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

Endpoint summary

Description of key information

A BCOP test showed that thioacid is not an eye irritant, having an in vitro irritancy score of 0.9.

A skin corrosivity test showed that thioacid is not corrosive to skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 October 2015 - 31 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Purity 98.76%
Batch No: 0000120575

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

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 ultra-structure 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:

Validation studies have shown that tests employing human skin models are able to reliably distinguish between known skin corrosives and non-corrosives (Botham et al, 1995, Barratt et al, 1998 and Fentem et al, 1998). 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.

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. 20 mg of the test item was 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. It was important to ensure the test item and the control items uniformly covered the tissue surface.

Duration of treatment / exposure:

3 minutes and 60 minutes

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.

At the end of the 3-hour MTT incubation period, the tissue culture inserts were blotted and transferred to pre-labeled 24-well plates for MTT extraction. 1.5 mL of MTT extractant solution (isopropanol) was used to completely immerse each tissue insert, and the plate was covered with a suitable plate sealer to inhibit isopropanol evaporation. The sealed plates (covered to protect from light) stood overnight at room temperature to extract the reduced MTT (formazan dye) from within the MTT loaded tissue.

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minute

Value:

ca. 93.1

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

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minute

Value:

ca. 98.3

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%.

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 : 93.1% 60 minutes exposure : 98.3%

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2015 -

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement

Specific details on test material used for the study:

Batch: 0000120575
Purity: 98.76%
Expiry date: 5 July 2016

Species:

other: bovine corneas

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Vehicle:

other: 0.9% w/v sodium chloride solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

20% w/v of test material in 0.9% w/v sodium chloride solution. Test item was formulated within 2 hours of being applied to the system.
20% w/v of Imidazole in 0.9% w/v sodium chloride solution was used as positive control.

Duration of treatment / exposure:

The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea.

Observation period (in vivo):

Not applicable

Duration of post- treatment incubation (in vitro):

240 minutes at 32+/-1 degrees

Number of animals or in vitro replicates:

3 corneas for test item, 3 corneas for negative control and 3 corneas for positive control

Details on study design:

All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading for average opacity was taken for each cornea using a calibrated opacitometer. Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492 nm (OD492) was measured using the Anthos 2001 microplate reader.

The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

ca. 0.9

Negative controls validity:

valid

Remarks:

negative control score: 1.8

Positive controls validity:

valid

Remarks:

positive control score: 77.6

Other effects / acceptance of results:

The positive control In Vitro Irritancy Score was within the range of 66.9 to 101.4. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity of ≤4.1 and permeability ≤0.105. The negative control acceptance criteria were therefore satisfied.

Tables with individual corneal results can be found in attached "BCOP Tables".

Interpretation of results:

GHS criteria not met

Conclusions:

No category. Not requiring classification to UN GHS or EU CLP.

Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS) and EU Classification, Labelling and Packaging (CLP) of chemicals (Regulation (EC) No 1272/2008), and it was therefore endorsed as scientifically valid for both purposes. Test items inducing serious eye damage are classified as UN GHS and EU CLP Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS/ EU CLP Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS/EU CLP No Category.

Method

The test item was applied at a concentration of 20% w/v in 0.9% w/v sodium chloride solution for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Interpretation

The test item is classified according to the prediction model below:

IVIS	Classification
≤ 3	No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55	No prediction of eye irritation can be made
> 55	Category 1. UN GHS or EU CLP Causes serious eye damage

Results

The in vitro irritancy scores are summarised as follows:

Treatment	In vitro irritancy score
Test item	0.9
Negative control	1.8
Positive control	77.6

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based upon the negative results obtained in one in vitro study performed in accordance with OECD guideline 431, Method B.40bis of Commission Regulation 440/2008 and GLP, the substance does not fulfil the criteria for classification as skin corrosive within the meaning of CLP.

Similarly, the results of the Bovine Corneal Opacity Study, conducted in accordance with OECD guideline 437 and GLP, were also negative. Therefore, the substance does not fulfil the criteria for classification as eye irritant, according to CLP.