Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5 January 2011 to 7 January 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, ECVAM Skin Irritation Validation Study: Validation of the EpiSkin™ test method 15 min - 42 hours for the prediction of acute skin irritation of chemicals.
Version / remarks:
Version 1.8 (February 2009)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Storage: room temperature 15-25 °C, continuously protected from all light

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
Recommended in international guidelines
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ Small Model (EpiSkin™SM)
- Source: Skinethic, Nice, France
- Tissue batch number(s): 11-EKIN-001
- Expiry date: 10 January 2011
- EpiSkin™ Small Model (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. 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.

KIT RECEPTION
- The colour of the agar medium used for transport was checked for its pH: orange colour = good or 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 indicator changes from white to grey at 40°C.
- The kit was found to be in good order at reception.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Check-method for possible direct MTT reduction with test material: Approximately 10 mg of test material was added to 2 mL MTT ready to use solution and mixed. The mixture was incubated for three hours at room temperature protected from light and then any colour change was observed: Test materials which do not interact with MTT turn yellow and test materials interacting with MTT turn blue or purple. If the MTT solution colour becomes blue or purple, the test material interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).
- Additional controls for MTT direct interacting chemicals: For an MTT-interacting test mateiral previously detected, and in addition to the normal procedure, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation in one run (untreated killed tissues may exhibit little residual NADH and dehydrogenase associated activity). The batch of killed tissues can be different than the batches of the living tissues. The same treatment steps are followed at these tissues as the living tissues.
- Killed epidermis for MTT-interaction substances: Living epidermis are placed at -20°C (or -80°C) for at least 48 hours. Before use, the killed tissues are de-frozen at room temperature (app. 1 hour in 2.2 mL of assay medium). Further use of killed tissues is similar to living tissues.
- Check-method to detect the colouring potential of test materials: Prior to treatment, chemicals were evaluated for their intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). An amount of 10 mg of the test material was added to 90 μL of water. The mixture was shaken for about 15 minutes and then colour checked (unaided visual assessment). If a coloured solution is detected or the test material has an intrinsic colour, the Non Specific Colour % (NSC %) is determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissue(s).
- Additional control(s) for dyes and chemicals able to colour the tissue: For dyes or chemicals able to colour the tissue previously detected, in addition to the normal procedure, at least 1 additional chemical-treated tissue is used for the nonspecific OD evaluation. This tissue follows the same treatment steps than other tissues except for the MTT step: MTT incubation is replaced by incubation with fresh assay medium. OD readings are made following the same conditions as for other tissues.

PERFORMANCE OF THE STUDY
PRE-INCUBATION (DAY [-1])
- The “maintenance medium” was pre-warmed to 37°C. The appropriate number of an assay plate wells 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.

APPLICATION AND RINSING (DAY 0)
- 10 mg of test material was applied evenly to the epidermal surface of each of the three test skin units, then 30 μL distilled water was added to the test material to ensure good contact with the epidermis. 10 μL PBS was added to each of the three negative control skin units and 10 μL SDS was added to each of the three positive control skin units
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 minutes) at room temperature (18 - 28°C).

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 1x solution (0.9%) to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with a suitable pipette tip linked to a vacuum source care was taken to avoid the damage of 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 (± 1 hour) at 37°C in an incubator with 5% CO2.

MTT TEST AFTER 42 HOURS INCUBATION (DAY 2)
- After the 42 hours incubation the EPISKIN-SM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37°C in an incubator with 5% CO2 protected from light.

FORMAZAN EXTRACTION (DAY 2)
- At the end of incubation with MTT a formazan extraction was undertaken: A disk of epidermis was cut from the unit (this involves 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 of 500 μL acidified isopropanol (one tube corresponding to one well of the tissue culture plate).
- The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material with the acidified isopropanol then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.

NUMBER OF REPLICATE TISSUES: 3

CELL VIABILITY MEASUREMENTS (DAY 2)
- Following the formazan extraction, 2×200 μL sample from each tube was placed into the wells of a 96-well plate (labelled appropriately) and read the OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer at 540 nm using acidified isopropanol solution as the blank (6×200 μL).


CALCULATIONS OF VIABILITY PERCENTAGES

Data calculation for normal test materials
Blank:
– The mean of the 6 blank OD values is calculated
Negative control:
– Individual negative control OD values are corrected with the mean blank OD:
OD Negative Control (ODNC) = ODNCraw – ODblank mean
– The corrected mean OD of the 3 negative control values are calculated: this corresponds to 100 % viability
Positive control:
– Individual positive control OD values are corrected with the mean blank OD:
OD Positive Control (ODPC) = ODPCraw – ODblank mean
– The corrected mean OD of the 3 positive control values are calculated
– The % viability for each positive control replicate is calculated relative to the mean negative control:
% Positive Control 1 = (ODPC1 / mean ODNC) ×100
% Positive Control 2 = (ODPC2 / mean ODNC) ×100
% Positive Control 3 = (ODPC3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for positive control is calculated:
Mean PC % = (%PC1 + %PC2 + %PC3) / 3
Test material:
– Individual test material OD values are corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
– The corrected mean OD of the 3 test material values are calculated
– The % viability for each test material replicate is calculated relative to the mean negative control:
% Treated Tissue 1 = (ODTT1 / mean ODNC) ×100
% Treated Tissue 2 = (ODTT2 / mean ODNC) ×100
% Treated Tissue 3 = (ODTT3 / mean ODNC) ×100
– The mean value of the 3 individual viability % for test material is calculated
Mean TT % = (%TT1 + %TT2 + %TT3) / 3


INTERPRETATION OF TEST RESULTS
- A test material is considered to be irritant to skin (R38), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
- Mean tissue viability % is ≤ 50 %: Irritant (I) R38
- Mean tissue viability % is > 50 %: Non Irritant (NI)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg with 30 μL distilled water to ensure good contact with the epidermis

NEGATIVE CONTROL
- Amount(s) applied: 10 µL

POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5%
Duration of treatment / exposure:
15 minutes (± 0.5 minutes)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
3 replicates per test material, 3 negative controls and 3 positive controls were used

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
93
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
VALIDITY OF THE TEST
- The mean OD value of the three negative control tissues was 0.795. The positive control result showed 25% viability. Each standard deviation value (SD) of the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

INDICATOR FOR POTENTIAL FALSE VIABILITY
- Possible direct MTT reduction with test material: No colour change was observed after three hours of incubation. The test material did not interact with the MTT, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colouring potential of test material: The test material showed no ability to become coloured in contact with water, therefore additional controls and data calculations were not necessary. A false estimation of viability can be precluded.

CELL VIABILITY
- The test material treated tissue viability was 93% after treatment.

Any other information on results incl. tables

Table 1: Optical density (OD) measured at 540 nm of each replicate and calculated % viability of the cells

Treatment

Optical Density (OD)

Viability (%)

Negative Control

1

0.859

108

2

0.661

83

3

0.865

109

Mean

0.795

100

Standard deviation

14.59

Positive Control

1

0.176

22

2

0.197

25

3

0.223

28

Mean

0.198

25

Standard deviation

11.86

Test Material

1

0.856

108

2

0.713

90

3

0.648

82

Mean

0.739

93

Standard deviation

14.37

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU Criteria
Conclusions:
Under the conditions of this study, the test material is not irritating to the skin.
Executive summary:

The skin irritation potential of the test material was investigated in accordance with the standardised guideline OECD 439, under GLP conditions.

Disks of EPISKIN (three units / treatment) were treated with the test material and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution (0.9%). Epidermis units were then incubated at 37°C for 42 hours. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically.

SDS 5% and PBS treated epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test material is considered to be irritant to skin, if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.

All validity criteria were within acceptable limits and therefore the study can be considered as valid. The test material treated tissues had a mean tissue viability of 93% after treatment.

Under the conditions of this study, the test material is not irritating to the skin.