[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:

22 September 2020 - 28 September 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

20 July 2012

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

18 June 2019

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N1,N3‐bis(3‐methylphenyl)‐5‐[(3‐methylphenyl)sulfamoyl]benzene‐1,3‐dicarboxamide

Molecular formula:

C29H27N3O4S

IUPAC Name:

N1,N3‐bis(3‐methylphenyl)‐5‐[(3‐methylphenyl)sulfamoyl]benzene‐1,3‐dicarboxamide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder
- Storage condition: At room temperature protected from light

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: SkinEthic Laboratories, Lyon, France.

Source strain:

other: not applicable.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm^2)
- Tissue batch number(s): 20 EKIN 039

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

TEST FOR DIRECT MTT REDUCTION AND COLOUR INTERFERENCE
The test item has been tested previously for possible direct MTT reduction and color interference in the Skin corrosion test using Epiderm as a skin model (Test Facility Study No. 20256931).

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Microplate reader: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

EVALUATION
The corrected OD (ODc) for each sample or control will be calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw).
ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u+MTT).
The %Viability for each sample and positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100

PREDICTION MODEL / DECISION CRITERIA: see Table 1.

ACCEPTABILITY CRITERIA:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the acceptance limits of OECD439 (lower acceptance limit ≥0.6 and upper acceptance limit ≤ 1.5) and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
d) The %NSCliving should be ≤ 30% relative to the negative control OD.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 15.1 to 24.8 mg (moistened with 5 µL Milli-Q water)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL Phosphate buffered saline (PBS)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 μL 5% (aq) Sodium dodecyl sulfate (SDS)

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

96

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100

Positive controls validity:

valid

Remarks:

14

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: yes
In addition to the normal procedure, three tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.66% of the negative control tissues. The OD of the tissues incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 14%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was ≤ 14%, indicating that the test system functioned properly.

Any other information on results incl. tables

Table 1           
Mean Absorption in the In Vitro Skin Irritation Test with Pergafast 425

	A (OD570)	B (OD570)	C (OD570)	Mean (OD570)		SD
Negative control	0.976	0.906	1.172	1.018	±	0.138
Test item	1.080	0.890	0.972	0.980	±	0.095
Positive control	0.219	0.113	0.100	0.144	±	0.065

OD = optical density

SD = Standard deviation

⁽¹⁾ The test item values are corrected for the non-specific color interference.

Triplicate exposures are indicated by A, B and C.

In this table the values are corrected for background absorption (0.0431). Isopropanol was used to measure the background absorption.

Table 2           
Mean Tissue Viability in the In Vitro Skin Irritation Test with Pergafast 425

	Mean tissue viability (percentage of control)	Standard deviation (percentage)
Negative control	100	14
Test item	96	9.3
Positive control	14	6.4

 

Applicant's summary and conclusion

Interpretation of results:

other: Not a skin irritant. According to Regulation (EC) No. 1272/2008 and its amendments.

Conclusions:

The in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles.
Since the mean relative tissue viability for the substance was above 50%, the substance is considered to be not irritant.

Executive summary:

The substance was tested in triplicate in an in vitro skin irritation test according to OECD TG 439 test guideline and GLP principles. Tissues were exposed to the substance, a negative control (PBS) and a positive control (5% SDS) for 15 minutes. The substance was tested for direct MTT reduction and colour interference.

The solutions did not turn blue / purple, nor a blue / purple precipitate was observed. The OD for the test item solution was >0.08, it was concluded that the test item did interfere with the MTT endpoint. In addition to the normal procedure, three tissues were treated with test item. Instead of MTT solution these tissues were incubated with assay medium. The non-specific color by the test item was 4.66% of the negative control tissues. The OD of the tissues incubated with assay medium was subtracted from the ODs of the test item treated tissues incubated with MTT medium.

Acceptability criteria for the negative control, positive control and variability between measurements were met.
The cell viability of the tissues exposed to the substance was 96% for 15 minutes exposure. Since the mean relative tissue viability for the substance was above 50%, the substance is considered to be not irritant.