bis(2,2,6,6-tetramethyl-1-(phenylthio)piperidin-4-yl)carbonate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-13 to 2018-12-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYÉI- National Institute of Pharmacy and Nutrition (21.04.2016)

Test material

Test material information

Constituent 1

Reference substance name:

bis[2,2,6,6-tetramethyl-1-(phenylsulfanyl)piperidin-4-yl] carbonate

EC Number:

827-277-8

Cas Number:

1771689-37-2

Molecular formula:

C31H44N2O3S2

IUPAC Name:

bis[2,2,6,6-tetramethyl-1-(phenylsulfanyl)piperidin-4-yl] carbonate

Test material form:

solid: particulate/powder

Remarks:

White powder

Specific details on test material used for the study:

Storage 15-25 °C

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: 10-KERA-003 + 10-KERA-004 (normal human keratinocytes)

Source strain:

other: human

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 (STATEMENT ON THE SCIENTIFIC VALIDITY OF THE EPISKINTM TEST (AN IN VITRO TEST FOR SKIN CORROSIVITY); ECVAM JRC Environment Institute, European Commission; Ispra; 03 April 1998).

Vehicle:

physiological saline

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: Episkin Small Model
- Tissue batch number(s): 18-EKIN-050

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (18-28°C)
- Temperature of post-treatment incubation (if applicable): 37±1°C in an incubator with 5±1% CO2 protected from light, ≥95% humidified atmosphere.

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

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 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 (1xPBS). The MTT stock solution was diluted with pre-warmed “assay medium” to a final concentration of 0.3 mg/mL.
- Incubation time: 3 hours (± 15 min)
- Spectrophotometer: Thermo Scientific; Multiscan FC
- Wavelength: at 570 nm (±10 nm; Read out range: 0-3.5 Abs)
- Linear OD range of spectrophotometer: Linearity range: 0.2136 – 3.1752

NUMBER OF REPLICATE TISSUES: 2

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the mean tissue viability is < 35 % after 3 min exposure (1A), the mean tissue viability is ≥ 35 % after 3 min exposure and < 35 % after 1 hour exposure or the mean tissue viability is ≥ 35 % after 1 hour exposure and < 35 % after 4 hours exposure (1B)
- The test substance is considered to be non-corrosive to skin if the mean tissue viability is ≥ 35 % after 4 hours exposure

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): An amount of 20 mg test item was applied evenly to the epidermal surface of the two test skin units/exposure times respectively. Subsequently, 100 μL NaCl solution (9 g/L saline) was added topically (to each unit) to ensure good contact with the epidermis. The test item was spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

NEGATIVE CONTROL / POSITIVE CONTROL
- Amount(s) applied (volume or weight): A volume of 50 μL positive control (glacial acetic acid) or negative control (NaCl 9 g/L) was applied on the skin surface by using a suitable pipette. Chemicals were spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

The plates with the treated epidermis units were incubated for the exposure time of 4 hours (±10 min) at room temperature (18-28°C).

Duration of post-treatment incubation (if applicable):

After the exposure of test item was terminated by rinsing with PBS, the EpiSkin 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 (± 15 min) at 37±1°C in an incubator with 5±1 % CO2 protected from light, ≥95% humidified atmosphere.

Number of replicates:

Two replicates were used for the test item and control(s) respectively.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

run 1 replicate 1

Value:

93

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

run 1 replicate 2

Value:

111

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Positive and negative controls showed the expected cell viability values within acceptable limits.
All assay acceptance criteria were met, the experiment was considered to be valid.

Any other information on results incl. tables

Table 1: OD values and cell viability percentages of the positive and negative control

Controls	Optical Density (OD)		Viability(%)	Δ%
Negative Control:NaCl (9 g/L saline)	1	0.756	99	1.1
	2	0.765	101
	mean	0.761	100
Positive Control: Glacial acetic acid	1	0.008	1	0.7
	2	0.014	2
	mean	0.011	1

Table 2: OD values and viability percentages of the test item

Test Item	Optical Density (OD)		Viability(%)	Δ%
Test substance	1	0.705	93	18.5
	2	0.846	111
	mean	0.776	102

Applicant's summary and conclusion

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The average test item treated tissue viability was 102 % at 4 hours of exposure. The test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.

Executive summary:

EpiSkin^TM SM test of the test item has been performed to predict its corrosion potential by measurement of its cytotoxic effect, as reflected in the MTT assay according to the OECD Test Guideline No. 431 (29 July 2016) following GLP.

Disks of EPISKIN (two units / incubation time) were treated with test item and incubated for 4 hours (±10 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. The viability of each disk was assessed by incubating the tissues for 3 hours (±15 min) with MTT solution at 37±1°C in an incubator with 5±1 % CO₂ in a ≥ 95% humidified atmosphere and protected from light. The formazan precipitated was then extracted using acidified isopropanol and quantified spectrophotometrically. NaCl (9 g/L saline) and glacial acetic acid treated epidermis were used as negative and positive controls respectively. For each treated tissue viability was expressed as a % relative to negative control. The test item is considered to be non-corrosive to skin, if the mean relative viability after 4 hours of exposure is above or equal 35 % of the negative control.

The test item did not show significantly reduced cell viability in comparison to the negative control after 4 hours of exposure. The average test item treated tissue viability was 102 % at 4 hours of exposure. Thus, the test item treated tissue viability was above 35 % of the mean negative control value after 4 hours of exposure. Positive and negative controls showed the expected cell viability values within acceptable limits. All assay acceptance criteria were met, the experiment was considered to be valid.

The results obtained from this in vitro skin corrosion test, using the EPISKIN model, indicated that the test item reveals no skin corrosion potential under the utilised testing conditions.