Androsta-1,4-diene-17-carbothioic acid, 6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-(1-oxopropoxy)-, (6.alpha.,11.beta.,16.alpha.,17.alpha.)-

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17th November 2015 - 19th November 2015

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

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

(6ALPHA,11BETA,16ALPHA,17ALPHA)-6,9-DIFLUORO-11-HYDRO XY-16-METHYL-3-OXO-17- (1-OXOPROPOXY)ANDROSTA-1,4-DIENE-CARBOTHIOIC ACID

EC Number:

617-084-5

Cas Number:

80474-45-9

Molecular formula:

C24H30F2O5S

IUPAC Name:

(6ALPHA,11BETA,16ALPHA,17ALPHA)-6,9-DIFLUORO-11-HYDRO XY-16-METHYL-3-OXO-17- (1-OXOPROPOXY)ANDROSTA-1,4-DIENE-CARBOTHIOIC ACID

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Thio Acid Propionate
Batch: 0000135240
Purity: 97.76%
Physical state/Appearance: Off white solid
Expiry Date: 26 July 2017
Storage Conditions: 4°C in the dark, over silica gel

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: In Vitro Reconstructed Human Epidermal (RHE, SkinEthic Laboratories, Lyon, France)

Details on animal used as source of test system:

The model consists of an airlifted, living, multilayered epidermal tissue construct (surface 0.5cm2) reconstructed of normal human epidermal keratinocytes for 17 days, produced in polycarbonate inserts in serum-free and chemically defined medium, featuring normal ultrastructure and functionally equivalent to human epidermis in vivo. The three-dimensional human epidermal model constructs contain basal, spinous and granular layers along with a functional stratum corneum.

Justification for test system used:

On the basis of peer review (ESAC, 2006) of the results of an inter-laboratory study (Kandarova et al, 2006) with the SkinEthic RHE Model, the ECVAM Scientific Advisory Committee (ESAC) endorsed the conclusion that the SkinEthic RHE Model can be used for distinguishing between skin corrosives and non-corrosive chemicals.

Vehicle:

unchanged (no vehicle)

Details on test system:

On arrival, the SkinEthic RHE tissues (Day 18 cultures), were stored at room temperature prior to transferring into 24-well plates designated ‘arrival plates’ containing 1000 µL of maintenance medium. It was important to ensure that there were no air bubbles present under the tissue inserts. The tissues were incubated overnight at 37 °C, 5% CO2 in air.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

20 mg of test item were applied. 20 μl of sterile distilled water was added for wetting of the solid test item to ensure adequate contact with the tissue culture surface.

Duration of treatment / exposure:

3 minutes and 60 minutes
Using sterile techniques 0.9 mL of maintenance medium, at room temperature, was dispensed into each well of two pre-labeled 6-well plates designated as treatment plates. One plate contained the tissues to be used in the 3 minute experiment whilst the other plate contained tissues to be used in the 60 minute experiment. The tissues were aseptically transferred into the treatment plates prior to dosing.
One plate was placed into the incubator, whilst the tissues in the other plate were being treated. 40 µL of sterile distilled water (negative control) was applied to the first two tissues, and the timer started. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure, and to ensure that each tissue received an equal exposure time. The test item and the positive control item (8.0N Potassium Hydroxide) were also applied to the corresponding duplicate tissues.
Using sterile techniques 0.9 mL of maintenance medium, at room temperature, was dispensed into each well of two pre-labeled 6-well plates designated as treatment plates. One plate contained the tissues to be used in the 3 minute experiment whilst the other plate contained tissues to be used in the 60 minute experiment. The tissues were aseptically transferred into the treatment plates prior to dosing. One plate was placed into the incubator, whilst the tissues in the other plate were being treated. 40 µL of sterile distilled water (negative control) was applied to the first two tissues, and the timer started. The tissues were dosed at regular intervals to allow for the time taken to rinse each tissue following exposure, and to ensure that each tissue received an equal exposure time. The test item and the positive control item (8.0N Potassium Hydroxide) were also applied to the corresponding duplicate tissues.

Duration of post-treatment incubation (if applicable):

The same treatment procedure was used for both exposure times. After treatment the plate for the 1 hour exposure experiment was placed into the incubator until rinsing. Due to the short exposure time the plate for the 3 minute exposure experiment remained in the safety cabinet during the exposure period. At the end of each exposure period, each tissue was removed from the well of the treatment plate using forceps and rinsed using a wash bottle containing PBS. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Excess PBS was removed by blotting the bottom of the tissue culture insert with absorbent paper. The rinsed tissues were placed into a holding plate containing 300 µL of maintenance medium in each well until all tissues had been rinsed, after which each tissue was blotted and transferred to a 24-well plate containing 300 µL of 0.5 mg/mL (v/v) MTT solution in each well for MTT loading. The plate was placed in an incubator at 37 °C, 5% CO2 in air for 3 hours.

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

ca. 94.1

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

The mean OD562 for the negative control treated tissues was 1.497 for the 3-Minute exposure period and 1.449 for the 60-Minute exposure period.

Positive controls validity:

valid

Remarks:

The relative mean tissue viability for the positive control treated tissues was 0.6% relative to the negative control treated tissues following the 60-Minute exposure period.

Remarks on result:

other: The test item was considered to be non-corrosive to the skin

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute

Value:

ca. 97.7

Vehicle controls validity:

not examined

Negative controls validity:

valid

Remarks:

The mean OD562 for the negative control treated tissues was 1.497 for the 3-Minute exposure period and 1.449 for the 60-Minute exposure period.

Positive controls validity:

valid

Remarks:

The relative mean tissue viability for the positive control treated tissues was 0.6% relative to the negative control treated tissues following the 60-Minute exposure period.

Remarks on result:

other:

Remarks:

not corrosive to skin

Other effects / acceptance of results:

The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The absolute OD562 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD562 of the two negative control tissues should be ≥ 0.8 and ≤ 3.0 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control: Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criteria if the mean relative tissue viability of the 60 minute positive control is <15%.
Coefficient of Variation: In the range of 20 to 100% viability and for Optical Densities ≥ 0.3, the difference in viability between the two tissue replicates should not exceed 30%.

In vivo

Results

Results 1

Irritation parameter:

other: OD562

Time point:

other: 3 minutes

Score:

ca. 94.1

Results 2

Irritation parameter:

other: OD562

Time point:

other: 60 minutes

Score:

ca. 97.7

Irritant / corrosive response data:

The relative mean viability of the test item treated tissues was as follows:

3 minutes exposure : 94.1%
60 minutes exposure : 97.7%

Any other information on results incl. tables

Classification of corrosivity potential was based on relative viabilities for each exposure time according to the following prediction model:

Mean tissue viability (% negative control)	Prediction (Corrosive/Non-Corrosive)
3 minute exposure: <50	Corrosive
3 minute exposure: ≥50	Corrosive
60 minute exposure: <15	Corrosive
3 minute exposure:  ≥50	Non-corrosive
60 minute exposure:  ≥50	Non-corrosive

Applicant's summary and conclusion

Interpretation of results:

other: Not corrosive to skin

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the SkinEthic in vitro Reconstructed Human Epidermal (RHE, SkinEthic Laboratories, Lyon, France) model after treatment periods of 3 and 60 minutes. Corrosive test items are able to penetrate the stratum corneum and are sufficiently cytotoxic to cause cell death in the underlying cell layers. Toxicity is determined by the metabolic reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase by viable cells in the test item treated tissues relative to the negative control treated tissues.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 1.5 mL Isopropanol for MTT extraction. At the end of the formazan extraction period the tissues were mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562). Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities of the test item treated tissues were: 3 minutes exposure : 94.1% 60 minutes exposure : 97.7%

Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.