Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 March 2015 to 15 June 2015
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:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Appearance: Violet powder
- Storage conditions: Controlled room temperature (15 to 25 °C, below 70 RH %), protected from light and humidity
- Safety precautions: Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to assure personnel health and safety

Test animals

Species:
other: EPISKIN MODEL
Strain:
other: Reconstructed Human Epidermis
Details on test animals and environmental conditions:
TEST SYSTEM:
HUMAN SKIN
- EPISKIN-SM is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are 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 (Tinois et al. 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.
- For killed epidermis, living epidermis units were placed in a 12 well plate with 2 mL of distilled water, then incubated at 37 °C in an incubator with 5 % CO₂, in a >95 % humidified atmosphere for 48 hours (±1 hour). At the end of the incubation the water was discarded and the dead epidermis units were frozen on 28 November 2014. Before use, the killed tissues were thawed at room temperature (at least 30 minutes in 2 mL of Assay Medium). Further use of killed tissues was similar to living tissues.

QUALITY CONTROL
- EPISKIN-SM kits are manufactured according to defined quality assurance procedures (certified ISO 9001). All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma. The quality of the final product is assessed by undertaking a MTT cell viability test and a cytotoxicity test with sodium dodecylsulphate (SDS).

JUSTIFICATION FOR SELECTION OF THE TEST SYSTEM
- The EPISKIN-SM model has been validated for irritation testing in an international validation study [9] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.

KIT CONTENTS
- Units: EPISKIN-SM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm₂) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
- Plate: 12-well assay plate
- Punch: EPISKIN-SM biopsy punch for easy sampling of epidermis
- Medium: Flask of sterile “Maintenance Medium”
- Flask of sterile “Assay Medium”

NUMBER OF REPLICATE WELLS
- In this assay, three replicates were used for the test material. Three negative controls and three positive controls were also run in the assay. As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. Furthermore, as the test material might have an MTT interacting potential, three additional test material-treated killed epidermis and three negative control treated killed epidermis were used in the study.

KIT RECEPTION
The pH of the agar medium used for transport was checked by checking the colour of the medium:
- Orange colour = good
- Yellow or violet colour = not acceptable
The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C (the colour change is irreversible, independent of the length of the period above 40 °C):
- White colour = good
- Grey or black colour = not acceptable
The kits were found to be in good order at reception.

STORAGE
- The EPISKIN-SM kit was kept in their packaging at 37 °C, the Assay Medium and Maintenance Medium supplied with the kits were stored at 2 to 8 °C until the initiation of the test.

Test system

Type of coverage:
not specified
Preparation of test site:
not specified
Vehicle:
unchanged (no vehicle)
Controls:
other: Positive and negative controls were included in the experiment
Amount / concentration applied:
TEST MATERIAL
- No formulation was required; the test material was applied as supplied.
- As the test material was solid, 10 μL distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis, then 20 mg of test material were applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.

CONTROLS
Positive and negative controls were included in the experiment. Furthermore, as the test material was coloured, one additional control tissue sample was used in the experiment for determination of the non-specific colour. Additional controls on killed epidermis (three test material treated and three negative control treated skin units) were also used in the study to determine the MTT interacting potential of the test material.
- Negative Control: Phosphate Buffered Saline (PBS), (outsourced)
- Positive Control: 5 % (w/v) Sodium Dodecyl Sulphate solution (SDS), prepared freshly in laboratory
- 50 μL of positive control (5 % (w/v) SDS solution) or negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
Duration of treatment / exposure:
The disks were treated with test material and incubated for 15 minutes; the test material was then rinsed. The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO₂. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37 °C in an incubator with 5% CO₂ protected from light.
Details on study design:
TEST SITE
- Area of exposure: Disks of EPISKIN (three units)

SCORING SYSTEM
- Optical Density (OD) measured, reported as percentage relative viability.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS)
- Time after start of exposure: The disks were treated with test material and incubated for 15 minutes; the test material was then rinsed with Phosphate Buffer Saline (PBS).

PERFORMANCE OF THE STUDY:
PRE-INCUBATION (DAY -1)
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate were filled with the pre-warmed medium (2 mL per well). The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight at 37 °C in an incubator with 5 % CO₂, in a >95 % humidified atmosphere.

APPLICATION AND RINSING (DAY 0)
- Test Material
As the test material was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis, then 20 mg of test material were applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a curved flat spatula (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
- Positive and negative control
50 μL of positive control (5 % (w/v) SDS solution) or negative control (PBS) were added to each skin unit by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary (without damaging the epidermis).
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (24.4 to 25.5 °C).
The test material was showed to be an MTT-interacting substance, in addition to the normal procedure, three test material treated killed epidermis and three negative control treated killed epidermis was used in the study (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues was different than the batch of the living tissues. The same treatment steps were followed in case of the killed tissues as in case of the living tissues.
After the incubation time, the EPISKIN-SM units were removed and rinsed thoroughly with PBS to remove any remaining material from the epidermal surface as much as possible. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
After rinsing the units were placed into the plate wells with fresh pre-warmed Maintenance Medium (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37 °C in an incubator with 5 % CO₂.

MTT TEST (DAY 2)
After the 42 hours incubation, all EPISKIN-SM units (except of one colour control unit) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). Then, all transferred EPISKIN-SM units were incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO₂ protected from light.

FORMAZAN EXTRACTION (DAY 2)
After the incubation with MTT, a formazan extraction was undertaken. A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit).
The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).
The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

CELL VIABILITY MEASUREMENTS (DAY 2)
Following the formazan extraction, 2 × 200 μL sample from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (optical density or absorbance) of the samples were measured using a plate reader at 570 ±30 nm and 690 nm (the test material has a violet colour, but this wavelength was considered to be suitable for this purpose based on the observed spectral results of the MTT checking test). The mean of 6 wells of acidified isopropanol solution (200 μL / well) was used as blank.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other:
Value:
73
Remarks on result:
other:
Remarks:
Basis: mean. Time point: Assessed after 42 hour incubation period. (migrated information)

In vivo

Irritant / corrosive response data:
As the test material was coloured, one additional test material-treated tissue was used for the non-specific OD evaluation. The optical density (measured at 570 nm) of this tissue was 0.011, Non Specific Colour % was calculated as 1.6 %. This value was below 5 %, therefore additional data calculation was not necessary.
As colour change (blue) was observed after three hours of incubation of the test material in MTT working solution, thus the test material might interact with MTT (this fact could not be properly determined due to the interfering colour of the test material). Therefore, additional controls and data calculations were necessary to exclude the false estimation of viability. Based on these observed (negative) OD value, the calculated NSMTT was considered to be insignificant, thus additional data calculation was not necessary.
The OD values for the test material treated skin samples showed 73.0 % relative viability.

Any other information on results incl. tables

Table 1: Optical Density (OD) and the calculated relative viability % of the samples

Substance

Optical Density (OD)

Viability (% RV)

 

Measured

Blank corrected

Negative Control: Phosphate buffered saline

1

0.713

0.668

93.8

2

0.795

0.749

105.2

3

0.765

0.720

101.1

mean

 

0.712

100.0

Postive Control: 5 % (w/v) SDS solution

1

0.077

0.032

4.5

2

0.092

0.046

6.5

3

0.093

0.047

6.6

mean

 -

0.042

5.9

Test Material

1

0.443

0.398

55.8

2

0.604

0.559

78.5

3

0.650

0.604

84.8

mean

0.520

73.0

Notes:

1. Mean blank value was 0.045.

2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

3. Based on the absorbance values measured at 690 nm, no significant absorbance due to possible test material remaining on the Episkin units could be detected compared to the negative control samples (the test material appeared to stain permanently the epidermis and matrix, but this fact did not interfere with the formazan extraction and absorbance reading, and was considered not to adversely affect the results or intergrity of the study).

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Following exposure with the test material, the mean cell viability was 73.0 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin.
Executive summary:

The in vitro irritation test of the test material was investigated in accordance with the standardised guidelines OECD Guideline 439 (In Vitro Skin Irritation) and EU Method B.46 (In Vitro Skin Irritation: reconstructed human epidermis model test) under GLP conditions. The study was awarded a reliability score of 1 in accordance with the principles for assessing data quality set forth by Klimisch et. al. (1997).

Disks of EPISKIN (three units) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of the test material was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37 °C for 42 hours in an incubator with 5 % CO₂. PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). An additional disk was used to provide an estimate of colour contribution from the test material. Following the exposure with the test material, the mean cell viability was compared to the negative control.

Under the conditions study, the test material was determined to be not irritating to the skin.