estr-4-ene-3,11,17-trione cyclic 3-(1,2-ethanediylmercaptole)

Endpoint summary

Description of key information

An Episkin study determined that the test item was not irritating to skin
A BCOP study determind  that the test item was not an ocular corrosive or severe irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed at a registered GLP site and reported a high standard andis in compliance with recognised OECD standards with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EPISKIN™ model

Strain:

not specified

Type of coverage:

open

Preparation of test site:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg

Duration of treatment / exposure:

15 minutes

Observation period:

6 days

Number of animals:

0

Details on study design:

Pre-Test - Assessment of Direct Test Item Reduction of MTT
MTT dye metabolism, cell viability assay
The MTT assay, a colourimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a blue formazan dye by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, each test item is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37°C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue, the test item is presumed to have reduced the MTT.

Pre-Incubation (Day 0: tissue arrival)
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37°C, 5% CO2 in air overnight.

Main Test - Application of Test Item and Rinsing (Day 1)
2 ml of maintenance medium, warmed to approximately 37°C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis. Triplicate tissues treated with 10 µl of DPBS served as the negative controls and triplicate tissues treated with 10 µl of SOS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SOS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). After 7-Minutes contact time the SOS solution was re-spread with a pipette tip to maintain the distribution of the SOS for the remainder of the contact period. The plate(s) were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37°C, 5% CO2 in air for 42 hours.

MTT Loading/Formazan Extraction (Day 3)
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenise the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30°C for possible inflammatory mediator determination.
2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37°C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN™ biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl of acidified isopropanol, ensuring that
both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10°C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as 'blanks'. The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

Irritation / corrosion parameter:

other: other: relative mean viability of treated tissues

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 15 minute. Reversibility: no data. Remarks: 119.3%. (migrated information)

Irritant / corrosive response data:

The relative mean viability of the test item treated tissues was 119.3% after a 15-Minute exposure period following quantitatively correction of the results for direct reduction of MTT.

RESULTS

Direct MTT Reduction

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Test Item, Positive Control Item and Negative Control Item

The individual and mean OD₅₄₀ values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in the table belwo. The mean viabilities and standard deviations of the test item and positive control, relative to the

negative control are also given in the table below.

The relative mean viability of the test item treated tissues was 119.3% after a 15-Minute exposure period following quantitatively correction of the results for direct reduction of MTT.

Quality Criteria

The relative mean tissue viability for the positive control treated tissues was 9.7% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 1.1 %. The positive control acceptance criterion was therefore satisfied.

The mean OD₅₄₀ for the negative control treated tissues was 0.734 and the standard deviation value of the percentage viability was 4.5%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 4.0%. The test item acceptance criterion was therefore satisfied.

Mean OD₅₄₀ Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540 oftissues	Mean OD540of triplicatetissues	± SD of OD540	Relativeindividualtissueviability (%)	Relativemeanviability (%)	±SD ofRelativemeanviability (%)
NegativeControl Item	0.740	0.734	0.033	100.8	100 +	4.5
	0.071			95.2
	0.764			10.41
PositiveControl Item	0.067	0.071	0.008	9.1	9.1	1.1
	0.50			10.9
	0.66			9.0
Test item	0.907	0.876	0.029	123.6	119.3	4.0
	0.849			115.5
	0.871			118.7

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be Non-Irritant (NI).

Executive summary:

Introduction:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1a in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline nonirritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the following:

• GECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010)

• Method 8.46 of Commission Regulation (EC) No. 440/2008/EC.

Method:

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. 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 viability of the test item treated tissues was 119.3% after the 15-Minute exposure period. Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion:

The test item was considered to be Non-Irritant (NI).

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed at a registered GLP site and reported a high standard andis in compliance with recognised OECD standards with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Principles of method if other than guideline:

No analysis was carried out to determine the homogeneity, concentration or stability of the test item formulation. The test item was formulated within two hours of being applied to the test system; it is assumed that the formulation was stable for this duration. This is an exception with regard to GLP, but is considered not to affect the purpose or integrity of the study.

GLP compliance:

yes (incl. QA statement)

Species:

other: Bovine Eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Source of Bovine Eyes
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 hours of receipt.

Preparation of Corneas
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 (epithelial side uppermost) 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 minimum essential medium (MEM) and plugged. The holders were incubated at 32 ±1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

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

Vehicle:

other: 20% w/v dilution in 0.9% w/v sodium chloride solution.

Controls:

other: Negative Control and Positive Control (20% w/v Imidazole)

Amount / concentration applied:

TEST MATERIAL
- Concentration : 20% w/v

VEHICLE
- Concentration : 0.9% w/v sodium chloride solution.

Duration of treatment / exposure:

240 minutes Treatment

Observation period (in vivo):

Initial post treatment observations to assess Opacity.
Further 90 minutes for Permeability assessement

Number of animals or in vitro replicates:

number of corneas - 9

Details on study design:

REMOVAL OF TEST SUBSTANCE
Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. 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°C for 240 minutes.
At the end of the exposure period the test item preparation 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. The anterior and posterior chambers were refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.
Application of Sodium Fluorescein
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°C for 90 minutes

Permeability Determinations
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 492nm (OD492) was measured.
If values greater than 1500 OD₄₉₂ were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.


SCORING SYSTEM:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity Measurement
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement
The corrected OD₄₉₂ was calculated by subtracting the mean OD₄₉₂ of the negative control corneas from the OD₄₉₂ value of each treated cornea. The OD₄₉₂ value of each treatment group was calculated by averaging the corrected OD₄₉₂ values of the treated corneas for the treatment group.
In Vitro Irritancy Score
The following formula was used to determine the In Vitro Irritancy Score:
                 In Vitro Irritancy Score    =   mean opacity value    +    (15 x mean OD₄₉₂ value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DATA INTERPRETATION
A test item that induces an In Vitro Irritancy Score 255.1 is defined as an ocular corrosive or severe irritant.
Criterion for an Acceptable Test
For an acceptable test the following positive control criterion must be achieved:
200% w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 55.8 to 126.1.

Irritation parameter:

other: In Vitro Irritancy Score

Basis:

mean

Score:

5.7

Irritant / corrosive response data:

Treatment                    In Vitro Irritancy Score
Test Item                              5.7
Negative Control                3.3
Positive Control                  94.7

Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in the table below entitled Individual and Mean Corneal Opacity and Permeability Measurements.

Criterion for an Acceptable Test
The positive control In Vitro Irritancy Score was within the range 55.8 to 126.1. The positive control acceptance criterion was therefore satisfied.

Other effects:

Corneal Epithelium Condition
The condition of each cornea post treatment is given in the table below entitled Corneal Epithelium Condition Post Treatment.
The corneas treated with the test item were slightly cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD)		In vitro Irritancy Score
		Pre-treatment	Post-treatment	Post-treatment - Pre-treatment	Corrected Value		Corrected Value
Negative Control	1	1	4	3		0.015
	2	3	6	3		0.011
	3	2	5	3		0.024
	mean			3.0		0.017		3.3
Positive Control	4	3	68	65	62.0	2.48	2.463
	5	2	48	46	43.0	2.315	2.298
	6	3	73	70	67.0	2.72	2.703
	mean				57.3		2.488	94.7
Test Item	7	2	12	10	7.0	0.069	0.052
	8	2	10	8	5.0	0.031	0.014
	9	2	9	7	4.0	0.025	0.008
	mean				5.3		0.025	5.7

Corneal Epithelium Condition Post Treatment

Treatment	Cornea Number	Observation Post Treatment
Negative control	1	clear
	2	clear
	3	clear
Positive Control	4	cloudy
	5	cloudy
	6	cloudy
Test Item	7	slightly cloudy
	8	slightly cloudy
	9	slightly cloudy

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered not to be an ocular corrosive or severe irritant.

Executive summary:

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).

A test item that induces an In Vitro Irritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant.

The in vitro Irritancy scores are summarised as follows:

Treatment                    In Vitro Irritancy Score

Test Item                            5.7

Negative Control                3.3

Positive Control                  94.7

Therefore the test item was considered not to be an ocular corrosive or severe irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 119.3% after the 15-Minute exposure period. The quality criteria required for acceptance of results in the test were satisfied. Therefore the test item was considered to be Non-Irritant (NI).

Eye Irritation

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).

A test item that induces an In Vitro Irritancy Score ≥55.1 is defined as an ocular corrosive or severe irritant.

The in vitro Irritancy scores are summarised as follows:

Treatment                    In Vitro Irritancy Score

Test Item                            5.7

Negative Control                3.3

Positive Control                  94.7

Therefore the test item was considered not to be an ocular corrosive or severe irritant.

Justification for selection of skin irritation / corrosion endpoint:
An Episkin study determined that the test item was not irritating to skin

Justification for selection of eye irritation endpoint:
A BCOP study determind that the test item was not an ocular corrosive or severe irritant.

Justification for classification or non-classification

In Vitro data has shown no potential for irritation to both the eye and skin. As such the material is not being considered for classification as an irritant via the assessed routes.