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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:
key study
Study period:
From 5th Feb 2013 to 17th Apr 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

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
GLP compliance:
yes

Test material

Constituent 1
Reference substance name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
EC Number:
294-430-8
EC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Cas Number:
91722-28-0
IUPAC Name:
Sulfonic acids, C16-18-alkane hydroxy and C16-18-alkene, sodium salts
Test material form:
liquid
Details on test material:
Identification: Internal Olefin Sulfonate
Description: pale yellow liquid
Storage conditions: approximately 4°C in the dark
The test item was heated to approximately 30°C and homogenized prior to use.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:
-Bovine Eyes
Bovine eyes were obtained from a local abattoir as a by-product from freshly slaughtered animals (J.W. TREUTH & SONS, Inc., Baltimore, MD). The eyes were excised and then placed in Hanks' Balanced Salt Solution, containing Penicillin/Streptomycin (HBSS), and transported to the laboratory on ice packs. Immediately upon receipt of the eyes into the laboratory, preparation of the corneas was initiated.

-Preparation of Corneas
The eyes were grossly examined for damage and those exhibiting defects were discarded. 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 until they 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 (without phenol red)). 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 ± 1ºC for a minimum of 1 hour.

Test system

Vehicle:
water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. An aliquot of 750 µL of each test article dilution, positive control (ethanol), or negative control (deionized water) was introduced into the anterior chamber.

Duration of treatment / exposure:
Three corneas were incubated in the presence of the positive control at 32 ± 1ºC for 10 minutes. Three corneas were incubated in the presence of the negative control at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of each test article dilution at 32 ± 1ºC for 10 minutes.
Number of animals or in vitro replicates:
Three corneas were incubated in the presence of the positive and negative control.
Five corneas were incubated in the presence of each test article dilution.
Details on study design:
-Controls
The positive control used in this study was ethanol (Pharmco). The negative control used in this study was sterile, deionized water (Quality Biological).

-Test Article Preparation
The test article was administered to the test system as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. The test article was supplied at a concentration of 35% and was warmed to 37°C prior to dilution preparation. The 20% (v/v) test article dilution was prepared by adding 4 mL of test article to 3 mL of water in a prelabeled conical tube. The 2% (v/v) test article dilution was prepared by adding 0.4 mL of test article to 6.6 mL of water in a prelabeled conical tube. The dilutions were vortexed for approximately 1 minute prior to application. The 20% (v/v) dilution was described as a slightly cloudy yellow semi-viscous liquid. The 2% (v/v) dilution was described as a clear colorless non-viscous solution.

-Bovine Corneal Opacity and Permeability Assay
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 (without phenol red). The initial opacity was determined for each cornea using an Electro Design OP-KIT opacitometer. Any cornea whose initial opacity was greater than 7 was not used in the assay. The treatment of each cornea was identified with the test article number written in permanent marker on colored tape, affixed to each holder. The medium was then removed from the anterior chamber and replaced with the test article, positive control, or negative control.

-Method for Testing Liquid or Surfactant Materials
The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. An aliquot of 750 µL of each test article dilution, positive control, or negative control was introduced into the anterior chamber while slightly rotating the holder to ensure uniform distribution over the cornea. Due to its viscous nature, the 20% (v/v) dilution of the test article was administered by direct addition onto the epithelial surface of the cornea using a positive displacement pipet (open chamber window technique). In the open chamber window technique, the glass window was removed from the anterior chamber immediately prior to treatment. Each cornea was completely covered with test article. After dosing, the glass window was replaced on the anterior chamber. Three corneas were incubated in the presence of the positive control at 32 ± 1ºC for 10 minutes. Three corneas were incubated in the presence of the negative control at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of each test article dilution at 32 ± 1ºC for 10 minutes. After the 10-minute exposure time, the control or test article 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 articles. The corneas were then given a final rinse with Complete MEM (without phenol red). For the corneas treated using the open chamber window technique, the glass window was removed from the anterior chamber, and the test article was rinsed from the treated cornea and the anterior chamber with Complete MEM (with phenol red). Care was taken not to spray the corneas directly. The chamber windows were returned to the chambers when most or all of the test article had been removed. The rinsing process continued in the same manner as the positive and negative control corneas. 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 hours 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 (without phenol red) and 1 mL of a 4 mg/mL fluorescein solution was added to the anterior chamber. The corneas were then incubated in a horizontal position (anterior side up) for approximately 90 minutes at 32 ± 1ºC. At the end of the 90-minute incubation period, the medium was removed from the posterior chambers and placed into tubes numbered corresponding to chamber number. Aliquots of 360 µL from the numbered tubes were placed into their designated wells on a 96 well plate. The optical density at 490 nm (OD490) was determined using a Molecular Devices Vmax kinetic microplate reader. If the OD490 value of a control or test article sample was greater than 1.500, a 1:5 dilution of the sample was prepared in Complete MEM (without phenol red) (to bring the OD490 value within the linear range of the platereader). A 360 µL sample of each 1:5 dilution was transferred to its specified well on the 96 well plate. The plate was read again and the final reading was saved to a designated print file.

-Criteria for Determination of a Valid Test
The BCOP assay was accepted when the positive control (ethanol) caused an in vitro score that fell within two standard deviations of the historical mean.

-Bovine Corneal Opacity and Permeability Assay
According to OECD TG 437, a substance that induces an in vitro score of ≥55.1 would be predicted to be a corrosive or severe eye irritant. If the substance is not predicted to be an ocular corrosive or severe irritant, additional testing should be conducted for regulatory classification or labeling purposes.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
20% of the test item
Value:
4.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Run / experiment:
2% of the test item
Value:
12.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Since the results of the positive control fell within two standard deviations of the historical mean (within a range of 39.2 to 63.9), the assay was considered valid.

Any other information on results incl. tables

Table 1 summarizes the opacity, permeability, and in vitro score for each test article dilution. Table 2 summarizes the opacity, permeability, and in vitro score for the positive control.

 

Table 1

BCOP Results of the Test Article

 

Assay

Date

IIVS Test Article Number

Conc. (v/v)

Exposure

Time

Mean Opacity

Value

Mean OD490

Value

In Vitro Score

pH

14 Feb 2013

13AA85

20%

10 minutes

-0.9

0.357

4.5

8.5

2%

10 minutes

4.5

0.534

12.6

6.0

 

Table 2

BCOP Results of the Positive Control

Assay

Date

Positive

Control

Exposure

Time

Mean Opacity

Value

Mean OD490

Value

In Vitro

Score

14 Feb 2013

Ethanol

10 minutes

32.0

0.941

46.1

 

Applicant's summary and conclusion

Interpretation of results:
other: The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant. However for the classification purpose, further testing is required.
Conclusions:
According to OECD TG 437, a substance that induces an in vitro score of ≥55.1 would be predicted to be a corrosive or severe eye irritant. The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant.
Executive summary:

The purpose of this study was to evaluate the potential ocular irritancy of the test article as measured by changes in opacity and permeability (to fluorescein) in isolated bovine corneas.

 

The test article was tested as a 2% (v/v) and a 20% (v/v) dilution in sterile, deionized water. Three corneas were incubated in the presence of the positive control and the negative control each at 32 ± 1ºC for 10 minutes. Five corneas were incubated in the presence of each test article dilution at 32 ± 1ºC for 10 minutes. After the 10-minute exposure time, the control or test article treatments were removed then the corneas were returned to the incubator for approximately 2 hours. An in vitro score was determined for each test article based on the induction of opacity and permeability (to fluorescein) in the isolated bovine corneas. According to OECD TG 437, a substance that induces an in vitro score of ≥55.1 would be predicted to be a corrosive or severe eye irritant.

 

The in vitro scores of a test substance were below 55.1. Therefore the test article is not predicted to be an ocular corrosive or severe irritant.