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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 28 September 2017 to 29 September 2017
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
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Deviations:
not specified
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Selenium disulphide
EC Number:
231-303-8
EC Name:
Selenium disulphide
Cas Number:
7488-56-4
Molecular formula:
S2Se
IUPAC Name:
selenium disulphide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Name: Selenium Disulphide
CAS Number: 7488-56-4
Batch number: PMC / 285 / 16
Appearance: Orange/yellow powder
Expiry date: 24 November 2019
Purity: 100%
Storage conditions: Room temperature (between 5oC and 30oC), protected from light Safety precautions: Enhanced safety precautions were applied considering the supplied safety data sheet to assure personnel health and safety.

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Adult donors
Source strain:
not specified
Details on animal used as source of test system:
Human Skin
EPISKINTM(SM) (Manufacturer: SkinEthic, France, Batch No.: 17-EKIN-039, Expiry Date: 02 October 2017) 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.
Justification for test system used:
The EPISKINTM(SM) model has been validated for corrosivity testing in an international trial (Fentem, 1998) and its use is recommended by the relevant OECD guideline for corrosivity testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
Quality Control
EPISKINTM(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). These quality control experiments were conducted at SkinEthic laboratories (supplier of the EpiSkinTM(SM) Test Kits used in the present study).

Number of Replicate Wells
In this assay, two replicates per test item were used. Two negative controls and two positive controls were also run in this assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Kit Reception
In each case, 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 EPISKINTM(SM) kits were 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.

MTT solution
MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1] was diluted in phosphate buffered saline (PBS) at a final concentration of 3 mg/mL (MTT stock solution). The obtained stock solution (prepared on 27 September 2017) was stored in refrigerator (2-8°C) protected from light. It was diluted with pre-warmed (37°C) Assay Medium to a final concentration of 0.3 mg/mL (MTT working solution) immediately before use.

Acidified isopropanol
Isopropanol was acidified with HCl acid to achieve a final concentration of 0.04N HCl (1.8 mL of 12N HCl acid was diluted in 500 mL isopropanol, or similar ratio was applied). The solution was prepared on the day of use.
Control samples:
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
- 20 mg of test item was applied evenly to the epidermal surface of each of two test item treated skin units and each additional control skin units and then 100 μL
physiological saline was added to the test item to ensure good contact with the epidermis.
- 50 μL of physiological saline was added to each of the two negative control skin units.
- 50 μL of glacial acetic acid was added to each of the two positive control skin units.
Duration of treatment / exposure:
4 hours (+/- 10 minutes)
Duration of post-treatment incubation (if applicable):
3 hours (+/- 15 minutes)
Number of replicates:
2 replicates per test or control (positive and negative)

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Selenium Disulphide Run 1
Value:
91.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Selenium Disulphide Run 2
Value:
94.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Selenium Disulphide Mean
Value:
92.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ADDITIONAL CONTROLS
As the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation. The mean optical density (measured at 570 nm) of these tissues was determined as 0.002, Non Specific Colour% (NSCliving%) was calculated as 0.3%. This is below the threshold of 5%, therefore correction due to colouring potential was not necessary.

As no colour change was observed after three hours of incubation of the test item in MTT solution, thus the test material did not interact with MTT. Therefore, additional
controls and data calculations were not necessary to exclude the false estimation of viability.

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 2. The mean OD value for
the test item treated skin samples showed an 92.7% relative viability compared to the negative control.

VALIDITY OF THE TEST
After receipt, the two indicators of the delivered kit were checked in each case. Based on the observed colours, the epidermis units were in proper conditions.
The mean OD value of the two negative control tissues was in the recommended range (0.758).
The two positive control treated tissues showed 1.0 % viability demonstrating the proper performance of the assay.
The difference of viability between the two test item-treated tissue samples in the MTT assay was 3.1%.
The difference of viability between the two negative control tissue samples in the MTT assay was 0.9%.
The mean OD value of the blank samples (acidified isopropanol) was 0.046.
All these parameters were within acceptable limits and therefore the study was considered to be valid.

Any other information on results incl. tables

Optical Density (OD) and the calculated Non Specific Colour % (NSCliving%) of the Additional Control Tissues

Additional control

Optical density (OD)

NSC% (living)

Treated with selenium disulphide

Number

Measured

Blank corrected

1

0.049

0.002

0.3

2

0.045

-0.001*

Mean

--

0.002

Notes:

1. Mean blank value was 0.046.

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

3. * OD value was excluded from the corrected calculation (negative value).

 

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

Substance

Optical density (OD)

Viability (% RV)

Number

Measured

Blank corrected

Negative control (physiological saline 0.9 %(w/v) NaCl

1

0.808

0.761

100.4

2

0.801

0.755

99.6

Mean

--

0.758

100.0

Positive control Glacial acetic acid

1

0.054

0.008

1.0

2

0.054

0.008

1.0

Mean

--

0.008

1.0

Selenium disulphide

1

0.738

0.692

91.3

2

0.760

0.713

94.1

Mean

--

0.702

92.7

Notes:

1. Mean blank value was 0.046.

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

HISTORICAL CONTROL DATA

 

Negative control

(Physiological saline)

Positive control

(Glacial acetic acid)

Minimum optical density (OD)

0.611

0.005

Maximum optical density (OD)

1.516

0.051

Mean optical density (OD)

0.871

0.017

Standard Deviation (SD)

0.164

0.010

Number of cases

81

81

 

Note: All optical density (OD) values measured are background corrected values (measured at 570±30 nm)

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Following exposure with Selenium Disulphide, the mean cell viability was 92.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EPISKIN™ (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-corrosive to the skin.
Executive summary:

An in vitro skin corrosivity test of Selenium Disulphide test item was performed in a reconstructed human epidermis model. EPISKINTM(SM) is designed to predict and classify the corrosive 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 corrosivity of the test item was evaluated according to the OECD No. 431 guideline.

Disks of EPISKINTM(SM) (two units) were treated with Selenium Disulphide test item and incubated for 4 hours at room temperature. Exposure of test material was terminated by rinsing with Phosphate Buffered Saline solution. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically.

Physiological saline (0.9% (w/v) NaCl solution) and glacial acetic acid treated epidermis were used as negative and positive controls, respectively (two units / control). Two additional disks were used to provide an estimate of colour contribution (NSCliving%) from the test item. For each treated tissue viability was expressed as a % relative to the negative control. If the mean relative viability after 4 hours of exposure is below 35% of the negative control, the test item is considered to be corrosive to skin.

Following exposure with Selenium Disulphide, the mean cell viability was 92.7% compared to the negative control. This is above the threshold of 35%, therefore the test item was considered as being non-corrosive. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in thisin vitroEPISKIN™ (SM) model test with Selenium Disulphide (Batch number: PMC / 285 / 16), the results indicate that the test item is non-corrosive to the skin.