Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 November 2011 to 11 November 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Substance name: Reactive Yellow F01-0555

In vitro test system

Test system:
human skin model
Remarks:
reconstructed human epidermis (RhE)
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN model has been validated for corrosivity testing in an international trial, it is considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
EpiSkinTM Small Model (EpiSkinTMSM) (Source: SkinEthic, France, Batch No.:11-EKIN-041, Expiry date: 14 November 2011) 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.

Source: Skinethic, Nice, France.
Batch No.: 11-EKIN-041
Expiry date: 14 November 2011

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 an MTT cell viability test and a cytotoxicity test with sodium dodecyl sulphate (SDS).

DEMONSTRATION OF PROFICIENCY
Prior to routine use of the method CiToxLAB Hungary Ltd. demonstrated technical proficiency, using the ten Proficiency Chemicals according to OECD Test Guideline No. 439.

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: A flask of sterile “Maintenance Medium” for incubations. (Batch No.: 11-MAIN3-053; Exp. Date: 16 November 2011)
A flask of sterile “Assay Medium” for use in for use in MTT assays. (Batch No.: 11-ESSC-043; Exp. Date: 16 November 2011)

Kit Reception Quality Check
The colour of the agar medium used for transport was checked for its pH:
-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 indicator changes from white to grey at 40°C
The kit was found to be in good order at reception.

Storage
The EPISKIN-SM kit was kept in its packaging at 37°C and the assay and maintenance medium supplied with the kit was stored at 2-8°C until the initiation of the test.

ADDITIONAL MATERIALS
MTT stock solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was dissolved to a final concentration of 3 mg/mL in saline buffer (PBS). The obtained stock solution can be stored in a refrigerator (2-8°C), protected from light up to 15 days.

MTT ready to use solution
The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL. The obtained solution was used within one hour.

Acidified Isopropanol
Isopropanol was diluted with HCl acid to a final concentration of 0.04N HCl.
The obtained solution can be stored in a refrigerator (2-8°C), protected from light for one month.

INDICATOR FOR POTENTIAL FALSE VIABILITY
Optical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test substance is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.

Check-method for possible direct MTT reduction with test substance
An amount of 10 mg test item 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 assessed:
- Test substances which do not interact with MTT: yellow
- Test substances interacting with MTT: blue or purple
If the MTT solution colour becomes blue or purple, the test substance 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).
The test item showed no direct interaction with MTT.

Check-method to detect the colouring potential of test-substances
As the test item has an intrinsic colour, further evaluation to detect colouring potential was necessary. Non Specific Colour % (NSC %) was determined in order to evaluate the ability of test substance to stain the epidermis by using additional control tissues.


Additional control(s) for dyes and chemicals able to colour the tissue
In addition to the normal procedure, one additional chemical-treated tissue was used for the non specific OD evaluation. This tissue followed the same test item application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh assay medium. This is to mimic the amount of colour from the test item that may be present in the test disks. OD readings were made following the same conditions as for the other tissues.


PERFORMANCE OF THE STUDY
Procedures described in sections 3.6.1., 3.6.2. and 3.6.3. were performed under axenic conditions.

Pre-incubation (Day [-1])
The “maintenance medium” was pre-warmed to 37°C. The appropriate number of 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)
First 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface of each of the three test skin units.
10 µl PBS was added to each of the three negative control skin units
10 µl SDS was added to each of the three positive control skin units
For additional controls for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface.

The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).

After the incubation time the EPISKIN-SM units were removed and rinsed thoroughly with PBS 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 (± 1h) at 37°C in an incubator with 5% CO2.

MTT test after 42 hours incubation (day 2)
After the 42 hours incubation all EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The one additional control for coloured substances was transferred to wells filled with fresh assay medium. 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)
At the end of incubation with MTT a formazan extraction was undertaken:

A disk of epidermis from each replicate 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.

Cell viability measurements (Day 2)
Following the formazan extraction, 2×200 µL samples from each tube were placed into the wells of a 96-well plate (labelled appropriately). The OD (Absorbance / Optical Density) of the samples in a 96-well plate spectrophotometer was read at 540 nm using acidified isopropanol solution blank (6×200 µL).
The validity of the microplate reader was verified with a standard verification plate daily before use. The standard plate was calibrated yearly by the manufacturer.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
First 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface of each of the three test skin units.
10 µl PBS was added to each of the three negative control skin units
10 µl SDS was added to each of the three positive control skin units
For additional controls for staining effects of the test item, 10 µl distilled water was applied to the epidermal surface to ensure good contact with the epidermis, then 20 mg of test item was applied evenly to the epidermal surface.
Duration of treatment / exposure:
The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature (18-28°C).
Duration of post-treatment incubation (if applicable):
After the 42 hours incubation all EPISKIN-SM units except the one staining control were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well). The one additional control for coloured substances was transferred to wells filled with fresh assay medium. 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.
Number of replicates:
In this assay 3 replicates for the test item and 3 negative controls + 3 positive controls were used. Furthermore one additional control for coloured substance was used.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of 3 replicates
Value:
98
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
VALIDITY OF THE TEST

The mean OD value of the three negative control tissues was 0.949. The positive control result showed 14% 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 Substance

No colour change was observed after three hours of incubation of the test item in MTT solution. 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 substances

As the test item has an intrinsic colour, one additional chemical-treated tissue was used for the non specific OD evaluation. Mean OD (measured at 540 nm) of this tissue was determined as 0.024, Non Specific Colour % was calculated as 2.5%. Therefore additional data calculation was not necessary.

Any other information on results incl. tables

CELL VIABILITY

 The results of the optical density (OD) measured at 540 nm of each replicate and the calculated % viability of the cells is presented below:

 

Substance

Optical Density (OD)

Viability (%)

Negative Control:

PBS

 

1

0.954

101

2

0.899

95

3

0.994

105

mean

0.949

100

standard deviation (SD)

5.03

Positive Control:

SDS 5% aq. solution

 

1

0.169

18

2

0.111

12

3

0.110

12

mean

0.130

14

standard deviation (SD)

3.46

Test Item:

Reactive Yellow F01-0555

 

1

0.892

94

2

0.894

94

3

1.019

107

mean

0.935

98

standard deviation (SD)

7.51

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Disks of EPISKIN (three units / chemical) were treated with Reactive Yellow F01-0555 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. 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 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified.
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 substance 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.


In this in vitro skin irritation test in the EPISKIN model with Reactive Yellow F01-0555 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].
All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Executive summary:

Disks of EPISKIN (three units / chemical) were treated with Reactive Yellow F01-0555 and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS. 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 5% CO2 protected from light. The formazan extract in acidified isopropanol was then spectrophotometrically evaluated for optical density (OD) and quantified. 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 substance 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.

In this in vitro skin irritation test in the EPISKIN model with Reactive Yellow F01-0555 the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

All validity criteria were within acceptable limits and therefore the study can be considered as valid.