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Diss Factsheets

Administrative data

Description of key information

Fluocortolone-21-valerate is neither corrosive nor irritant to the skin or irritant to the eyes based on in vitro tests with the read-across substance fluocortolone (reconstructed human epidermis - Leidenfrost, 2017a+b; BCOP - Rauh, 2016; HET-CAM - Leidenfrost, 2017c; HCE - Leidenfrost, 2017d).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
commercially available test method
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany)
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature)
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

NUMBER OF TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION
- The optical density of the isopropanol-extracts of 3 insert was determined by duplicate per insert = 6 OD values.

PREDICTION MODEL / DECISION CRITERIA
- The mean optical density (OD) values obtained with the test item were used to calculate the percentage of viability relative to the negative control, which is set at 100 %.
- According to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post treatment incubation is less than or equal (≤ ) to 50 %.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg (plus 30 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 0.9% NaCl in water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µl
- Concentration (if solution): 5% SDS in physiological saline
Duration of treatment / exposure:
20 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
cell viability after 20 min [%]
Value:
101.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- Reliability of the test was previously confirmed by interlaboratory validation

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, mean OD negative control≥1.0 and ≤ 2.8
- Acceptance criteria met for positive control: Yes, the relative viability of the positive control is ≤ 20%
- Acceptance criteria met for variability between replicate measurements: Yes, the mean viability of the 3 replicates is > 20% and the coefficient of variation (CV) does not exceed 0.3.

Table 1: Tabular summary of the results    

 Sample No. Test item  OD mean *  Std Dev  % Viability 
 1 - 3

 Negative control NaCl 0.9 %

2.30

0.08

100.00

 4 - 6

 Positive control SDS 5 % 

0.02

0.00

0.97 

 7 - 9

Fluocortolon 

 2.32

0.05

101.10

* 6 values

Interpretation of results:
GHS criteria not met
Conclusions:
A study for predicting a skin irritation potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439. After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 101.1 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, Fluocortolon is considered to have no skin irritation category as defined in the UN GHS.
Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 439 (In Vitro Skin Irritation, 2015), Fluocortolon (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 20 minutes in triplicates. 30 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. 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 20 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 42 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 20 minutes treatment with Fluocortolon compared to the negative control tissues was 101.10%. Since the mean relative tissue viability for the test substance was above 50%, Fluocortolon is identified to be not irritating.


 

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
commercially available test method
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: epiCS® (CellSystems, Troisdorf, Germany).
- Cat.-No: CS-1001

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: RT (room temperature) for 3 min and incubator (37°C, CO2 5%) for 60 min
- Temperature of post-treatment incubation (if applicable): Incubator temperature: 37 ± 2° C (CO2 gas concentration: 5 %; Humidity: maximum)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1mg/ml
- Incubation time: 3 hours
- Spectrophotometer: EL808, Bio-Tek
- Wavelength: 570 nm

PREDICTION MODEL / DECISION CRITERIA:
- Corrosivity potential of test materials is predicted from the cell viabilities obtained after 3 min and 60 min treatment compared to the negative control. A chemical is classified "corrosive" (sub-category 1A) if the cell viability after 3 min treatment is decreased by more than 50 %. If cell viability after 3 min exposure is ≥ 50 %, while it is below 15 % after 60 min exposure the substance is also classified as corrosive, but sub-category 1 B/1 C. If cell viability after 3 min exposure is ≥ 50 % and after 60 min exposure ≥ 15 %, the substance is classified as non-corrosive.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
Reliability of the test was previously confirmed by interlaboratory validation

NUMBER OF REPLICATE TISSUES: triplicate


REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Volume and number not reported: After the exposure of the test item the inserts were washed carefully in PBS.
- Observable damage in the tissue due to washing: No


Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (plus 50 µl 0.9% NaCl to moisten and ensure good contact with the epidermis surface)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl
- Concentration (if solution): 0.9% NaCl
Duration of treatment / exposure:
3 and 60 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
cell viability after 3 min [%]
Value:
95.94
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
cell viability after 60 min [%]
Value:
104.65
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
not applicable
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not reported
- Direct-MTT reduction: not reported
- Colour interference with MTT: not reported

Table 1: Tabular summary of the results   

 Sample No.  Test item  Time (min)  OD mean *  Std Dev  % Viability
 1 - 3  Negative control NaCl 0.9 %  60   2.09 0.04  100.00 
 7 - 9 Fluocortolon  60   2.19 0.15 104.65 
10 - 12 Negative control NaCl 0.9 %   3  2.28 0.06   100.00
16 - 18  Fluocortolon  3  2.18 0.14  95.94

* 6 values

Interpretation of results:
GHS criteria not met
Conclusions:
A study for predicting a non-specific, corrosive potential of the test item by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431. For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 95.94 % viability; 60 min.: 104.65 % viability) showed, that fluocortolon has no corrosive property under the conditions of the assay used.
Executive summary:

In a dermal irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion, 2015), Fluocortolon (100% a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 and 60 minutes in triplicates. 50 μL of 0.9% NaCl were topically applied to the epidermal surface in order to improve further contact between the solid and the epidermis. Each approximately 25 mg of the test item were applied to the wetted tissues. The test item was spread to match the surface of the tissue.


 


After 3 minutes exposure at room temperature or 60 minutes exposure in the incubator, the tissues were washed with phosphate buffered saline to remove any residual test material. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.


 


The 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 3 and 60 minutes treatment with Fluocortolon compared to the negative control tissues was 95.94 and 104.65%, respectively. Since the mean relative tissue viability for the test substance was above 50%, Fluocortolon is identified to be not corrosive.


 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting 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
Qualifier:
according to guideline
Guideline:
other: Appendix B3 of "ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products", NIH Publication No. 10-7553, September 2010.
Version / remarks:
2010
Principles of method if other than guideline:
- Principle of test:
The Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) is a test method which implies the use of a complete tissue constituted of blood vessels and proteins that is capable of responding to chemical injury with an inflammatory process similar to the one occuring in the conjunctival tissue of the eye.

- Short description of test conditions: The test substance is applied directly to the chorioallantoic membrane (CAM) of fertilized chicken eggs

- Parameters analysed / observed: acute effects on haemorrhage, lysis of blood vessels and coagulation
GLP compliance:
yes (incl. QA statement)
Species:
chicken
Strain:
other: Lohmann Brown hen eggs
Details on test animals or tissues and environmental conditions:
SOURCE OF FERTILIZED CHICKEN EGGS
- Source: Josef Brinkschulte GmbH & Co.KG, 48308 Senden, Germany
- Number of eggs: 4
- Characteristics of donor animals: fertile Lohmann Brown hens
- Treatment conditions of eggs prior initiating testing: day 1-7: an incubator with an automatic rotating device (e.g. Ehret GmbH), optimum temperature : 37.5 °C, relative humidity 63%; day 8: with the large end
upward and not rotated for ensuring accessibility to the Chorioallantoic membrane (CAM) region
- Time interval prior to initiating testing: 8 days
- Indication of any existing defects or lesions in eggs: after 7 days of incubation, all eggs were candled in order to discard those that were defect and to mark the air bubble
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 300 μL/egg (corresponding to an average amount of 103.5 mg)


Duration of treatment / exposure:
300 sec
Duration of post- treatment incubation (in vitro):
not applicable
Number of animals or in vitro replicates:
4 eggs
Details on study design:
At day 8 of incubation the sections marked for the air bubble were sawed out of the shell. The inner membrane was moistened with NaCl 0.9 % and carefully removed with forceps. Only eggs with normally developed embryos and blood vessel systems were used for testing. Undilutet test item was applied directly onto the Chorioallantoic membrane (CAM) of each egg in a volume of 300 µL undiluted test item. 4 eggs each were used for the test item, negative and positive controls.

Observations of effects to the blood vessels, albumen or embryo over a period of 300 seconds after substance application are determined for each single egg. The time to the appearance of each of the observations mentioned above has been monitored and recorded. If no effect appeared during the observation period of 300 seconds (observation = 0) the result was assigned as negative for the related endpoint, and the factor set to 0 for this endpoint when calculating the Irritation Score (IS).

Scoring criteria for the acute effects and calculation of Irritation Score (IS):
0 = no effect
1 = vasodilatation, slight haemorrhage (H)
2 = vessel lysis, strong haemorrhage (L)
3 = blood-coagulation, albumen-coagulation (C)

IS = 5 x (301-sec H)/300 + 7 x (301- sec L)/300 + 9 x (301- sec C)/ 300
H= observed start of haemorrhage reactions in seconds; L= observed start of vessel lysis, strong haemorrhage in seconds; C= observed start of blood- oagulation, albumen-coagulation in seconds

Data Interpretation of Irritation Score (IS):
0 - 0.9 -> Non-irritant
1 - 4.9 -> Slight irritant
5 - 8.9 -> Moderate irritant
9 - 21 -> Strong irritant

A test substance is considered to cause severe irritation when the IC value is greater than nine.
Irritation parameter:
other: irritation score (IS)
Run / experiment:
mean after observation period 300 sec
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Table 1: Individual irritation scores for the test item fluocortolon

 Egg

 Effect

 Effect detected after [sec]

 Irritation Score (IS)

 9

 1

> 300

 

 

 2

> 300

3

> 300

 10

 1

> 300

 

 

 2

> 300

 

 3

> 300

 11

 1

> 300

 

 

 2

> 300

 

 3

> 300

 12

 1

> 300

 

 

 2

> 300

 

 3

> 300

Effect :

1 = vasodilatation, slight haemorrhage

2 = vessel lysis, strong haemorrhage

3 = blood-coagulation, albumen-coagulation

Table 2: Tabular summary of the irritation scores for test item fluocortolon, negative and positive control

 Test item  Irritation Score (IS)  Classification
Negative control NaCl 0.9 %  non-irritant
Positive control SDS 1 %  11   strong irritant
Fluocortolon   0 non-irritant 
Interpretation of results:
GHS criteria not met
Conclusions:
Fluocortolon was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method. This is a method that makes use of the chorioallantoic mambrane of fertilized chicken eggs. For determination of acute effects on haemorrhage, lysis of blood vessels and coagulation 300 µL of the undiluted test item per egg was directly applied onto the chorioallantoic membrane for 5 minutes. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non-irritant. The results of the positive (SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.
Executive summary:

In an irritation study performed according to ICCVAM Test Method Evaluation Report: Current Validation Status of In Vitro Test Methods Proposed for Identifying Eye Injury Hazard Potential of Chemicals and Products. NIH Publication No. 10-7553. Research Triangle Park, NC: National Institute of Environmental Health Sciences, 2010, Fluocortolon (100% a.i.) was applied to the CAM of 4 fertilized Lohmann Brown hen eggs for 300sec. A volume of 300 μl of the undiluted test item was applied on to the CAM (4 eggs each), ensuring that at least 50 % of the CAM surface area is covered. In case of Fluocortolon this volume corresponded to an amount from an average of 103.5 mg.


Effects are measured by the onset of haemorrhage, vessel lysis or coagulation during the first 300 seconds after application. Times till appearance of each of these endpoints are used to calculate an irritation score.


The positive (1% 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 irritation score obtained after 300 sec treatment with Fluocortolon compared to the negative control tissues was 0. Thus, Fluocortolon is identified to be not irritating.


 

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
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)
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.

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%.
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

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

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.


 

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
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)
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.
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.
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

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

- 2.5 0.002 - 2.5 - 0.4 2.0 

(20 % Fluocortolon)

- 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.

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.


 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

For fluocortolone-21-valerate (CAS No. 36130-02-6) no skin and eye irritation/corrosion data are available. Therefore, in vitro skin and eye irritation/corrosion data of fluocortolone (CAS No. 152-97-6) were used since these data are regarded as representative as most likely ester cleavage of fluocortolone-21-valerate occurs under physiological conditions. A search for structure-analogue substances using the QSAR OECD Toolbox 3.4 recommended fluocortolone as one out of 4 category substances for a read-across approach (for additional information see QSAR OECD Toolbox Report on Fluocortolon-21-Valerat in "Attached justification").

A study for predicting a non-specific, corrosive potential of the test item fluocortolone by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 431 (Leidenfrost, 2017a). For the determination of time related cytotoxic effects the incubation periods were 3 min. and 60 min. The MTT (Methylthiazoletetrazolium) viability test results (3 min.: 95.94 % viability; 60 min.: 104.65 % viability) showed, that fluocortolone has no corrosive property under the conditions of the assay used.

In addition, a study for predicting a skin irritation potential of the test item fluocortolone by using reconstructed human epidermis (test method epiCS®) was performed according to OECD TG 439 (Leidenfrost, 2017b). After an exposure period of 20 minutes, followed by a 42 hours post-treatment incubation period, the mean value of cell viability was measured to be 101.1 % in the MTT (Methylthiazoletetrazolium) conversion assay. Thus, fluocortolone is considered to have no skin irritation category as defined in the UN GHS.

Fluocortolone was investigated in the Bovine Corneal Opacity and Permeability (BCOP) test according to OECD TG 437 (Rauh, 2016). 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 fluocortolone was characterized by having no potential to seriously damage the eye.

In addition, an in vitro study for assessing ocular irritation of the test item fluocortolone was conducted in a human corneal epithelial (HCE) cell model (Leidenfrost, 2017c). 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), fluocortolone was considered to be non-irritant to the eye.

Furthermore, fluocortolone was tested in the in vitro assay applying the Hen´s Egg Test on the Chorio-Allantoic Membrane (HET-CAM) test method (Leidenfrost, 2017d). This is a method that makes use of the chorioallantoic mambrane of fertilized chicken eggs. For determination of acute effects on haemorrhage, lysis of blood vessels and coagulation 300 µL of the undiluted test item per egg was directly applied onto the chorioallantoic membrane for 5 minutes. The Irritation Score (IS) value was calculated to be 0 for the test item which was interpreted as being non-irritant. The results of the positive (SDS 1% in physiologic saline) and negative (physiologic saline solution) controls confirmed the validity of the test system.

Justification for classification or non-classification

Based on the study results of the in vitro skin and eye irritation/corrosion tests with the read-across substance fluocortolone classification of fluocortolone-21-valerate is not required according to Regulation (EC) No. 1272/2008 (CLP).