Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 October 2017 to ****
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 guideline
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS number: Not yet known
EC Number: Not yet known
Specific details on test material used for the study:
Physical state/Appearance: Dark amber viscous liquid
Purity: >93%

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Fresh eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics. They were transported to the test facility over ice packs on the day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.75 mL of the test item or control items were applied to the appropriate corneas
Duration of treatment / exposure:
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Three
Details on study design:
Preparation of Corneas
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling, and 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 chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red, plugged and 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.

Treatment of Corneas
The medium from both chambers of each holder was replaced with fresh complete EMEM and a pre treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. The EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the appropriate corneas and each holder was incubated at 32 ± 1 ºC for 10 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The chamber was then refilled with fresh complete EMEM without phenol red. A post treatment opacity reading was taken and each cornea was visually observed.
The holders were incubated at 32 ± 1 ºC for 120 minutes. After incubation, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

Application of Sodium Fluorescein
Following the final opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium was removed and replaced with sodium fluorescein solution (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 µL of media representing each cornea was dispensed into the appropriate wells of a pre labeled 96 well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader and LT-com software.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
1.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
Corneal Epithelium Condition
The corneas treated with the test item were clear post treatment and clear post incubation. The corneas treated with the negative control item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation.

Criteria for an Acceptable Test
The positive control acceptance criterion was therefore satisfied. The negative control gave opacity of ≤3.0 and permeability ≤0.077. The negative control acceptance criteria were therefore satisfied

Test Item
The in vitro irritancy score of the test item was 1.9, based on this result, the test item in not an eye irritant.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The in vitro irritancy score of the test item was 1.9. Based on this result, the test item in not an eye irritant.
Executive summary:

The Bovine Corneal Opacity and Permeability (BCOP) test was performed to assess the potential of the test material to induce serious eye damage and to identify those not requiring classification for eye irritation or serious eye damage.

The undiluted test material was applied for 10 minutes followed by an incubation period of 120 minutes. Negative (0.9% (w/v) Sodium Chloride) and positive (Neat Ethanol) control items were tested concurrently. Quantitative measurements of changes in corneal opacity (assessed as decreased light transmission through the cornea) and permeability (assessed as increased passage of sodium fluorescein dye through the cornea) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The In Vitro irritancy scores are summarized as follows:

Test material: 1.9

Negative Control: 0.8

Positive Control: 43.1

The corneas treated with the test item were clear post treatment and clear post incubation with an IVIS of 1.9, which did not meet classification criteria (≤ 3; No Category; Not classified for irritation).

The corneas treated with the negative control item were clear post treatment and post incubation. The negative control gave opacity of ≤3.0, permeability of ≤0.077, and met acceptance criteria.

The corneas treated with the positive control item were cloudy post treatment and post incubation. The IVIS was within the historical range of 31.6 to 58.7 and satisfied the acceptance criterion.

Based on the results of the BCOP test, the test material is not classified for serious eye damage or eye irritation according to GHS criteria.