Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-04-06 to 2017-05-22
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015-07-28
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)
EC Number:
236-793-7
EC Name:
N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)
Cas Number:
13483-58-4
Molecular formula:
C43H87N3O2
IUPAC Name:
N-{3-[methyl(3-octadecanamidopropyl)amino]propyl}octadecanamide

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
recommended by the OECD testing guideline 439
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
EPISKIN™ - 0.38 cm2, Supplier SkinEthic Laboratories (4, A. Fleming, 69366 Lyon, France), Batch 17-EKIN-020, arrived at RTC on 16 May 2017.
The test system EPISKIN™is a reconstructed human epidermis (RhE) model, which in its overall design (the use of human derived epidermis keratinocytes as cell source and use of representative tissue and cytoarchitecture) closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e., the epidermis.
The test system was shipped onMonday and received on Tuesday. According to the supplier procedure, at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2 mL/well SkinEthic Maintenance Medium. Culture plates were placed in the incubator at 37 °C, 5% CO2 and saturated humidity for approximately 24 hours.

REMOVAL OF TEST SUBSTANCE
At the end of the exposure, each tissue was rinsed with approximately 25 mL of sterile D-PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2 mL/well of maintenance medium.

PRELIMINARY TEST FOR DIRECT MTT REDUCTION
Non-specific reduction of MTT was evaluated as follows: 2 mL of MTT ready-to-use solution (0.3 mg/mL) was incubated with 20 ± 2mg of test item at 37 °C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

PRELIMINARY TEST FOR COLOURING POTENTIAL
The test item's colouring potential was assessed for potential interaction with the test system. 20 ± 2mg of the test item was added to 180 µL of distilled water in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. Suspension appearance was recorded.

MTT STAINING FOR MAIN ASSAY
Each tissue insert was incubated with 2mL/well of MTT ready-to-use solution. Plates were incubated for approximately 3 hours at 37 °C, 5% CO2 and saturated humidity, simulating test conditions. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
Observation of blue or purple appearance of the solution at the end of the incubation time was carried out as follows: The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were preserved for approximately 3 days at 4 °C to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (13000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at 595 nm. Optical Density (OD) values were recorded. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank. In order to ensure the spectrophotometer linear range, on the day of spectrophotometer analysis, an MTT formazan calibration curve was performed and adequate results were obtained.

PREDICTION MODEL / DECISION CRITERIA
A test substance is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test substance is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST ITEM: 20 ± 2 mg/epidermis unit, each one measuring 0.38cm2 (treatment level: 53mg/cm2)
NEGATIVE CONTROL: 20 µL/epidermis unit
POSITIVE CONTROL: 20 µL/epidermis unit
Duration of treatment / exposure:
15 min (at room temperature)
Duration of post-treatment incubation (if applicable):
42 h (at 37 °C, 5% CO2 and saturated humidity)
Number of replicates:
triplicates

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item
Value:
91
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:
In a first pre-test, the test item was assayed for the ability of reducing MTT per se. A white precipitate was noted. No interaction was recorded between the test item and MTT in test conditions similar to those of the Main Assay.
In a second pre-test, the test item was assayed for the ability of colouring water per se. No colouring potential was recorded, thus evaluation by spectral analysis at 595 nm was not performed.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for blank: yes
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Any other information on results incl. tables

The results of the main assay are:

Group

OD

595 nm

Tissue 1

OD

595 nm

Tissue 2

OD

595 nm

Tissue 3

Mean OD

all Tissues

Standard Deviation

Viability [%]

Negative Control

0.6245

0.6505

0.6240

0.6

 

0.02

100

Positive

Control

0.0410

0.0295

0.0465

0.04

0.01

 6

Test item

0.6100

0.5455

0.5770

0.6

0.03

91

The mean Optical Density (OD) of Blank Controls was 0.037, lower than the maximum acceptable value (0.1).

The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability lower or equal to 18), in agreement with guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability.

Positive control results indicated an appropriate cell death with an acceptable relative cell viability (6% of the negative control value). Variability between replicates gave also the expected value (SD of %viability=1.4).

Based on the stated criteria, mean viability, expressed as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.

The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 91%, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability=5.1 lower than 18).

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was not irritating in the in vitro skin irritation test.
Executive summary:

The potential of the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) to be irritant to the skin was investigated according to OECD Guideline 439 (28 July 2015) through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.

The test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) (100% a.i.) was tested for its ability to impair cell viability. The test item was applied as supplied by the sponsor in three tissue replicates at the treatment level of 20 ± 2 mg/epidermis unit, each one measuring 0.38cm2(treatment level: 53mg/cm2).

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. No interaction was recorded between the test item and MTT in test conditions similar to those of theMain Assay. In a second step, the test item was assayed for the ability of colouring water per se. No colouring potential was recorded. Based on these results, no additional control was added in theMain Assay.

After an exposure period of 15 minutes at room temperature, each tissue was rinsed with approximately 25 mL of sterile D-PBS to remove any residual test material. Subsequently, the tissues were incubated for 42 h at 37 °C. Cytotoxicity (irritancy) was expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

The positive controls [sodium dodecyl sulphate 5% (w/v) solution in water] and the negative controls [Dulbecco’s phosphate buffered saline (D-PBS)] were concurrently tested, in the same number of replicates and test conditions as the test item. The controls were tested at the treatment level of 20 µL/epidermis unit and gave responses that were within the acceptance criteria and as such demonstrated the validity of the study.

The test item did not induce cell death in any replicate. When compared to the negative control, the mean cell viability of the tissues treated with the test item was 91% after the blank subtraction.

Based on the results obtained, and since the mean cell viability of the treated tissues was above 50%, the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) is classified as not irritant to the skin (UN GHS No Category).

Categories Display