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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:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
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:
2017
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Rosin, reaction products with acrylic acid
EC Number:
280-192-2
EC Name:
Rosin, reaction products with acrylic acid
Cas Number:
83137-13-7
Molecular formula:
N.A. - UVCB substance
IUPAC Name:
Rosin, reaction products with acrylic acid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 3M Company, Lot 646729
- Expiration date of the lot/batch: 19 November 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature and humidity

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: 1.000 g of material was brought to a total volume of 5 mL with 0.9% saline and mixed

FORM AS APPLIED IN THE TEST (if different from that of starting material): Test material dissolved in 0.9% saline

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Spear Products (recieved 13 July 2017); provided in a Hanks' Balance Salt Solution (HBSS) with penicillin-streptomycin.
- Number of animals: No data
- Characteristics of donor animals (e.g. age, sex, weight): No data
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported in Hanks' Balanced Salt Solution with penicillin-streptomycin in a refrigerated container.
- Time interval prior to initiating testing: No data
- Indication of any existing defects or lesions in ocular tissue samples: Corneas from eyes that were free of defects were chosen for the study.
- Indication of any antibiotics used: penicillin-streptomycin

Test system

Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): 20% (200 mg/mL) test article in 0.9% saline

VEHICLE
- Lot/batch no. (if required): Lot No. J6P916
Duration of treatment / exposure:
4 hours (+/- 10 minutes)
Duration of post- treatment incubation (in vitro):
90 (+/- 5 minutes)
Number of animals or in vitro replicates:
12 bovine corneas (4/group)
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: Bovine eyes (at least six months old) were obtained from an abattoir and were transported to the laboratory containing Hanks’ Balanced Salt solution Hanks’ Balanced Salt Solution (HBSS) and penicillin-streptomycin in a refrigerated container. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in specially designed holders that were separated into anterior and posterior chambers and filled separately. Each cornea was mounted allowing the epithelium of the cornea to project into the anterior chamber. The posterior chamber was filled with MEM solution ensuring contact with the endothelium. The anterior chamber was filled with MEM solution, ensuring contact with the epithelium. Each cornea was visually inspected again to ensure there were no defects.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. A pre-exposure determination of opacity was made for each cornea by measuring each against the blank supplied by the opacitometer. Any cornea with a value greater than 7 units was discarded.

NUMBER OF REPLICATES: 12 bovine corneas (4/group)

NEGATIVE CONTROL USED: Minimal Essential Media (MEM), Lot No. SLBQ8741V

SOLVENT CONTROL USED (if applicable): 0.9% Sodium Chloride Irrigation (saline), Lot No. J6P916

POSITIVE CONTROL USED: Imidazole, Lot No. B0139549

APPLICATION DOSE AND EXPOSURE TIME: 0.75 ml of the 20% (w/v) test article formulation in saline, 20% (w/v) imidazole formulation in saline, MEM solution or 0.9% saline was applied to the epithelium of each of each of the three test article corneas, three positive control corneas, three negative control corneas and three vehicle control corneas in a manner which ensured that the entire cornea was covered. Exposure time was 4 hours.

TREATMENT METHOD: The test article corneas were dosed via the open-chamber method. The negative, positive, and vehicle controls were dosed via the closed-chamber method.

POST-INCUBATION PERIOD: 90 (+/ 5) minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2 (MEM with and without phenol red)
- POST-EXPOSURE INCUBATION: Incubated for 90 (+/- 5) minutes in 1.0 mL of 0.5% sodium fluorescene solution in Dulbecco's PBS at 32C in the horizontal position to ensure the fluorescein contacted the cornea

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity measured following the 4 hour exposure period. Differences in light transmission through the control and treated cornea will be determined using an OP-KIT opacitometer produced by Electro-Design Corporation of Riom, France. Each treated cornea will be scored in comparison to the blanks provided with the OP-KIT opacitometer .
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The IVIS for the test article and vehicle control was calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density, as shown by the equation below. The calculation to obtain an IVIS for the positive control was performed in the same manner as the test article.

In Vitro Irritancy Score (IVIS) = Corrected Mean Opacity Score + (15 X Corrected Mean Optical Density Score)

The calculation to obtain an IVIS for the negative control was performed by adding the mean opacity score to fifteen times the mean optical density, as shown by the equation below.

In Vitro Irritancy Score (IVIS) = Mean Opacity Score + (15 X Mean Optical Density Score)

Based on the IVIS score, the test article was classified according to the prediction model described in DB-ALM Protocol No. 1271, a modification of the prediction model suggested by Gautheron, et al., (1994). Results from test situations should be compared to known materials tested under similar conditions. This
classification system is the most consistent with recent regulatory reviews of the assay performance. Test substances can be classified as ocular corrosive or severe irritants (i.e., In Vitro Irritation Score (IVIS) >55) or may be considered not classified for UN GHS1 (i.e., IVIS ≤3). A substance that tests equivocal per OECD Guideline 437 criteria (i.e., IVIS >3 and ≤55) may need further testing.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Test Material
Value:
-1.56
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
0.9% Saline (Vehicle Control)
Value:
-1.97
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Imidazole in 0.9% Saline (Positive Control)
Value:
87.04
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
MEM (Negative Control)
Value:
1.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Positive Control Historical Data mean IVIS of 97.32 (+/- 19.69) (n = 29).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the IVIS score of -1.56, the test article is not considered to be an eye irritant (mean IVIS 0 to 3) in the Bovine Corneal Opacity and Permeability (BCOP) test.
Executive summary:

The ocular irritation and corrosion potential of the test article was tested in the Bovine Corneal Opacity Permeability (BCOP) test. The test method was based on OECD 437 and was performed in compliance with GLP. The eyes were examined prior to use on the day of dosing. Any eye with a cornea exhibiting evidence of vascularization, pigmentation, opacity or scratches was discarded. Corneas from eyes that were free of defects were dissected from the surrounding tissues. A 2-3 mm rim of sclera was left attached to each cornea. The corneas were then placed in a container of fresh HBSS. The dissected corneas were mounted in holders and placed in chambers filled with MEM solution. The entire holder was incubated at 32°C for at least 1 hour but not longer than 2 hours. Corneas (3/group) were treated with 0.75 mL of the 20% (w/v) test article formulation in 0.9% saline and incubated in the horizontal position for 4 hours at 32°C. A positive control (20% w/v imidazole formulation in 0.9% saline), negative control (MEM solution), and a vehicle control (0.9% saline) were tested in parallel with the test material. At the end of the exposure period, the corneas were rinsed with a MEM solution containing phenol red; a final rinse was made with MEM without phenol red. Opacity was evaluated using the OP-KIT following the 4-hour exposure. Following opacity readings, the cell culture medium was replaced with 1.0 ml of 0.5% sodium fluorescein solution in Dulbecco's phosphate buffered saline (PBS) and incubated for approximately 90 minutes at 32°C. Following the 90-minute sodium fluorescein exposure, permeability was measured as the optical density at 490 nm by a spectrophotometer. The In Vitro Irritancy Score (IVIS) for the test material, vehicle control, and positive control was calculated by adding the corrected mean opacity score to fifteen times the corrected mean optical density; the IVIS for negative control was performed by adding the mean opacity score to fifteen times the mean optical density. Based on the IVIS score, the test article was classified according to the prediction model described in DB-ALM Protocol No. 127, a modification of the prediction model suggested by Gautheron, et al. (1994). The mean IVIS for the test article was -1.56. The 4-hour mean corrected opacity score was -1.66 and the corrected mean optical density score was 0.007. Controls performed as expected. Based on the IVIS score of -1.56, the test article is not considered to be an eye irritant (mean IVIS 0 to 3) in the Bovine Corneal Opacity and Permeability (BCOP) Test.