Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-06-04 to 2018-06-20
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
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)
Version / remarks:
October 2017
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse.
Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The solid test item was suspended in a 0.9% sodium chloride solution with a final test substance concentration of 20% as recommended in the test guideline 437 for non-surfactant solids.
750 μL were added to completely cover the cornea’s epithelium in the anterior chamber.
Duration of treatment / exposure:
240 min (recommended exposure time for non-surfactant solids)
Duration of post- treatment incubation (in vitro):
at 32 ± 1 °C for 90 ± 5 minutes
Number of animals or in vitro replicates:
3 corneas per group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse.
To minimise deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL.
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation.
Only corneas from eyes free of defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : 0.9% sodium chloride solution
Positive control item: 20% Imidazole (CAS no. 288-32-4)6 in 0.9%
sodium chloride solution

POSITIVE CONTROL USED
20% Imidazole (CAS no. 288-32-4)6 in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME :
750 μL of the test or control items were added to completely cover the cornea’s epithelium in the anterior chamber.
Exposure period: 240 minutes (recommended exposure time for non-surfactant solids)

TREATMENT METHOD:The open-chamber method was used.

REMOVAL OF TEST SUBSTANCE
Subsequently, the epithelium was washed with EMEM containing phenol red at least three times.
Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible.
The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer (BASF 2011-13; 67063 Ludwigshafen am Rhein, Germany)
resulting in opacity values measured on a continuous scale.
- Corneal permeability:
To determine the corneal permeability 1 mL sodium fluorescein9 solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface)
was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes.
The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 7.2).
Measurements at 490 nm were recorded as optical density (OD490).
The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Experiment 1
Value:
50.035
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no information reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.585 ± 1.163 and a mean permeability value of 0.023 ± 0.013.
The calculated IVIS value of 0.925 ± 1.294 was within two standard deviations of the current historical mean and well below the cut-off value of 3 (UN GHS no category).

- Acceptance criteria met for positive control:
The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 57.702 ± 9.732 and a mean permeability value of 2.496 ± 0.384 compared to the solvent control.
The calculated IVIS value of 95.137 ± 14.345 was within two standard deviations of the current historical mean and well above the cut-off value of 55.

The acceptance criteria for the test were fulfilled.

Applicant's summary and conclusion

Conclusions:
The calculated IVIS for Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine is 50.035 ± 4.921.
Executive summary:

The purpose of this study was to determine a possible potency of Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine of being 'ocular corrosive and severe irritant' employing an in vitro system. The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro.

 

In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from

bovine eyes.Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

 

Three corneas were used for each treatment group (test item, solvent control and positive control).

The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% test item as recommended in the test guideline 437 for non-surfactant solids.

0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

 

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the

controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The acceptance criteria of validity were fulfilled in this test.

 

Following treatment with test substance a mean opacity of 48.645 ± 4.760 and a mean permeability value of 0.093 ± 0.015 compared to the negative control were determined.

The calculated IVIS of 50.035 ± 4.921 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, (identifying test substances as inducing serious eye damage UN GHS Category 1).

According to criteria of the guideline no prediction concerning irritating potential of the test item can be made.