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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018-01-31
Reliability:
other: A reliability rating is not applicable. A pre-test on applicability of the test system (in accordance with OECD 437) was performed in an external laboratory with expertise in the respective GLP studies. Thus,results are assumed being reliable.

Data source

Reference
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
no guideline required
Principles of method if other than guideline:
a pre-test on the applicability of the test system (in accordance with OECD 437) was conducted.

Due to the physical form of the test items scheduled for the testing in the bovine corneal opacity and permeability assay, being pasty and sticky semi-liquid, a feasibility test was performed on a representative test substance (Bismuth (3+) neodecanoate) to assess whether the system is compatible with the substance fatty acids, tall oil, manganese salts displaying similar properties.

The assay uses isolated corneas obtained as a by-product from animals freshly slaughtered at an abattoir, e.g. A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes are collected from the slaughterhouse and are transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation is initiated.
The tissue surrounding the eyeball is carefully pulled away and the cornea is excised leaving a 2 to 3 mm rim of sclera. The corneas are mounted in corneal holders.
The chambers of the corneal holder are then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The corneas are incubated for one hour at 32 ± 1 °C for equilibration. After the equilibration period, the medium is removed from both chambers and replaced with fresh complete RPMI. An initial measurement is performed on each of the corneas using the opacitometer. Afterwards the medium is removed from the anterior chamber and replaced with the test item or control. After incubation the test item or control substance is removed and the epithelium washed at least three times with MEM (containing phenol red). Once the medium is free of test substance, the cornea is finally be rinsed with complete RPMI (without phenol red). Second illuminance measurements are performed.
After the illuminance measurement medium is removed from both chambers of the holder. The posterior chamber is refilled with fresh complete RPMI and is incubated for 90 minutes at 32 ± 1 °C. Afterwards optical density is determined, using a spectrophotometer.
GLP compliance:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: highly viscous, sticky
Details on test material:
- State of aggregation at room temperature: highly viscous, sticky, light yellow
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in a sealed container


OTHER SPECIFICS: The pre-test was conducted with a representative sample displaying pasty and sticky semi-liquid properties.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: abattoir A. Moksel AG, Buchloe, Germany
- Storage, temperature and transport conditions of ocular tissue: fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories
- Time interval prior to initiating testing: immediately after arrival of the eyes.

Test system

Vehicle:
not specified
Controls:
not required
Amount / concentration applied:
not specified
Duration of treatment / exposure:
not specified
Duration of post- treatment incubation (in vitro):
not required, since only a pre-test on applicability was conducted
Number of animals or in vitro replicates:
1 cornea was incubated with the test material (pre-test on applicability)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
- tissue surrounding the eyeball was pulled away and the cornea was excised.
- isolated corneas were mounted in corneal holders
- the chambers of the corneal holder were then filled with pre-warmed RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI).
- corneas were incubated for one hour at 32 ± 1 °C for equilibration in an incubator.SELECTION AND PREPARATION OF CORNEAS

QUALITY CHECK OF THE ISOLATED CORNEAS
- after the equilibration period, the medium was removed from both chambers and replaced with fresh complete RPMI.
- an initial illuminance measurement was performed on the cornea using the opacitometer.

APPLICATION DOSE AND EXPOSURE TIME
not specified

REMOVAL OF TEST SUBSTANCE/CONTROL SUBSTANCES
- epithelium was washed at least three times with MEM (containing phenol red).
- once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red).

METHODS FOR MEASURED ENDPOINTS
-the applicability of the test system was evaluated visually. In case the test material cannot be completely removed from the cornea without damaging the cornea, testing is considered technically not feasible.

Results and discussion

In vitro

Results
Remarks on result:
not determinable because of methodological limitations
Remarks:
Since the physical form of the ffatty acids, tall oil, manganese salts was identified as being intrinsically sticky, a pre-test was conducted in order to evaluate the feasibility of testing: A representative test item displaying similar properties (intrinsically sticky) was tested in a pre-test on one single tissue. The test material was examined for its suitability for the eye irritation testing using the BCOP assay. As the test material is highly sticky, it was not possible to remove the test material from the corneal chamber and to wash it off from the epithelium after incubation. Therefore, remaining test item would have an impact on subsequent opacity and permeability measurements. Due to the intrinsic properties (sticky) of the test item, testing was technically not feasible.
Other effects / acceptance of results:
Due to technical limitations, the study cannot be performed according to the guideline OECD439 and therefore the study is cancelled. Please refer to the field "Attached full study report".

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Since the physical form of the fatty acids, tall oil, manganese salts was identified as being intrinsically sticky, a pre-test was conducted in order to evaluate the feasibility of testing: A representative test item displaying similar properties (intrinsically sticky) was tested in a pre-test on one single tissue.

Pre-test with one bovine cornea was performed with Bismuth neodecanoate. Due to the test items characteristics it was not possible to remove the substance from the corneal chamber and to wash it off from the epithelium after incubation. Therefore, remaining test item would have an impact on subsequent opacity and permeability measurements and BCOP-Assay is not feasible for this test substance.

The test item, fatty acids, tall oil, manganese salts was examined by the respective study director. The study director did not recommend BCOP-Assay for classification of fatty acids, tall oil, manganese salts due to the physical properties (sticky).
Sticky Substances will adhere to the cornea and it will not be possible to remove it without damaging the cornea. Due to these technical limitations the BCOP assay is not recommended for these substances and therefore the studies were cancelled because the studies cannot be performed according to the guideline OECD437.