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Eye irritation

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eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Sep 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to 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)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
17-hydroxy-19-norpregn-4-ene-3,20-dione 17-acetate
EC Number:
EC Name:
17-hydroxy-19-norpregn-4-ene-3,20-dione 17-acetate
Cas Number:
Molecular formula:
(1R,3aS,3bR,9aR,9bS,11aS)-1-acetyl-11a-methyl-7-oxo-1H,2H,3H,3aH,3bH,4H,5H,7H,8H,9H,9aH,9bH,10H,11H,11aH-cyclopenta[a]phenanthren-1-yl acetate
Specific details on test material used for the study:
- 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: The test item was suspended shortly prior to application in isotonic saline solution to achieve
a concentration of 20% (w/v).
- 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:
Bovine eyes of slaughtered cattle were extracted and transferred in containers with Hank’s salt solution (HSS) with 1% penicillin/streptomycin solution. For transportation the containers were ice cooled.
Eyes with defects were sorted out and disposed of, eyes without any defects were transferred into fresh HSS supplemented with 1% penicillin/streptomycin solution and 1 % fetal bovine serum (FBS) and stored overnight at 2-8 °C. On the next day (day of testing) the containers with the eyes were placed in an incubator at 32 ° C (± 1 ° C) for about 2 hours before preparation of the corneas.

Test system

physiological saline
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
- Amount(s) applied (volume or weight with unit): 750 μL per cornea and chamber
- Concentration (if solution): 20 % (w/v)

- 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.

TREATMENT METHOD: closed chamber


- 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

Irritation parameter:
in vitro irritation score
Run / experiment:
Vehicle controls validity:
Negative controls validity:
not applicable
Positive controls validity:

Applicant's summary and conclusion

Interpretation of results:
other: non corrosive
Executive summary:

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 0.7 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.