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

Skin irritation / corrosion

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

11-KN

IUPAC Name:

11-KN

Constituent 2

Reference substance name:

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

Molecular formula:

C20H26O2S2

IUPAC Name:

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

Constituent 3

Reference substance name:

920-684-2

EC Number:

920-684-2

IUPAC Name:

920-684-2

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 11-KN
- Physical state: White powder
- Lot/batch No.: CGG238K1
- Date received: 12 February 2013
- Expiration date of the lot/batch: 12 February 2014
- Storage condition of test material: room temperature in the dark

Test animals

Species:

other: EPISKIN™ model

Strain:

not specified

Test system

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.

Results and discussion

In vitro

Results

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)

In vivo

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).