Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 28th
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report date:
2011

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
The bovine corneal opacity and permeability assay (BCOP) was used to assess the potential ocular irritancy of the test substance to isolated bovine corneas. Bovine corneas, obtained as a by-product from freshly slaughtered animals, were mounted in special holders and exposed to the test substance. An in vitro score was determined for the test substance based on the induction of opacity and permeability (to fluorescein).
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate
EC Number:
700-652-2
Cas Number:
1259300-69-0
Molecular formula:
C20H34O8
IUPAC Name:
4-{[3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoyl]oxy}butyl 3-(2,4-dimethyl-1,3-dioxolan-2-yl)propanoate

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: from a local abattoir as a by-product from freshly slaughtered animals (J.W. Treuth & Sons, Inc. Baltimore, MD).
- Storage, temperature and transport conditions of ocular tissue: the eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS) and transported to the laboratory on ice packs.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 750 µl
Duration of treatment / exposure:
10 min
Number of animals or in vitro replicates:
- 5 corneas exposed to test substance
- 3 corneas exposed to positive control and negative control
Details on study design:
QUALITY CHECK OF THE ISOLATED CORNEAS: the eyes were grossly examined for damage and those exhibiting defects were discarded

PREPARATION OF CORNEAS: immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated. The tissue surrounding the eyeball was carefully pulled away and the cornea was excised such that a 2 to 3 mm rim of sclera was present around the cornea. The isolated corneas were then stored in a petri dish containing HBSS and were mounted in a corneal holder. The corneas were mounted in the holders with the endothelial side against the O-ring of the posterior chamber. The anterior chamber was then positioned on top of the cornea and the screws were tightened. Starting with the posterior chamber, the two chambers were then filled with Minimum Essential Medium (EMEM) without phenol red, containing 1 % fetal bovine serum and 2 mM L-glutamine (Complete MEM). Each corneal holder was uniquely identified with a number written in permanent marker, on both the anterior and posterior chambers. The corneal holders were incubated at 32 ± 1 °C for a minimum of 1 hour.

pH DETERMINATION
The pH of the test substance was determined using 0 -14 pH paper.

SELECTION OF CORNEAS: after a minimum of 1 hour of incubation, the corneas were removed from the incubator. The medium was removed from both chambers and replaced with fresh Complete MEM. The initial opacity was determined for each cornea using a OP-Kit opacitometer. Any cornea with an initial opacity greater than 7 was not used in the assay. Three corneas, whose initial opacity readings were close to the median opacity for all the corneas, were selected as the negative control corneas. The medium was then removed from the anterior chamber and replaced with the test article, positive control or negative control.

TREATMENT METHOD (Method for Testing Liquid or Surfactant Materials)
750 µl of the test substance, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Each treated cornea was completely covered with the test substance. Three corneas were incubated in the presence of the positive control at 32 ± 1 °C for 10 min. Three corneas were incubated in the presence of the negative control at 32 ± 1 °C for 30 min. Five corneas were incubated in the presence of the test substance at 32 ±1 °C for 10 min. After the 10 -min exposure time, the control or test treatments were removed. The epithelial side of the corneas was washed at least three times with Complete MEM (containing phenol red) to ensure total removal of the control or test substance. The corneas were then given a final rinse with Complete MEM (without phenol red).The anterior chambers were refilled with fresh Complete MEM (without phenol red) and an opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hrs after which a final measure of opacity was obtained. After the final opacity measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was filled with fresh Complete MEM and 1 ml of 4 mg/ml fluorescin solution was added to the anterior chamber. The corneas were then incubated in a horizontal position for approximately 90 min at 32 ± 1 °C. At the end of the incubation period, medium was removed from posterior chambers and placed into tubes numbered with corresponding chamber numbers. Aliquots of 360 µl from the numbered tubes were placed into their designated wells on a 96-well plate. The optical density was determined at 490 nm (OD490). If the OD490 value of a control or test article was 1.500 or above, a 1:5 dilution of the sample was prepared in Complete MEM (or bring the OD490 value within the linear range of platereader). A 360 μl sample of each a 1:5 dilution was transferred to its specified well on the 96-well plate. The plate was read again.

CALCULATIONS
- Corneal opacity: the change in opacity for each cornea (including the negative control corneas) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting from each the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of each cornea for that treatment condition.
- Corneal permeability: the mean OD490 value for the blank wells was calculated. The mean blank OD490 value was then substracted from the raw OD490 value of each well (corrected OD490). Any dilutions that were made to bring the OD490 readings into the linear range of the platereader (OD490 should be less than 1.500) had each diluted OD490 reading multiplied by the dilution factor. The final corrected OD490 values of the test article and the positive control was then calculated by subtracting the average corrected OD490 value of the negative control corneas from the corrected OD490 value of each treated cornea.
Final corrected OD490 = (raw OD490-mean blank OD490)-average corrected negative control OD490.
The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.
In Vitro Score = Mean Opacity Value + (15 x Mean OD490 Value)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
- The following classification was established by Sina et.al. (1995) based on studies with a wide range of test substances:
In Vitro Score:
from 0 to 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = severe irritant

Vanparys et.al. (1993) propsosed a modification of Sina classification system based on 50 pharmaceutical and commercially-available industrial compounds
In Vitro Score:
from 0 to 3 = non-irritant
from 3.1 to 25 = mild irritant
from 25.1 to 55 = moderate irritant
from 55.1 and above = corrosive/severe irritant

Sina et.al. (1995). A collaborative evaluation of seven alternatives to the Draize eye irritation test using pharmaceutical itermediates. Fundamental and Applied Toxicology 26:20-31
Vanparys et.al. (1993). Evaluation of the bovine opacity-permeability assay as an in vitro alternative to the Draize eye irritation test. Toxicol. In Vitro 7, 471-476

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
-1.2
Negative controls validity:
not specified
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: the positive control caused an in vitro score that fell within 2 standard deviations of the historical mean, the test was considered valid.

Applicant's summary and conclusion

Interpretation of results:
other: not classified as an eye irritant according to the CLP Regulation (EC) No.1272/2008
Conclusions:
Non-eye irritant
Executive summary:

The eye irritation potential of the test substance to induce opacity and permeability to fluorescein in an isolated bovine cornea was evaluated in the in vitro BCOP test according to the OECD Guideline 437.

Bovine corneas were mounted in special holders and exposed to the test substance. Five corneas were exposed to the test substance, three to the positive control ethanol and three to the negative control deionized water. The test and positive control corneas were exposed at 32 ± 1 °C for 10 min instead the negative control corneas for 30 min, after which the control or test treatments were removed and the corneas were rinsed; the opacity measurement was performed. The corneas were returned to the incubator for approximately 2 hrs after which a final measure of opacity was obtained. The posterior chamber was filled with fresh Complete MEM and 1 ml of 4 mg/ml fluorescin solution was added to the anterior chamber. The corneas were then incubated for 90 min at 32 ± 1 °C, after which aliquots were placed into their wells on a 96 -well plate and the optical density was determined at 490 nm (OD490).

The mean IVIS for the test substance was -1.2. Based on the classification criteria, the test substance is considered to be non-irritating to eyes.