male/female, approximately 7 weeks of age and 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue: Transport in small plastic boxes on tissues moistened with isotonic saline at ambient temperature
- Time interval prior to initiating testing: Within 2 hours after kill
- Indication of any existing defects or lesions in ocular tissue samples: Only undamaged eyes used
- Indication of any antibiotics used: No data

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- 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.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured to determine the zero reference value for corneal swelling calculations.

OBSERVATION PERIOD
Examination of the eyes was performed after 0, 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eyes were rinsed with 20 mL saline after 10 seconds exposure

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity, fluorescein retention and swelling: slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland)
- Macroscopic morphological damage to the surface: microscope
- Others: histopathology (preserved with 4% formaldehyde and stained with PAS)

SCORING SYSTEM:
- Mean corneal swelling (%), mean maximum opacity score and mean fluorescein retention score at 30 minutes post-treatment: According to criteria specified in OECD TG 438

DECISION CRITERIA: According to OECD TG 438.

Results and discussion

Results of ex vivo / in vitro studyopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
Slit-lamp examination
Value:
13
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean score
Irritation parameter:
cornea opacity score
Run / experiment:
Slit-lamp examination
Value:
3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean score
Irritation parameter:
fluorescein retention score
Run / experiment:
Slit-lamp examination
Value:
3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Microscopic examination of the corneas treated with Peomosa revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. 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, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea).

DEMONSTRATION OF TECHNICAL PROFICIENCY: This OECD TG was developed at this CRO and therefore the test has been performed for many years.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
- Acceptance criteria met for positive control: The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 (serious eye damage)
Remarks:
According to EU CLP 1272/2008 and its amendments.
Conclusions:
Under the test conditions (OECD 438 and GLP), the test substance is considered to cause serious damage to the eye and should be classified as such (Eye Dam. 1 / H318) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and its amendments.
Executive summary:

In accordance with 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 13%), severe opacity (mean score of 3.0) and severe fluorescein retention (mean score of 3.0). In addition, loosening of epithelium was observed. Microscopic examination of the corneas revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea). Based on these results, the test substance is considered to cause serious damage to the eye and should be classified as such (Eye Dam. 1 / H318) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC)

and its amendments

.