5,7-di-tert-butyl-3-[4-(2-hydroxyethoxy)phenyl]-1-heteropolycycl-2(3H)-one

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation / corrosion

Remarks:

other: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2011 - 20 December 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Method B.46 of Commission Regulation (EC) No. 440120081E0

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals

Species:

other: Reconstructed human epidermis model

Strain:

other: EPISKIN

Details on test animals or test system and environmental conditions:

EPISKIN MODEL KIT
Supplier: SkinEthic Laboratories, Nice, France

The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type 1 matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Test system

Type of coverage:

other: n/a

Preparation of test site:

other: n/a

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

10 mg of test material was applied to the epidermis surface which had previously been moistened with 5 µL of sterile distilled water.

Duration of treatment / exposure:

15 minutes.

Observation period:

42 hour post exposure observation period.

Number of animals:

n/a

Details on study design:

NEGATIVE AND POSITIVE CONTROL ITEMS
Dulbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control.
Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.

Preparation of Negative and Positive Control Items, MTT and Acidified Isopropanol
-The negative control item was used as supplied.
-The positive control item was prepared as a 5% w/v aqueous dilution.
-A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required.
-A 0.04 N concentration of hydrochloric acid in isopropanol was prepared when required.

PROCEDURE
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-diphenyl-tetrazolium 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 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.

The test item was shown to directly reduce MTT in the direct MTT reduction test. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin irritation potential was performed in parallel on viable and water-killed tissues.

This step was a functional check which employs water-killed tissues that possess no metabolic activity but absorb and bind the test substance like viable tissues.

Water-killed tissues were prepared by placing untreated EPISKIN tissues in a 12-well plate containing 2.0 mL of sterile distilled water in each well. The tissues were incubated at 37°C, 5% CO2 in air for 48 ± 1 hours. At the end of the incubation the water was discarded. Once killed the tissues were stored in a freezer (-14 to -30°C) for up to 6 months. Before use each tissue was thawed by placing in 2.2 mL of maintenance medium for approximately 1 hour at room temperature.

In addition to the normal test procedure, each MTT reducing test substance was applied to a water-killed tissue. In addition, one water-killed tissue remained untreated. The untreated water-killed control showed a small amount of MTT reduction due to residual reducing enzymes within the killed tissue.


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 pi of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS 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 SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS 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 500µL 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.


INTERPRETATION OF RESULTS
Quantitative MTT Assessment (percentage tissue viability)
For the test item the relative mean tissue viabilities obtained after the 15-minute exposure period followed by the 42-hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3). The relative mean viabilities were calculated in the following way:

Relative mean viability %) = [(mean OD540 of test item) / (mean OD540 of negative control)] X 100

Classification of irritation potential is based upon relative mean tissue viability following the 15-minute exposure period followed by the 42-hour post-exposure incubation period according to the following:

Criteria for in vitro interpretation Classification
Relative Mean Tissue Viability is ≤50% Irritant (I) R38
Relative Mean Tissue Viability is >50% Non-Irritant (NI)*
The results were evaluated according to EU labelling regulations Commission Directive 20011591EC for classification and labelling of dangerous items.

*The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42-hour post-exposure incubation period may be determined for test items which are found to be borderline non-irritant based upon the MTT cell viability endpoint (mean tissue viability 51 to 60%). This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

QUALITY CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:

-Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was ≤40% relative to the negative control treated tissues, and the standard deviation value of the percentage viability is ≤18%.

-Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD540 for the negative control treated tissues was ≥0.6, and the standard deviation value of the percentage viability is ≤18%.

Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

105.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

111.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

88.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Direct MTT Reduction
An assessment found the test item was able to directly reduce MTT. Therefore, an additional procedure using water-killed tissues was performed during the determination of skin irritation potential. However, the results obtained showed no significant degree of interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the water-killed tissues for quantitative correction of results or for reporting purposes.

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 Table 1. The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.

The relative mean viability of the test item treated tissues was 102.1% after a 15-Minute exposure period.

Quality Criteria
The relative mean tissue viability for the positive control treated tissues was 4.5% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.8%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was 1.315 and the standard deviation value of the percentage viability was 6.7%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues was 11.7%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1  Mean OD540 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD540 of tissues	Mean OD540 of triplicate tissues	± SD of OD540	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative control item	1.415	1.315	0.088	107.6	100*	6.7
	1.250			95.1
	1.281			97.4
Positive control item	0.070	0.059	0.010	5.3	4.5	0.8
	0.056			4.3
	0.050			3.8
Test item	1.393	1.342	0.154	105.9	102.1	11.7
	10465			111.4
	1.169			88.9

*The mean viability of the negative control is at 100%

SD = Standard Deviation

Applicant's summary and conclusion

Interpretation of results:

other: Non-Irritant (NI)

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

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

Executive summary:

The test item was considered to be a Non-Irritant (NI). This method was designed to be compatible with the OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation" (adopted 22 July 2010) and the Method B.46 of Commission Regulation (EC) No. 44012008/EC