Fluocortolone

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

2013

Deviations:

yes

Remarks:

analytical determination of stability and homogeneity of the test item in the vehicle was not performed

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

6-fluoro-11,21-dihydroxy-16- methylpregna-1,4-diene-3,20-dione

Cas Number:

152-97-6

Molecular formula:

C22H29FO4

IUPAC Name:

6-fluoro-11,21-dihydroxy-16- methylpregna-1,4-diene-3,20-dione

Test animals / tissue source

Species:

other: isolated cornea from eyes of slaughtered cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Extraction: Staff of the slaughterhouse
- Transport: 1L containers with 500 mL HSS and 1 % penicillin/streptomycin solution; transport of the containers in coolers on ice
- Number of animals: three coneae were used
- Indication of any existing defects or lesions in ocular tissue samples: Eyes were examined after delivery to the laboratory for any damage (like opacity, scratches or neovascularization) on the day of slaughter (1 day before the experiment). Eyes without any visible defects were transferred into new containers with fresh HSS solution supplemented with 1 % penicillin / streptomycin solution and 1 % FBS and stored overnight at refrigerator temperature (2-8 °C). Eyes with defects were discarded.

- Selection and preparation of corneas: On the next day (day of experiment) the containers ith the eyes were transferred in an incubator at 32 °C (± 1 °C) for about 2 hours. For the preparation of the cornea the sclera of each eye was incised with a scalpel and cut by scissors. A 2-3 mm scleral edge was left around the cornea for further handling. The isolated
corneas were placed with the epithelium side down into a prepared beaker filled with MEM medium supplemented with 1 % penicillin / streptomycin solution and 1 % FBS. Each cornea was placed in a cornea holder with the endothelial side on the sealing ring of the posterior chamber. The anterior chamber was then fixed by screws on the other side. The chambers were filled with MEM medium, avoiding air bubbles. The holders so prepared were transferred for at least 1 hour into the incubator at 32 °C (± 1 °C). Following 1 hour in the incubator, the MEM medium was aspirated and the chambers were refilled with fresh MEM medium. For each cornea the reference opacity value was measured then. The mean and standard deviation of the measured values were calculated by using Microsoft Excel. The corneas with values within the range of mean ± standard deviation were selected for the actual test and assigned to the test groups.

Test system

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

750 μL per cornea

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

- DETERMINATION OF OPACITY: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). The validation of the opacitometer was carried out under the study number T8082017. Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- DETERMINATION OF PERMEABILITY: The medium in anterior chamber of each holder was replaced by 1 ml of fluorescein sodium solution (concentration 5 mg/mL). Afterwards the holders were incubated at 32 °C (± 1 °C) for about 90 minutes. After the incubation period, the medium of the posterior chamber was aspirated by a syringe and filled into a 10 mL tube. Three wells of a 96 well plate were filled with 300 µL of each tube (triplicate determination). In addition, a standard series of 5 mg/mL sodium fluorescein solution was prepared and also filled into the 96-well plate, in triplicates. The permeability was determined by measuring the amount of fluorescein sodium which diffused through all cell layers of the cornea. The measurement was carried out at a wavelength of 490 nm (OD490) by an ELISA- Reader (Bio-Tek EL 808, Software Gen5).
- CALCULATION AND EVALUATION OF IN VITRO IRRITANCY SCORE (IVIS): All parameters and the IVIS values were calculated by using Microsoft Excel. The validation of the Excel file was carried out under the study number T0082019.
The opacity values were calculated by applying the following formulae:
1) Opacity= (Io/I-0.9894)/0.0251
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity change= opacity change- mean opacity change NC
4) Mean opacity = mean of all corrected opacity changes per group
The permeability values were calculated by applying the following formulae:
1) OD49o change OD490 value - mean blank value OD490
2) Corrected OD490 change= OD490 change- mean OD490 change NC
3) Mean OD490 =mean of all corrected OD490 changes per group
Calculation of In Vitro Irritancy Score (IVIS):
1) IVIS per cornea= corrected opacity change+ (15 x corrected OD490 change)
2) IVIS per group mean of IVIS values per cornea in a group

Io = single value of the measurement of empty holder with medium but without cornea, measured l-2days before the experiment;
I = individual value of each opacity measurement before and after application 0.9894/0.0251 is a constant, which is required for calculation.

- DECISION CRITERIA: The IVIS cut-off values for identifying test chemicals as inducing serious eye damage (UN GHS Category 1) and test chemicals not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given hereafter:
IVIS <= 3 (No category), IVIS > 3 - <= 55 (No prediction can be made / No Category 1) or IVIS > 55 (Category 1)
NUMBER OF REPLICATES
Three coneae were used

NEGATIVE CONTROL USED: 0.9%NaCl

POSITIVE CONTROL USED: 20% Imidazole solution (w/v)

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20% (w/v) solution of the test item

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: At least three washing steps. During the final rinse cycle the corneas were rinsed again with pure MEM medium in order to remove residues of the dye. The anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. Before measuring opacity, fresh MEM medium was filled in the chambers.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The measurements of opacity were carried out using an opacitometer BASF OP3.0 (with integrated light meter testo 545 and Comfort 3.4 SP6 software from Testo AG, Lenzkirch). Before each measurement the opacitometer was adjusted to about 1000 LUX and a filter calibration measurement was carried out by using 3 different filters.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader(OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS):
All parameters and the IVIS values were calculated by using Microsoft Excel. The validation
of the Excel file was carried out under the study number T0082019.
The opacity values were calculated by applying the following formulae:
1) Opacity = (I 0 /I-0.9894)/0.0251
2) Opacity change = opacity after application - opacity before application
3) Corrected opacity change = opacity change - mean opacity change NC
4) Mean opacity = mean of all corrected opacity changes per group

The permeability values were calculated by applying the following formulae:
1) OD 490 change = OD 490 value - mean blank value OD 490
2) Corrected OD 490 change = OD 490 change - mean OD 490 change NC
3) Mean OD 490 = mean of all corrected OD 490 changes per group

Calculation of In Vitro Irritancy Score (IVIS):
1) IVIS per cornea = corrected opacity change + (15 x corrected OD 490 change)
2) IVIS per group = mean of IVIS values per cornea in a group
I 0 = single value of the measurement of empty holder with medium but without cornea, measured 1-2days before the experiment
I = individual value of each opacity measurement before and after application
0.9894 / 0.0251 is a constant, which is required for calculation.

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Irritation parameter:

other: in vitro irritancy score (IVIS)

Run / experiment:

4 hrs

Value:

< 0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

Any other information on results incl. tables

Table 1: Tabular in vitro irritancy scores (IVIS)

	Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group mean	SD
Vehicle control	1	- 0.5	0.010	- 0.3	3.7	4.4
(0.9 % NaCl)	2	3.0	0.010	3.2
	3	8.2	0.008	8.4
Positive control	4	47.4	1.468	69.4	95.1	27.9
(20 % Imidazole)	5	114.2	0.703	124.7
	6	69.9	1.418	91.2
Test item	7	- 2.5	0.002	- 2.5	- 0.4	2.0
(20 % Fluocortolon)	8	- 0.1	0.002	- 0.1
	9	1.5	0.000	1.5

No potential for serious eye damage was concluded from the study, as the IVIS was below 55 for the test item.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Fluocortolon was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437. The epithelial surface of the corneas was exposed to 750 µL of the test substance formulated in physiological saline. Measurement of corneal opacity and permeability after a 4 hours exposure followed by a post-treatment incubation of 90 minutes revealed an in vitro irritation score (IVIS) of - 0.4, well below the threshold for classification of serious eye damage (IVIS <= 55). The positive (20 % imidazole) and vehicle (physiological saline solution) controls confirmed the validity of the test. Thus, under the conditions of this test fluocortolon was characterized by having no potential to seriously damage the eye.

Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of Fluocortolon (20% in 0.9% NaCl) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, 2013.

 

The corneae were incubated with the test substance and controls for 4 h. After rinsing with saline, the measurement of Opacity and Permeability was conducted in triplicates. The in vitro irritancy score (IVIS) was calculated as mean opacity value + (15 x mean OD490 value); a substance that induces an IVIS ≥ 55.1 is defined as a corrosive or severe irritant.

 

A 20% dilution of the test substance in physiological saline caused no increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was -0.4.

 

The positive control (Imidazole (20% (w/v) in 0.9% NaCl) increased the opacity and permeability of the corneae (mean in vitro irritation score 95.1.

 

With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean in vitro irritation score 3.7). The solvent control olive control did not show relevant effects.

 

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of Fluocortolon in physiological saline is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.