Pregna-1,4-diene-3,20-dione, 6-fluoro-11-hydroxy-16-methyl-21-[(1-oxopentyl)oxy]-, (6.alpha.,11.beta.,16.alpha.)-

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:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

pre-guideline study

Deviations:

yes

Remarks:

SDS used as a positive control

Principles of method if other than guideline:

- Principle of test:
Assessment of ocular irritation potential of the test substance by determination of cytotoxic effects on a human corneal epithelium (HCE) cell model (similar to EpiOcular).
The HCE model is currently involved in the eye irritation validation conducted by COLIPA following ECVAM guidelines. Furthermore, it is routinely used by the major Cosmetic and Pharmaceutical companies, and has already been prevalidated in 2004 (van Goethem et. al., Tox in Vitro 20: 1-17, 2006). This model is recognized as the model of choice and scientifically relevant as documented by several publications (e.g. Alepee et. al., Toxicology in Vitro 34: 55–70, 2016).

A draft OECD test guideline (492B) is currently available until 07.12. 2021 for commenting. The study was performed similar to that guideline.

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 material form:

solid

Test animals / tissue source

Species:

human

Strain:

other: human corneal epithelial cells

Details on test animals or tissues and environmental conditions:

The experiment was carried out on a Human Corneal Epithelial (HCE) Model, which is standardized and commercially available (SkinEthicTM Human Corneal Epithelial Model (HCE), SkinEthic, France). Inserts were of 0.5 cm² size. When cultivated at the air-liquid interface in a chemically defined medium, the immortalized human cornea epithelial cells reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg per insert

Duration of treatment / exposure:

60 minutes

Duration of post- treatment incubation (in vitro):

16 hours

Number of animals or in vitro replicates:

3

Details on study design:

The irritation potential of the test item is assessed by determination of its cytotoxic effect on an reconstructed human ocular epithelia. The test principle is based on the MTT assay reflecting the cell viability after exposure of the cornea equivalent to topically applied test item. After the exposure period the inserts were washed carefully with PBS. After a post-exposure incubation of 16 hours in the incubator (37 +/- 2 °C, 5 % CO2, maximum humidity) MTT reduction was performed. Cell viability was measured by the amount of MTT reduction, i.e. an OD value following exposure to the negative or positive control substances or the test item. A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%) exposed to the test substance is ≤ 50 %.

Reconstructed tissues
The experiment was carried out on reconstituted Human Corneal Epithelium (HCE/S/5);
Reference HCEJ05; Episkin, France.
The tissue equivalents were shipped in 24 well cell culture plates on semi solid agar’s medium. The scope of supply contains maintenance medium for incubation (Episkin,
Reference MIMA/125). Inserts were of 0.5 cm² size.

EQUILIBRATION AND BASELINE RECORDINGS: Upon receipt each insert was transferred from the packaging plate to 6 well culture plates containing 1ml of fresh maintenance medium per well. The HCE inserts were incubated for at least 2 hours (5% CO2, 37°C, max humidity). Afterwards a media change was performed and the HCE inserts were continuing adapted overnight to the recommended tissue culture conditions (5% CO2, 37°C, max humidity).

NUMBER OF REPLICATES: Triplicates

NEGATIVE CONTROL USED: 30 µL PBS

POSITIVE CONTROL USED: SDS 0.5% (30 µL)

APPLICATION DOSE AND EXPOSURE TIME: For testing of chemically induced eye irritation the HCE inserts were exposed to 30 mg (plus 30 μL PBS to moisten and ensure good contact with the tissue) of the test item for 60 min (RT; three inserts).

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After the exposure period of 60 minutes the inserts were washed carefully with PBS. After a post-exposure incubation of 16h in the incubator MTT reduction assay was performed.


METHODS FOR MEASURED ENDPOINTS:
- Measurement of Viability: For viability testing the inserts were placed in new 24 well plates containing 300 μL of MTT solution (37°C, 0.5 mg/mL in Maintenance medium). The tissues were incubated for about 3 hours under cell culture conditions (5% CO2, 37°C, max humidity). The extraction of blue formazan was performed in isopropanol (24 well plates, 1.5 mL per insert) on a vertical shaker (for at least 2 hours). For determination of cell viability per insert the absorption of the isopropanol-extracts were measured in duplicates at 570 nm in an automatic reader (EL808, Bio-Tek; 96 well format, 200 μL).

Acceptance criteria
The following acceptance criteria determined the validity of an assay:
- mean OD negative control ≥ 0.7
- mean relative viability of the positive control is ≤ 50 %
- If the mean viability of the 3 replicates > 20% the coefficient of variation (CV) should
not exceed 0.3.

DECISION CRITERIA: Cytotoxic indices (viability decrease measured by MTT)
A test substance is predicted to be an ocular irritant if the mean relative tissue viability (%)
exposed to the test substance is ≤ 50%.

Results and discussion

In vitro

Results

Irritation parameter:

other: % cell viability

Run / experiment:

cell viability after 60 min [%]

Value:

102.53

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Table 1: Tabular summary of the results

 

Sample No.	Test item	OD mean*	Std Dev	% Viability
1 - 3	Negative control PBS	1.12	0.09	100.00
4 - 6	Positive control SDS 0.5 %	0.03	0.00	2.60
13 - 15	Fluocortolon	1.14	0.05	102.53

*6 values

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

An in vitro study for assessing ocular irritation was conducted in a human corneal epithelial (HCE) cell model. This model is recognized in the scientific community as a highly valuable model for the identification of substances that do not require classification for serious eye damage/eye irritancy (e.g. Cotovio et al., Toxicol In Vitro 24: 523-537, 2010), and is routinely used by cosmetic and pharmaceutical companies. Undiluted test item was applied topically to the reconstructed HCE tissue (30 mg per insert plus 30 µl PBS to moisten and ensure good contact to the tissue). After an exposure period of 60 minutes, followed by a 16 hours post-treatment incubation period, the cell viability was 102.53 % as measured by a MTT conversion assay. As the cut-off for a non-irritant to the eye is 50 % (a test substance is predicted to be an ocular irritant if the relative tissue viability (%) exposed to the test substance is ≤ 50), fluocortolon was considered to be non-irritant to the eye.

Executive summary:

In an eye irritation study performed similar to draft OECD Guideline 492B (2021), Fluocortolon (100% a.i.) was applied to human cornea epithelial cells from the cell line HCE reconstruct a corneal epithelial tissue (mucosa), without a stratum corneum, ultra-structurally (tissue morphology and thickness) similar to the corneal mucosa of the human eye for an exposure period of 60 minutes in triplicates. 30 μL of 0.9% NaCl were topically applied in order to improve further contact between the solid and the inserts. Each approximately 30 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.

 

After 60 minutes exposure at room temperature, the tissues were washed with phosphate buffered saline to remove any residual test material. Subsequently the tissue constructs were incubated for 16 h at 37°C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

 

The positive (5% SDS) and negative (0.9% NaCl) control gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

 

The relative mean tissue viability obtained after 60 minutes treatment with Fluocortolon compared to the negative control tissues was 102.53%. Since the mean relative tissue viability for the test substance was above 50%, Fluocortolon is identified to be not irritating.