[No public or meaningful name is available]

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 26th to October 30th, 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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted June14th, 2019

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis[(1Z)-3-oxo-1,3-diphenyl-1-propen-1-olate]

Cas Number:

21333-45-9

Molecular formula:

C30H22O4.Zn

IUPAC Name:

Zinc bis[(1Z)-3-oxo-1,3-diphenyl-1-propen-1-olate]

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE/Human Epidermis (RHE/S/17)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from multiple donors

Details on test system:

The test system used for the in vitro skin corrosion test was the reconstructed human epidermis (SkinEthicTM RhE) as recommended by the OECD 431 guideline. The SkinEthicTM RhE model consists of normal human keratinocytes cultured for 17-days on a 0.5 cm2 polycarbonate filter insert at the air-liquid interface in a chemically defined growth medium. The cells form a multi-layered, highly differentiated, and stratified epidermis model of the human epidermis that consists of a main basal, suprabasal, spinous, and granular layers and a functional stratum corneum.

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthicTM RhE
- Tissue batch number(s): Batch# 21-RHE-168
- Production date:
- Shipping date:
- Delivery date:
- Date of initiation of testing: October 26, 2021

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
- Observable damage in the tissue due to washing:

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 180 min
- Spectrophotometer: absorbance SynergyH1 microplate reader (BioTek Instruments, USA)
- Wavelength: 570 nm
- Filter: polycarbonate
- Filter bandwidth: 0.5 cm2
- Linear OD range of spectrophotometer: 570 nm

NUMBER OF REPLICATE TISSUES: three replicates were used per exposure for the test item, positive control and negative control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues
- Procedure used to prepare the killed tissues (if applicable): Killed tissues were prepared by incubating the viable tissues at -80 ± 5°C for at least 48 hours. For the treatment of negative control tissues, 40 µL/0.5 cm2 each of sterile distilled water and , for the treatment of freeze-killed positive control tissues, 40 µL/0.5 cm2 of 8N KOH was applied for 60 minutes of exposure time. Nylon mesh was used for the uniform spreading of liquid materials.
- N. of replicates : two
- Method of calculation used: Non-specific MTT reduction calculation (NSMTT)
ODKu: Untreated killed tissues OD
ODKT: Test item treated killed tissues OD
NSMTT = [(ODKT - ODKU) / mean ODNC] x 100

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test material: 20 mg ± 2/0.5 cm2
Negative control (sterile distilled water): 40 µL/0.5 cm2
Positive control (8N KOH): 40 µL/0.5 cm2

Duration of treatment / exposure:

3 and 60 minutes (37 ± 1 °C in 5 ± 1% CO2)

Duration of post-treatment incubation (if applicable):

180 minutes (37±1 °C in 5±1% CO2 in a 95% humidified incubator)

Number of replicates:

3

Test system

Details on study design:

After exposure, tissues were rinsed and dried with cotton buds. Treated tissues were rinsed 20 times in a constant soft stream of 1 mL DPBS at a 5-8 cm distance from the insert to remove all residual test items from the epidermal surface. Mesh (applied on negative control, positive control treated tissues) was removed by washing all tissues. The bottoms of the tissue inserts were dried on sterile absorbent paper (Kim wipes) for 1-2 seconds. The surface of the stratum corneum was gently swept using both ends of a cotton tip (5-6 turns per end).

After rinsing and drying, the MTT test was performed. Tissues were placed in 300 µL of MTT (1.0 mg/mL) solution and incubated for 180 minutes at 37±1 °C in 5±1% CO2 in a 95% humidified incubator. At the end of the MTT test, tissues were observed for MTT reduction.
At the end of the MTT incubation period, the blue formazan salt was extracted by submerging tissues in 1.5 mL isopropanol in a 24-well plate. Tissues were incubated (without shaking) overnight at room temperature, protected from light.

The optical density (OD) of the extracted formazan (200 μL/well of a 96-well plate) was determined in triplicate per tissue using an absorbance SynergyH1 microplate reader (BioTek Instruments, USA) at 570 nm.
The data from blanks, negative controls, positive controls, treated tissues and non-specific MTT reduction were calculated as follows:
The OD mean from all blank replicates for each plate (ODBlank).

Negative Controls (NC)
- The blank corrected value: ODNC = ODNC – ODBlank
- The OD mean per tissue
- The mean OD for all tissues corresponds to 100% viability = mean ODNC
- The mean, standard deviation and percent coefficient of variation were calculated

Positive Control (PC)
- The blank corrected value: ODPC = ODPC – ODBlank
- The OD mean per tissue
- The viability per tissue: %PC = [ODPC / mean ODNC] x 100
- The mean viability for all tissues: Mean PC = Σ %PC / number of tissues
- The mean, standard deviation and percent coefficient of variation were calculated

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean % Viabiliy after 3min

Value:

93.14

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean % Viabiliy after 60 min

Value:

109.14

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No
Assay Acceptance Criteria

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. OD = 1,452; standard deviation = 0.00 (Negative control (NC) acceptance criteria: The NC data of mean optical density were within the OECD Guideline 431 range, i.e., ≥ 0.8 and ≤ 3.0 for each exposure time).
- Acceptance criteria met for positive control: yes. Mean viability = 0.46; standard deviation = 0.08 (Positive control (PC) acceptance criteria: Mean viability of tissues exposed for 60 minutes with the positive control (8N KOH), expressed as % of the negative control were < 15%).
- Test item: yes. Standard deviation = 1.24

Variation: In the range of 20-100% viability and for OD’s ≥ 0.3, the difference in viability between tissue replicates was not > 30%.

Applicant's summary and conclusion

Interpretation of results:

other: The substance shall not be classified as "skin corrosive" according to the CLP Regulation (EC) No. 1272/2008.

Conclusions:

Not corrosive

Executive summary:

The skin irritation potential of the test item on Reconstructed Human Epidermis (RHE SkinEthic) was investigated according to the OECD 431. Tissues were exposed to ZnDBM - dibenzoylmethanate zinc salt (test item) and sterile distilled water (negative control) for 3 minutes at room temperature and 60 minutes at 37 ± 1 °C in 5 ± 1% CO2 using three replicates/time point. Positive control tissues were exposed for 60 minutes at 37 ± 1 °C in 5 ± 1% CO2. Killed negative control (60 minutes), and positive control (60 minutes) treated tissues were also exposed in the same manner using two replicates to correct the direct MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide] reduction obtained by positive control.

The test item, under the conditions of the test, showed a mean cell survival of 109.14% and therefore it resulted to be non skin corrosive as its cell viability is higher than 50%. All the acceptance criteria were passed.