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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From July 28 to August 11, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:
2013
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2008
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Intercure 18
IUPAC Name:
Intercure 18
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: local abattoir as a by-product from freshly slaughtered animals
- Characteristics of donor animals (e.g. age, sex, weight): cattle typically 12 to 60 months old
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): the eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS). They were transported to the test facility over ice packs on the same day of slaughter.
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 μg/mL

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Test item: 0.75 ml (20 % w/v solution in 0.9 % w/v sodium chloride solution).
Negative control item: 0.75 ml (0.9 % w/v sodium chloride solution).
Positive control item: 0.75 ml (20 % w/v solution in 0.9 % w/v sodium chloride solution).
Duration of treatment / exposure:
240 min
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: sodium chloride 0.9% w/v

POSITIVE CONTROL USED: Imidazole

APPLICATION DOSE AND EXPOSURE TIME: the EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

REMOVAL OF TEST SUBSTANCE: Number of washing steps after exposure period: At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: visually observed
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of the Anthos 2001 microplate reader (OD492)
- Histopathology: the corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
ca. 65.9
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Corneal Epithelium Condition

The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

In Vitro Irritancy Score

The In Vitro irritancy scores are summarized as follows:

Treatment

In Vitro Irritancy Score

Test Item

65.9

Negative Control

1.2

Positive Control

87.5

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.

The negative control gave opacity of ≤ 4.1 and permeability ≤ 0.105. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 according to the OECD 437 criteria
Conclusions:
The test substance causes serious eye damage.
Executive summary:

Method

The potential eye irritation/corrosion of the test substance has been evaluated using the BCOP test ( “Bovine Corneal Opacity and Permeability Assay”) according to the OECD guideline No. 437 (updated 26 July 2013).

Observations

The IVIS score recorded for the test Item is 65.9

Conclusion

According to the classification reported in the OECD Guideline, if the IVIS Scor recorded is > 55, the test item shell be classified as Category 1. UN GHS or EU CLP Causes serious eye damage.

The test substance causes serious eye damage.