Diarsenic triselenide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2105

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Diarsenic triselenide

EC Number:

215-119-5

EC Name:

Diarsenic triselenide

Cas Number:

1303-36-2

Molecular formula:

As2Se3

IUPAC Name:

diarsenic triselenide

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Internal product code: 300000434
- Batch nr: E15-01025
- Expiration date of the lot/batch: 31 Jan 2035
- Purity: 100 %
- Appearance: black solid
- Date of production: Jan 2015


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: controlled room temperature (15-25°C)

In vitro test system

Test system:

artificial membrane barrier model

Remarks:

EPISKIN

Source species:

other:

Cell type:

other:

Cell source:

other:

Source strain:

other:

Vehicle:

unchanged (no vehicle)

Remarks:

the test item was applied as supplied. the test item was a fine powder; no additional manipulation step was necessary

Details on test system:

- pH of the agar medium used for transport was checked
- temperature indicator was inspected to verify that the kit has not been exposed to temperatures > 40°C

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

NEGATIVE CONTROL
- 3 negative controls

POSITIVE CONTROL
- 3 positive controls

Number of replicates:

3
+ 1 additional test item-treated tissue for non specific OD evaluation

Test animals

Species:

other: 3d human epidermis model

Strain:

not specified

Details on test animals or test system and environmental conditions:

EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:15-EKIN-015, Expiry Date: 20 April 2015) 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) [7]. 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.
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.

Test system

Preparation of test site:

other:

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Details on study design:

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” (Batch No.: 15 MAIN3 015; Exp. Date: 22 April 2015) / A flask of sterile “Assay Medium” (Batch No.: 15 ESSC 015; Exp. Date: 22 April 2015)


INDICATOR FOR POTENTIAL FALSE VIABILITY
Chemical action by the test material on MTT may mimic that of cellular metabolism leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material directly acts on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test item interference with the viability measurement. Methods of how to correct direct MTT reduction and interferences by colouring agents are detailed in the following paragraphs.


STUDY PERFORMANCE
Procedures were performed under aseptic conditons (in sterile hood using sterile equipment):

- 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)
As the test item was solid, first an appropriate amount (10 μL) distilled water was applied to the epidermal surface in order to improve further contact between test item and epidermis, then 20 mg of test item were applied evenly to the epidermal surface. If necessary, the test item 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.

Negative and positive controls
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 (25.6-26.8°C).
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 (if test item was stuck on the epidermal surface, the rinsing step was repeated up to three rinsing circles). 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. The mean of 6 wells of acidified isopropanol solution (200 μL/well) was used as blank. The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 02 September 2014, calibration is valid until September 2016) at the required wavelength on each day before use.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

viability (%RV)

Value:

86.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

In the present study, the irritancy potential of test substances is predicted by the mean tissue viability of tissues exposed to the test item. The test item is considered to be irritant to skin (Category 2, H315), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
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 (0.659). Standard deviation of the viability results for negative control samples was 5.3.
The positive control treated tissues showed 11.9% viability demonstrating the proper performance of the assay. The standard deviation value of the viability results for positive control samples was 5.3. The standard deviation of viability values of the three test item-treated tissue samples in the MTT assay was 4.4. All these parameters met the acceptability criteria, therefore the study was considered to be valid.

As the test item was coloured, one additional test item-treated tissue was used for the non specific OD evaluation. The optical density (measured at 570 nm) of this tissue was 0.026, Non Specific Colour % was calculated as 4.0%. This value was below 5%, therefore additional data calculation was not necessary. As no colour change (yellow mixture) was observed after three hours of incubation of the test item in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary; the false estimation of viability can be excluded.

Any other information on results incl. tables

Data calculation for normal test items

Blank:
– The mean of the 6 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 item:

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

Data calculation for MTT-interacting items

Test items 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 item 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– ODKU)]

ODTT: test item treated viable tissues

The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:

%RV1=[TODTT1/meanODNC]×100 %RV2=[TODTT2/meanODNC]×100 %RV3=[TODTT3/meanODNC]×100

The mean value of the 3 individual relative viability % for test item 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 item must be considered as incompatible with the test.

Data calculation for dyes and chemicals able to colour the tissue

For test items 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 % (NSC%):

NSC%=(ODCT/meanODNC)×100

ODCT: test item treated tissue (not incubated with MTT) ODNC: negative control OD (incubated with MTT)

If NSC %is ≤ 5% then the normal calculation mode is used (see 3.7.1).

If NSC% is > 5% and ≤30%, then true MTT metabolic conversion (TODTT) is undertaken as follows.

TODTT= [ODTV- ODCT]

ODTV: test item treated tissue (incubated with MTT) ODCT: test item treated tissue (not incubated with MTT)

The % relative viability (% RV) for each test item replicate is calculated relative to the mean negative control:

%RV1=[TODTT1/meanODNC]×100 %RV2=[TODTT2/meanODNC]×100 %RV3=[TODTT3/meanODNC]×100

The mean value of the 3 individual relative viability % for test item is calculated:

Mean Relative Viability % = (% RV 1 +% RV 2 +% RV 3) / 3

If NSC % is > 30% relative to the negative control, then additional steps must be undertaken if possible, or the test item must be considered as incompatible with the test.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with GASIR5, the mean relative viability was 86.1% compared to the negative control value. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
In conclusion, in this in vitro EPISKIN model test with GASIR5, the results indicate that the test item is non-irritant to skin.

Executive summary:

An in vitro skin irritation test of GASIR5 test item was performed in a reconstructed human epidermis 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 (detailed in 3.6. section). The irritation potential of the test item was evaluated according to the OECD No. 439 guideline [1].

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item 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. 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% CO2protected 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). An additional disk was used to provide an estimate of colour contribution from the test item. For each treated tissue, the viability was expressed as a % relative 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 testitem is considered to be irritant to skin.

Following exposure with GASIR5, the mean cell viability was 86.1% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN model test with GASIR5, the results indicate that the test item is non-irritant to skin.