Sodium 1,1,1,2,2,3,3,19,19,20,20,21,21,21-tetradecafluoro-11-({[1-(2,2,3,3,4,4,4-heptafluorobutoxy)propan-2-yl]oxy}carbonyl)-7,15-dimethyl-9,13-dioxo-5,8,14,17-tetraoxahenicosane-10-sulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 18 - Oct 19, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD TG 437.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

sodium 1,1,1,2,2,3,3,19,19,20,20,21,21,21-tetradecafluoro-11-({[1-(2,2,3,3,4,4,4-heptafluorobutoxy)propan-2-yl]oxy}carbonyl)-7,15-dimethyl-9,13-dioxo-5,8,14,17-tetraoxahenicosane-10-sulfonate

EC Number:

700-540-3

Molecular formula:

C27H28F21NaO12S

IUPAC Name:

sodium 1,1,1,2,2,3,3,19,19,20,20,21,21,21-tetradecafluoro-11-({[1-(2,2,3,3,4,4,4-heptafluorobutoxy)propan-2-yl]oxy}carbonyl)-7,15-dimethyl-9,13-dioxo-5,8,14,17-tetraoxahenicosane-10-sulfonate

Test material form:

semi-solid (amorphous): gel

Remarks:

migrated information: paste

Test system

Vehicle:

other: Polyethylenglycol 200

Amount / concentration applied:

750 µL of a 20% solution of the test material/control material in PEG 200

Duration of treatment / exposure:

incubation for 240 min

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS Score

Basis:

mean

Time point:

other: 240 min

Score:

35.1

Any other information on results incl. tables

Collection of bovine eyes

Freshly isolated bovine eyes of donor cattle were collected from the slaughterhouse. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium.

Preparation of corneas

The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the experiment were collected in incubation medium (EMEM, pre-warmed at 32 °C).

Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (0-ring) of the posterior part of the holder. The cornea was gently flattened over the 0-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium (EMEM). The posterior compartment was filled first to return the cornea to its natural convex form.

Opacity measurement before treatment

For equilibration, the corneas in the holder were incubated (incubator: Grumbach BSS 160) in a vertical position at 32 ± 1 °C for about one hour.
At the end of the incubation period, the incubation medium (EMEM) was removed from both compartments and replaced by fresh incubation medium (EMEM), and the baseline opacity was determined with an opacitometer (BASF-OP2.0).
The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value was recorded in a table and converted into an opacity value (baseline opacity values). The opacitometer was calibrated as described in the manual and the opacity of each cornea was determined by reading each holder placed in the photoreceptor compartment of the opacitometer.
Any corneas that showed macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or an opacity > 7 opacity units were discarded. The mean opacity of all equilibrated corneas was calculated. Three corneas with opacity values close to the median value for all corneas were selected as negative control corneas. The remaining corneas were distributed into treatment and positive control groups.

TREATMENT

Fresh incubation medium (EMEM) was placed in the posterior compartment, while the anterior compartment received the test material, negative or positive control on the surface of the corneas. Afterwards, the cornea holders were incubated at 32 ± 1 °C in an incubator (Grumbach BSS 160) in a horizontal position.

The test material was dissolved in Polyethylenglycol 200 to a concentration of 20%. As positive control a 20% Imidazole solution (in 0.9% sodium chloride solution) and as negative control Polyethylenglycol 200 were used. A volume of 750 µL each (negative control, positive control or test material) was introduced into the anterior chamber. After the exposure period, the test material, the negative control, or the positive control was removed from the epithelium. The corneal surface was washed at least three times (or until no visual evidence of the test substance was observed) with wash medium. Afterwards, incubation medium was used as final rinse to ensure removal of the phenol red from the anterior chamber prior to the opacity measurement. In a next step fresh incubation medium was replaced in both compartments and the opacity value after treatment was measured.

Permeability determination

In the second step of the assay, an increased permeability of the cornea possibly caused by the test material was determined as an indication of the integrity of the epithelial cell layers. Fresh incubation medium (EMEM) was added to the posterior compartment and 1 mL of a fluorescein solution, dissolved in DPBS (5 mg/mL) was placed in the anterior compartment.

The corneas were incubated again in a horizontal position for further 90 minutes at 32±1°C.

The amount of sodium fluorescein that crossed into the posterior chamber was measured spectrophotometrically (BioTek ELx800) at 490 nm (OD490). Therefore, 3 x 360 µL medium from the posterior chamber per cornea were well mixed and transferred into a 96-well plate.

Evaluation of results

Opacity

The change of the opacity value of each cornea treated with the test material, positive or negative control was calculated by subtracting the initial baseline opacity from the post treatment opacity reading, for each individual cornea The average change in opacity of the negative control corneas was calculated and this value was subtracted from the change in opacity of each cornea treated with the test material or positive control to obtain a corrected opacity.
The mean corrected opacity value of each treatment group was calculated from the individual corrected opacity values of the treated corneas.

Permeability

The corrected OD490 value of each cornea treated with the positive control or the test material was calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
The mean corrected permeability value of each treatment group was calculated from the individual corrected permeability values of the treated corneas.
In Vitro Irritancy Score (IVIS) calculation
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:

IVIS = mean opacity value + (15 x permeability OD490 value)

The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test materials:

IVIS = corrected opacity value + (15 x corrected permeability OD490 value)

The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values. A substance that induced an In Vitro Irritancy Score IVIS > 55.1 is defined as a corrosive or a severe irritant.

Validity criteria

The test is acceptable if the IVIS of the positive control falls within two standard deviations of the current historical mean (IVIS: 82.7 - 143.9, IVIS negative control: 2.3 - 8).

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: OECD GHS

Conclusions:

Under the given experimental conditions, the test material did not show an ocular severe irritant or corrosive potential.

Executive summary:

Study Design

This in vitro study was performed to assess the ocular severe irritant or corrosive potential of the test item by means of the BCOP (Bovine Corneal Opacity and Permeability Assay). Therefore, the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test material as a 20% solution in Polyethylenglycol 200. As negative control Polyethylenglycol 200 and as positive control 20% imidazole was used. 3 corneas were used per group (test material, negative and positive control group).

After a first opacity measurement of the fresh bovine corneas, 750 µL of the suspended test material, positive or negative control were applied on the corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase the test material, the positive, and the negative control were rinsed from the corneas and the opacity was measured again.

After the opacity measurements, the permeability of the corneas was determined by application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 1 °C. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically.

The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

Results

The treatment of the corneas with the negative control showed neither an increase of opacity nor an increase of permeability. The IVIS of 6.2 was within the range (two standard deviations) of the current historical mean of the negative controls (2.3 - 8.0). After treatment with the positive control (20% imidazole) the calculated IVIS was 94.7 and, thus, within two standard deviations of the current historical mean (90.2 - 148.5). Therefore, the study fulfilled the validity criteria.

The IVIS obtained after treatment with the test material was 35.1 and, thus, lower than 55.1. Therefore, the test material is not considered to possess an ocular severe irritant or corrosive potential.

Conclusions

Under the given experimental conditions, the test material did not show an ocular severe irritant or corrosive potential.