Registration Dossier

Administrative data

Endpoint:
skin irritation / corrosion
Remarks:
other: Method measures cytotoxicity in reconstituted human epidermal cell cultures following topical application
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
between 15 September and 21 September 2009
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
The principle of the study was based on the measurement of cytotoxicity in reconstituted human epidermal cultures following topical exposure to the test material 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-diphenyltetrazolium bromide) to a blue formazan salt )within the mitochondria of viable cells) in the test material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant 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. The EPISKIN(TM) model is a three-dimensional reconstituted 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 coreum.
GLP compliance:
yes (incl. QA statement)
Remarks:
Date of inspection 19th August 2008, Date of signature 4th March 2009

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Batch number: APB401608; Date Received: 10 August 2009; Storage conditions: room temperature in the dark; Purity =98.6-98.8%.
SMILES Notation: c2cc(OC)ccc2C(c1ccccc1)=C(C(#N))C(=O)OCCCCCCCCCC=C

Test animals

Species:
other: Human reconstituted epidermis
Strain:
other: not applicable
Details on test animals or test system and environmental conditions:
Not applicable for in vitro test.

Test system

Type of coverage:
other:
Preparation of test site:
other: Not applicable for in vitro test
Vehicle:
other: not applicable for in vitro test
Controls:
other: Not applicable to study.
Amount / concentration applied:
See free text in "Details on study design" section below.
Duration of treatment / exposure:
15 minutes direct exposure. See free text in "Details on study design" section below.
Observation period:
See free text in "Details on study design" section below.
Number of animals:
Not applicable to study
Details on study design:
The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstituted human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours.

Negative and Positive Control Materials:

Dulbecco’s phosphate buffered saline (PBS) with Ca++ and Mg++ was used as the negative control. Sodium dodecyl sulphate (SDS) was used as the positive control.

Preparation of Negative and Positive Control Materials, MTT and Acidified Isopropanol:

The negative control material was used as supplied.

The positive control material was prepared as a 5% w/v aqueous dilution.

A 3 mg/ml MTT stock solution was prepared in PBS. 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.

Pre-Test: Assessment of Direct Test Material Reduction of MTT:

MTT dye metabolism, cell viability assay:

The MTT assay, a colourimetric method of determining cell viability, is based on the reduction of the yellow tetrazolium salt (2-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) to a purple formazan dye by mitochondrial succinate dehydrogenase in viable cells.

Assessment of Direct Test Material Reduction of MTT:

One limitation of the assay is possible interference of the test substance with MTT. A test substance may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test substance is a problem only if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test substance 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 by the procedure described as follows:

Test for Direct MTT Reduction:

As specified, a test substance 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 substance is checked for the ability to directly reduce MTT according to the following procedure:

10 microliters of the test material 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 deg C, 5% CO2 in air for 3 hours. Untreated MTT solutions were used as a control.

If the MTT solution containing the test substance turns blue/purple, the test substance is presumed to have reduced the MTT.

Pre-Incubation (Day 0: tissue arrival):
2ml of maintenance medium, warmed to approx. 37 deg C, was pipetted into the first column of 3 wells of a pre-labeled 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 material and each control material. The tissues were incubated at 37 deg C, 5% CO2 in air for at least 24 hours.

Main Test:

Application of the Test Material and Rinsing (Day 1):

2 ml of maintenance medium, warmed to approx. 37 deg C, was pipetted into the second column of 3 wells of the 12-well plate.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 microliters of the test material was applied to the epidermis surface. Triplicate tissues treated with 10 microliters of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). 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 +-0.5 minutes.

At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing PBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approx. 40 seconds using a constant soft stream of PBS to gently remove any residual test material. 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 deg C, 5% CO2 in air for approximately 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+-2 minutes to homogenize the released mediators in the maintenance medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at –14 to –30 deg 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 deg C, 5% CO2 in air. At the end of the 3 hour incubation period each tissue was placed onto absorbent paper to dry. The tissues were examined and the degree of MTT staining evaluated (qualitative evaluation of cell viability) using the MTT Visual Scoring scheme. Following qualitative evaluation of tissue viability a total biopsy of the epidermis was made using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 ml micro tubes containing 500 microliters 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 deg 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 microliter samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 microliters 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 material the relative mean tissue viabilities obtained after the 15 minute treatment 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 = (mean OD540 of test material/mean OD540 of negative control) x 100
viability (%)

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

Criteria for in vitro interpretation Classification
Mean tissue viability is <=50% Irritant (I) R38
Mean tissue viability is >50% Non-Irritant (NI)●

● = The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation will be determined for test materials which are found to be borderline non-irritant based upon the MTT cell viability endpoint (mean tissue viability 51-60). 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 criterion is 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 (SD) value of the percentage viability is <20%.

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 SD value of the percentage viability is <20%.


Results and discussion

In vivo

Results
Irritation parameter:
other: Qualitative evaluation (see free text)
Basis:
other: Qualitative evaluation (see free text)
Time point:
other: Not applicable
Score:
0
Max. score:
0
Reversibility:
no data
Irritant / corrosive response data:
Direct MTT Reduction:

The MTT solution containing the test material did not turn blue/purple, which indicated that the test material did not directly reduce MTT.

Test Material, Positive Control Material and Negative Control Material:

The individual and mean OD540 values, standard deviations and tissue viabilities for the test material, negative control material and positive control material were determined and tabulated. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control were determined and tabulated.

The relative mean viability of the test material treated tissues was 105.8% after a 15 minute exposure.

The qualitative evaluation of tissue viability was determined visually and summarized in the table shown in the "Remarks on results including tables and figures" section below.. Following the 15 minute exposure the test material treated tissues appeared blue, which was considered indicative of viable tissue.

Quality Criteria:

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 was <20%. The positive control acceptance criterion was therefore satisfied.

The mean OD540 for the negative control treated tissues was >=0.6 and the SD value of the percentage viability was <=20%. The negative control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Quantitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative control material

-

-

-

Positive control material

+

+

+

Test material

-

-

-

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test material was considered to be Non-Irritant.
Executive summary:

Introduction. 

The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKINTMreconstituted 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 reconstituted human epidermal cultures following topical exposure to the test material 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 material treated tissues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 hour post-exposure incubation period is also determined for test materials which are found to be borderline non-irritant 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.

Triplicate tissues were treated with the test material for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for approximately 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 material treated tissues relative to negative control tissues).


Results: 

The relative mean viability of the test material treated tissues was105.8% after a 15‑minute exposure.

Quality criteria: 

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

Conclusion: 

The test material was considered to be Non-Irritant.