Estradiol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 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:

27 June 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Estradiol

EC Number:

200-023-8

EC Name:

Estradiol

Cas Number:

50-28-2

Molecular formula:

C18H24O2

IUPAC Name:

estra-1,3,5(10)-triene-3,17-diol

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: Analytical determination of stability and homogeneity of the test item in the vehicle was not performed specifically for this study.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: The usage of physiologic saline solution for formulation of the test item is plausible based on the current knowledge of the sponsor. No objections were seen particularly in regard to stability.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was suspended by using a mortar shortly prior to application in physiologic saline solution, the formulation was continuously stirred.
- Final preparation of a solid: 20% (w/v)

FORM AS APPLIED IN THE TEST (if different from that of starting material): The preparation was visually described as suspension.

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Laame, Buchenhofen 26, 42329 Wuppertal, Germany
- Number of animals: N/A
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transport: 1 L containers with 500 mL HSS and 1 % penicillin / streptomycin solution, transport of the containers in coolers on ice
- 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: 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).
- Quality check of the isolated corneas: 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:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 20 % (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 0.9% NaCl (w/v)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

no further incubation required

Number of animals or in vitro replicates:

3 cornea

Details on study design:

Immediately before application, the medium was aspirated from the anterior chamber. 750 μL of the test material formulations were applied to the corneas, each through the holes of the anterior chamber. The holes of both chambers were sealed with adhesive tape and the holders were kept with the front side up, so that the formulations covered the cornea sufficiently (closed chamber method). The holders were transferred into the incubator at 32 °C (± 1 °C) for the exposure time of 4 hours. After the exposure, the formulations were aspirated from the anterior chamber and the corneas were rinsed at least 3 times with phenol red containing MEM to show effectiveness of test substance removal. 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 and the holes were sealed with tape.

Positive control: 20 % Imidazole in physiologic saline (w/v; 750 µL)

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

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:The medium in anterior chamber of each holder was replaced by 1ml 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 fluoresceinsolution 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).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the OECD TG 437

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

2.3

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Individual values of opacity, permeability and IVIS

Cornea No.	Opacity per cornea	Permeability per cornea	IVIS per cornea	IVIS per group
				mean		SD
Vehicle control                    1	-0.6	0.006	-0.5
0.9 % NaCl                              2	-0.1	0.006	0.0	-0.9		1.2
3	-2.4	0.008	-2.3
Positive Control                     4	77.3	1.004	92.3
20 % Imidazole                      5	60.5	1.122	77.4	87.1		8.4
6	75.1	1.091	91.5
Test item                                10	-1.1	0.000	-1.1
20% Estradiol           11	1.7	-0.002	1.6	2.3		3.7
12	6.2	0.005	6.3

 

Applicant's summary and conclusion

Interpretation of results:

other: negative

Conclusions:

The test item (20 % (w/v) in physiologic saline) was tested in the BCOP test according to OECD TG 437. For determination of corneal damage opacity as well as tissue permeability was measured after a 4 hour exposure time. Based on these data, in comparison to the vehicle control, the In Vitro Irritancy Score (IVIS) value was calculated to be 2.3 which is below the IVIS cut-off threshold of 55 and thus identifying the test item as not inducing serious eye damage. The results of the positive (20 % imidazole solution) and vehicle (physiologic saline solution) controls confirmed the validity of the test system.

Executive summary:

This in vitro study was performed to assess them corneal irritation and damage potential of Estradiol (20% w/v in physiologic saline) by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted 27 June 2013.

The corneae were incubated with the test substance and controls for 4 h. After rinsing 3 times with phenol-red containing MEM the anterior chamber was then filled again with MEM medium to avoid drying out of the cornea. The test was performed in triplicates. Opacity and permeability were determined. 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 relevant increase of the corneal opacity and permeability. The calculated mean in vitro irritation score was 2.3.

The positive control (Imidazole 20%) increased the opacity and permeability of the corneae in both experiments (mean in vitro irritation score 92.3 (1st experiment) and 77.4 (2nd experiment)).

With the negative control (physiological saline) neither an increase of opacity nor permeability of the corneae could be observed in both experiments (mean in vitro irritation score -0.5 (1st experiment) and 0.0 (2nd experiment)).

Since the mean in vitro irritancy score of the test substance was <55.1, a 20% dilution of Estradiol 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.