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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 November 2016 to 18 November 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

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:
2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
Qualifier:
according to
Guideline:
other: EpiSkin™ SOP, Version 1.8 (February 2009), 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:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
-White to pale yellow
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The EPISKIN™ (SM) model has been validated for irritation testing in an international validation study 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.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ (SM)
- Manufacturer: SkinEthic, France
- Tissue batch number(s): 16-EKIN-046
- 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.
- Expiry date: 21 November 2016
- 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 - 8 °C until the initiation of the test.

CHECK FOR DIRECT MTT REDUCTION
Approximately 10 mg of test material was added to 2 mL MTT working solution and mixed. The mixture was incubated at 37 °C in a shaking water bath for 3 hours protected from light, and then any colour change was recorded:
Test materials which do not react with MTT: Yellow,
Test materials reacting with MTT: Blue or purple.
After three hours incubation, yellow colour of the mixture was detected in the test tube. Thus, the test material did not react with MTT and therefore the use of additional controls was not necessary.

CHECK FOR COLOURING POTENTIAL OF THE TEST MATERIAL
Prior to treatment, the test material was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol (simulating a tissue humid environment). As the test materials had an intrinsic colour, further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSCliving %) was determined in order to evaluate the ability of test material to stain the epidermis by using additional control tissues.
Therefore, in addition to the normal procedure, two additional test material-treated living tissues were used for the non specific OD evaluation. This tissue followed the same test material application and all steps as for the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test material that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

MAIN STUDY
PRE-INCUBATION (DAY -1)
The Maintenance Medium was pre-warmed to 37 °C. The appropriate number of wells in an assay plate was 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 (DAY 0)
As the test material was solid, 10 μL of distilled water was applied to the epidermal surface in order to improve further contact between test material and epidermis and then 10 mg of the test material was applied evenly to the epidermal surface. If necessary, the test material was spread gently on the skin surface with a pipette tip (or other appropriate tool) without damaging the epidermis. The amount was sufficient to cover the epidermal surface.
50 μL of negative control (PBS) or positive control (5 % (w/v) SDS solution) 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 (23.9 to 26.0 °C).


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (23.9 to 26.0 °C)
- Temperature of post-treatment incubation: 37 °C

NUMBER OF REPLICATES FOR THE TEST: 3

REMOVAL OF TEST MATERIAL AND CONTROLS
After the 15 minutes 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, in a > 95 % humidified atmosphere.

MTT TEST AND FORMAZAN EXTRACTION (DAY 2)
After the 42 hour incubation, all EPISKIN™ (SM) units (except the two living colour control units) were transferred into the MTT working solution filled wells (2 mL of 0.3 mg/mL MTT per well). The transferred EPISKIN™ (SM) units were incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO2, in a > 95 % humidified atmosphere, protected from light.
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. 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.
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 was measured using a plate reader at 570 nm. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank.

CALCULATIONS OF VIABILITY PERCENTAGES

FOR NORMAL TEST MATERIALS:
-Blank: The mean of the six blank OD values was calculated
-Negative control: Individual negative control OD values (NCraw) were corrected with the mean blank OD: OD Negative Control (ODNC) = ODNCraw – ODblank mean. The mean corrected OD of the 3 negative control samples were calculated: This value corresponds to 100 % viability
-Positive control: Individual positive control OD values (PCraw) were corrected with the mean blank
OD: OD Positive Control (ODPC) = ODPCraw – ODblank mean. The mean corrected OD of the 3 positive control samples were calculated. The % viability for each positive control replicate was 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 relative viability % for positive control was calculated:
Mean PC % = (%PC1 +%PC2 +%PC3) / 3

-Test material: Individual test material OD values (TTraw) were corrected with the mean blank OD:
OD Treated Tissue (ODTT) = ODTTraw – ODblank mean
The mean corrected OD of the 3 test material samples were calculated. The % viability for each test material replicate was 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 relative viability % for test material was calculated: Mean TT % = (%TT1 +%TT2 +%TT3) / 3

FOR TEST MATERIALS WITH MTT-INTERACTING POTENTIAL:
Test materials that interfere with MTT can produce non-specific reduction of the MTT. In this case, additional control samples are used to determine the OD value derived from non-specific reduction of the MTT. The measured OD value is corrected by the result of the additional controls before calculation of viability% as detailed below:
-Non-specific MTT reduction calculation (NSMTT%):NSMTT (%) = [(ODKT- ODKNC) / ODNC] × 100
ODKNC: Negative control killed tissues OD
ODKT: Test material treated killed tissues OD
ODNC: Negative control OD
If NSMTT% is ≤ 30 %, then true MTT metabolic conversion (TODTT) is undertaken as follows:
TODTT = [ODTT – (ODKT – ODKNC)]
ODTT: Test material treated viable tissues
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated:
Mean Relative Viability % = (% RV 1 + % RV 2 + % RV 3) / 3
If NSMTT% is > 30 % relative to the negative control, then additional steps must be undertaken if possible, or the test material must be considered as incompatible with the test.

FOR TEST MATERIALS WITH COLOURING POTENTIAL
For test materials detected as able to stain the tissues, the non-specific OD due to the residual chemical colour (unrelated to mitochondrial activity) is evaluated and subtracted before calculation of the “true” viability % as detailed below:
Non-specific Colour % with viable tissues (NSCliving %):= (mean ODCTV / mean ODNC)×100
ODCTV: Test substance treated viable tissues (not incubated with MTT)
ODNC: Negative control OD (incubated with MTT)
If NSCliving % is ≤ 5 % then the normal calculation mode is used.
If NSC % is > 5 % and ≤ 30 %, then true MTT metabolic conversion (TODTT) is undertaken as follows.
TODTT = [ODTT – mean ODCTV]
ODTT: Test substance treated viable tissue (incubated with MTT)
ODCTV: Test substance treated viable tissue (not incubated with MTT)
The % relative viability (% RV) for each test material replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test material is calculated: (% RV 1 +% RV 2 +% RV 3) / 3
If NSC living % is > 30 % relative to the negative control, additional steps must be undertaken if possible, or the test substance must be considered as incompatible with the test.

FOR TEST MATERIALS WITH BOTH MTT INTERACTING POTENTIAL AND COLOURING POTENTIAL
Test substances detected as able to stain the tissues and interfere with MTT also require a third set of controls before calculation of the “true” viability %.
Non-specific Colour % with killed tissues (NSCkilled%) = (mean ODCTK / mean ODNC)×100
ODCTK: Test substance treated killed tissues (not incubated with MTT)
ODNC: Negative control OD (incubated with MTT)
TODTT = [ODTT – (ODKT – ODKNC) – mean ODCTV +mean ODCTK]
ODTT: Test substance treated viable tissues (incubated with MTT)
ODKT: Test substance treated killed tissues OD
ODKNC: Negative control killed tissues OD
ODCTV: Test substance treated viable tissues (not incubated with MTT)
ODCTK: Test substance treated killed tissues (not incubated with MTT)
The % relative viability (% RV) for each test substance replicate is calculated relative to the mean negative control:
% RV 1 = [TODTT1 / mean ODNC] × 100
% RV 2 = [TODTT2 / mean ODNC] × 100
% RV 3 = [TODTT3 / mean ODNC] × 100
The mean value of the 3 individual relative viability % for test substance is calculated = (% RV 1 + % RV 2 + % RV 3) / 3

INTERPRETATION OF TEST RESULTS
-The test material considered to be irritant to skin (Category 2), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg applied with 10 µL distilled water.

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL
- Concentration: 5 % w/v (prepared in distilled water)
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 ± 1 hours
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
103.1
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
ADDITIONAL CONTROLS
-As no colour change was observed after three hours of incubation of the test material in MTT working solution, the test material did not interact with MTT and additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
-As the test material was coloured, two additional test material-treated tissues were used for the non-specific OD evaluation. The mean optical density of tissues were 0.006, Non-specific Colour % was calculated as 0.8 %. This value was below 5 %, therefore additional data calculation was not necessary.

VIABILITY RESULTS
The results of the optical density (OD) measured at 570 nm of each sample and the calculated relative viability % values are presented in Table 1. The OD values for the test material treated skin samples showed 103.1 % relative viability.

Any other information on results incl. tables

Validity of the Test

-After receipt, the two indicators of the delivered kit were checked. Based on the observed colours, the epidermis units were in proper conditions.

-The mean OD value of the three negative control tissues was in the recommended range (0.744). Standard deviation of the viability results for negative control samples was 4.7.

-The positive control treated tissues showed 6.3 % viability demonstrating the proper performance of the assay. The standard deviation of the viability results for positive control samples was 1.6.

-The standard deviation of viability values of the three test material-treated tissue samples in the MTT assay was 2.6.

-The mean OD value of the blank samples (acidified isopropanol) was 0.046.

-All these parameters met the acceptability criteria, therefore the study was considered to be valid.

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

Substance

Optical density

Viability (% RV)

 

Measured

Blank corrected

Negative control: PBS

1

0.790

0.744

100.1

2

0.754

0.708

95.2

3

0.824

0.778

104.7

Mean

-

0.744

100.0

Positive control: 5 % (w/v) SDS solution

1

0.087

0.041

5.5

2

0.085

0.039

5.3

3

0.107

0.061

8.2

Mean

-

0.047

6.3

Test material

1

0.832

0.786

105.7

2

0.811

0.765

102.8

3

0.794

0.748

100.6

Mean

-

0.767

103.1

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 non-irritant in the in vitro skin irritation test.
Executive summary:

The potential of the test material to cause skin irritation was determined in accordance with the standardised guidelines OECD 439, EU Method B.46 and EPISkin SOP Version 1.8 under, GLP conditions using a human skin model.

EPISKIN (SM) is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Disks of EPISKIN (SM) 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, in a > 95 % humidified atmosphere. The test was performed in triplicate.

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. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

PBS and 5 % (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving) from the test material. For each treated tissue, the viability was expressed as a % relative compared to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control, the test material is considered to be irritant to skin.

Following exposure to the test material, the mean cell viability was 103.1 % compared to the negative control. This is above the threshold of 50 %, therefore the test material was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, under the conditions of this study the results indicate that the test material is non-irritant to skin (No Category based on the skin corrosivity test), UN GHS Classification: Non-classified.