Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-09-01 till 2015-09-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD 438 guideline study in compliance with GLP, available as unpublished report, no restrictions, fully adequate for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Triskelion, Utrechtseweg 48, 3700 AV, Zeist

Test material

Reference
Name:
Unnamed
Test material form:
liquid

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: Phosphate buffered saline (PBS).
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
0, 30, 75, 120, 180, and 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.

TOOL USED TO ASSESS SCORE: All examinations were carried out with the hand-slit lamp microscope. Fluorescein retention was only scored at approximately 30 minutes after treatment. After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
mean 240 min
Value:
4
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum value during the test
Irritation parameter:
cornea opacity score
Run / experiment:
mean 180 min
Value:
0.7
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum value during the test
Irritation parameter:
fluorescein retention score
Run / experiment:
mean 30 min
Value:
0
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Slit-lamp examination:
The test substance caused very slight corneal swelling (4%), very slight or slight opacity (mean score of 0.7) and no fluorescein retention (mean score of 0.0). 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 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 erosion (two corneas) and slight necrosis (one cornea) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis.

Applicant's summary and conclusion

Interpretation of results:
other: Not an eye irritant
Remarks:
in accordance with EU CLP (EC 1272/2008 and its updates)
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is not considered to be an eye irritant.
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 very slight corneal swelling (4%), very slight or slight opacity (mean score of 0.7) and no fluorescein retention (mean score of 0.0). 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 severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with Pinyl Iso Butyraldehyde revealed very slight erosion (two corneas) and slight necrosis (one cornea) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities, apart from very slight vacuolation (top region) of the epithelium. The positive control BAC 5% caused moderate or severe erosion, slight vacuolation (one cornea; low region) of the epithelium, and endothelial necrosis. Based on these results the test substance is not considered to be an eye irritant.