Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008/10/21-2008/10/27
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting of methodological deficiancies, which do not affect the quality of relevant results. Study conducted according to GLP.

Data source

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

Materials and methods

Test guideline
Guideline:
other: No guideline followed.
Principles of method if other than guideline:
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 (1, 2, 3, 4). The principle of the assay is 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.
The EPISKIN 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 I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13 day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

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 reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium 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 only a problem, if at the time of the MTT test (after rinsing) there is 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 below.

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 procedure below:
10 mg of the test material was added to 2 ml of a 0.3 mg/ml MTT solution (v/v) freshly prepared in assay medium. The solution was incubated in the dark at 37ºC, 5% CO2 in air for 3 hours. Untreated MTT solution was 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)
2 ml of maintenance medium, warmed to approximately 37ºC, 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 each test material and control material. The tissues were incubated at 37ºC, 5% CO2in air for at least 24 hours.

Main Test
Application of Test Material and Rinsing (Day 1)
2 ml of maintenance medium, warmed to approximately 37ºC, 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. 10 ± 2 mg of the test material was applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl sterile distilled water to improve contact between the solid test material and the epidermis. Triplicate tissues, treated with 10 µl of PBS, were used to serve as negative controls. Triplicate tissues, treated with 10 µl of SDS 5% w/v, were used to serve as 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 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 ± 0.5 minutes.
At the end of the exposure period, each tissue was removed from the well of the treatnt plate using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++and Mg++. 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 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 37ºC, 5% CO2in 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 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 store in a freezer at ‑14 to ‑30ºC for possible inflammatory mediator determination.
2 ml of 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% CO2in 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 EPISKINTMbiopsy 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.
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP standards (Schedule 1, Good Laboratory Practice Regulations 1999 (SI 1999/3106 as amended by SI 2004/0994)).

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
other: reconstituted human epidermis
Details on test animals and environmental conditions:
Not applicable as the test was performed in vitro.

Test system

Type of coverage:
other: test material applied directly
Preparation of test site:
other: washed previous to testing
Vehicle:
water
Controls:
other: In Vitro test
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
10 ± 2 mg/l



VEHICLE
The epidermis surface had previously been moistened with 5 µl sterile distilled water to improve contact between the solid test material and the epidermis.
Duration of treatment / exposure:
15 minutes
Observation period:
Not applicable
Number of animals:
The test was conducted In Vitro. 3 replicate test tissues were used for the test material test.
Details on study design:
TEST SITE
- Area of exposure:
Whole of test tissue surface
- % coverage:
100%
- Type of wrap if used:
Not applicable


REMOVAL OF TEST SUBSTANCE
- Washing (if done):
At the end of the exposure period, each tissue was removed from the well of the treatment plate using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. 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 material.
- Time after start of exposure:
15 ± 0.5 minutes.

SCORING SYSTEM:
Not applicable.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean % viability
Value:
105
Remarks on result:
other:
Remarks:
Basis: mean. Time point: Day 6. Max. score: 114.5. Reversibility: other: not applicable. Remarks: Invitro result using the EPISKIN reconstituted human epidermis model.. (migrated information)

In vivo

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 negative control, test material and positive control are given in Table 1. The mean viabilities and standard deviations of the test material and positive control, relative to the negative control are also given in Table 1.
The relative mean viability of the test material treated tissues was 105.0% after a 15 minute exposure.
The qualitative evaluation of tissue viability is given in Table 2.
Following the 15-minute exposure the test material treated tissues appeared blue which was considered indicative of viable tissue.

Any other information on results incl. tables

Table1              Mean OD540Values and % Viabilities for the Negative Control Material, Positive Control Material and Test Material

Material

OD540of tissues

Mean OD540of triplicate tissues

± SD of OD540

Relative individual tissue viability

Relative mean % viability

± SD of % viability

Negative Control Material

0.567

0.752

0.160

75.4

100*

21.3

0.831

110.5

0.857

114.0

Positive Control Material

0.027

0.045

0.016

3.6

6.0

2.2

0.058

7.7

0.051

6.8

Test Material

0.861

0.789

0.109

114.5

105.0

14.5

0.664

88.3

0.843

112.1

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

Table2              Qualitative Evaluation of Tissue Viability (MTT uptake visual evaluation)

Material

Tissue 1

Tissue 2

Tissue 3

Negative Control Material

-

-

-

Positive Control Material

++

++

+

Test Material

-

-

-

MTT visual scoring scheme
-
          =         blue tissue (viable)
+
         =         blue/white tissue (semi-viable)
++
       =         tissue is completely white (dead)

 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: no direct reduction of MTT and colour of treated tissues after treatment period.
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 EPISKINTM reconstituted 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 store 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 % 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 was 105.0% after a 15‑minute exposure.

Quality criteria: 

The quality criteria required for acceptance of results in the test were satisfied with the exception that one negative control tissue was particularly lower that the other two negative control tissues when viabilities were measured. As the other two identically treated negative control tissues appeared satisfactory, and the test material was clearly a non-irritant, it was considered that the study was acceptable and a repeat test unnecessary.

Conclusion:  The test material was considered to be non-irritant.