[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 MAY 2021 - 20 JUL 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

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)

Version / remarks:

26 June 2020

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) No 2019/1390 of 31 July 2019 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

bis(2,2,6,6-tetramethyl-1-(1,3-benzothiazol-2-ylsulfanyl)piperidin-4-yl)carbonate

Cas Number:

2311845-49-3

Molecular formula:

C33H42N4O3S4

IUPAC Name:

bis(2,2,6,6-tetramethyl-1-(1,3-benzothiazol-2-ylsulfanyl)piperidin-4-yl)carbonate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

EpiSkinTM Small Model (EpiSkinTMSM)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: three-dimensional human skin model comprising a reconstructed epidermis with a functional stratum corneum

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no-skin irritating test substances (STATEMENT ON THE VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Remarks:

The test item was applied in its original form, no formulation was required.

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkinTM Small Model (EpiSkinTMSM)
- Tissue batch number(s): 21-EKIN-022
- Expiry date: 07 June 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature (22.9-23.7 °C).
- Temperature of post-treatment incubation (if applicable): The epidermis units were placed with the media below them, in contact with the epidermis into each prepared well and then incubated overnight (18-24h) at 37±1 °C in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere.

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL/each tissue 1x PBS solution to remove all of the test material from the epidermal surface. The rest of the 1x PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid damage of epidermis).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: The MTT stock solution was diluted with pre-warmed (37 °C) “assay medium” to a final concentration of 0.3 mg/mL.
- Incubation time: After the 42 hours (± 1h) incubation, the EpiSkinTMSM 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±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.
- Spectrophotometer: Varioskan™ LUX Type 3020
- Wavelength: 200 – 1000 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Approximately 10 mg test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C in an incubator with 5±1 % CO2, ≥ 95 % humidified atmosphere, protected from light and then any colour change observed (unaided eye assessment):
- Test items which do not interact with MTT: yellow
- Test items interacting with MTT: blue or purple
If the MTT solution colour becomes blue or purple, the test item 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).

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
3 replicate of negative control, positive control and test item.

PREDICTION MODEL / DECISION CRITERIA
-In case the test chemical is found to be non-corrosive, and the tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50 %, the test item is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered as non-irritant to skin in accordance with UN GHS No Category if the tissue viability after exposure and post-treatment incubation is more than (>) 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): The epidermal surface was first moistened with 5 μL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 10 μL negative control (1x PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 10 μL positive control (SDS 5 % aq.)

Additional control for dyes and chemicals able to colour the tissue:
In addition to the normal procedure, two additional test item-treated tissues were used for the non-specific OD evaluation (NSCliving).

Duration of treatment / exposure:

exposure time of 15 minutes (± 0.5 min)

Duration of post-treatment incubation (if applicable):

42 hours (± 1h) incubation

Number of replicates:

Three replicates were used for the test item and positive and negative controls, respectively. Furthermore, two replicates were used for the additional control.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ replicate 1

Value:

101

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ replicate 2

Value:

90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1/ replicate 3

Value:

91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

94

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: Colour change was not observed after three hours of incubation. Therefore, the test item was considered not to interact with the MTT, and additional controls and data calculations were not necessary. A false estimation of viability can be precluded.
- Colour interference with MTT: The test item showed no ability to become coloured in contact with water. The intrinsic colour of the test item is white and therefore considered not to be able to significantly stain the tissues and lead to a false estimate of viability. However, a white milky suspension was observed after the test item was mixed with isopropanol, so the test item showed interaction with isopropanol. Based on this information, two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.012. The Non Specific Colourliving % (NSCliving %) was calculated as 1 % (below 5 %). Therefore, additional data calculation was not necessary. A false estimation of viability can be precluded.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD value of the three negative control tissues should be between 0.6 and 1.5 and the standard deviation value (SD) of the % viability should be ≤ 18.
- Acceptance criteria met for positive control: Yes. The acceptable mean percentage viability for positive controls is < 40% and the standard deviation value (SD) of the % viability should be ≤ 18.
- Acceptance criteria met for variability between replicate measurements: Yes. For test chemicals (test item and controls), the standard deviation value (SD) of the % viability should be ≤ 18.
The mean OD value of the three negative control tissues was 1.310. The mean OD value obtained for the positive control was 0.059 and this result corresponds to 4 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative value: 94 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control.
Therefore, from this in vitro skin irritation test, using the EPISKIN model, indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

The In Vitro Skin Irritation test was performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD guideline 439 and under GLP compliance.

Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes (± 0.5 min) at room temperature. Exposure to the test material was terminated by rinsing with 1x PBS solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2, ≥95 % humidified atmosphere, protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically.
SDS (5 % aq.) and 1×PBS treated epidermis (three units / positive and negative control) were used as positive and negative controls, respectively. For each treated tissue viability was expressed as a percentage relative to negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control (mean relative value: 94 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore, the test item was considered to be non-irritant to skin.
Positive and negative controls showed the expected OD and cell viability values within acceptable limits. Standard deviation of all calculated viability values (test item and controls) was below 18. The experiment was considered to be valid.

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicate that the test item reveals no skin irritation potential under the utilised testing conditions. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).