N,N'-[(methylimino)dipropane-1,3-diyl]dioleamide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-10-24 to 2017-11-27

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)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-[(methylimino)dipropane-1,3-diyl]dioleamide

EC Number:

256-233-5

EC Name:

N,N'-[(methylimino)dipropane-1,3-diyl]dioleamide

Cas Number:

45320-65-8

Molecular formula:

C43H83N3O2

IUPAC Name:

(9Z)-N-{3-[methyl({3-[(9Z)-octadec-9-enamido]propyl})amino]propyl}octadec-9-enamide

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:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ - 0.38 cm2
- Tissue batch number(s): 17-EKIN-047 (alive tissues) and 17-EKIN-028 (killed tissues)
- Production date:
- Shipping date:
- Delivery date: 21 November 2017
- Date of initiation of testing:

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: each tissue was rinsed with approximately 25mL of sterile D-PBS
- Observable damage in the tissue due to washing:


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of MTT ready-to-use solution (0.3 mg/mL)
- Incubation time: 3 hours at 37 °C, 5% CO2
- Spectrophotometer: no further information
- Wavelength: 595 nm
- Linear OD range of spectrophotometer: yes

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
no data available

NUMBER OF REPLICATE TISSUES: 3 for live tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : yes
- Procedure used to prepare the killed tissues (if applicable): stored at -20°C.
- N. of replicates : 2 for killed tissues
- Method of calculation used:

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

PREDICTION MODEL / DECISION CRITERIA
Mean relative viability ≤ 50% UN GHS Category 2 or 1
Mean relative viability > 50% UN GHS No Category (for member states
that do not adopt optional category 3)

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 20±2mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): D-PBS 20 µL


POSITIVE CONTROL
- Amount(s) applied (volume or weight): 20 µL
- Concentration (if solution): 5% (w/v) SDS

Duration of treatment / exposure:

15±0.5 minutes

Duration of post-treatment incubation (if applicable):

42 ± 1 hour

Number of replicates:

triplicates

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

test item

Value:

92

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:
In a first step, the test item was assayed for the ability of reducing MTT per se. Purple precipitate was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT.

- Colour interference with MTT:
In a second step, the test item was assayed for the ability of colouring water per se.
A colourless, opaque suspension was observed, indicating that the test item has no potential interfering ability.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Amounts of 20 mg or 20 μL of test item or positive control item were used for treatment during the demonstration of laboratory proficiency, in order to uniformly cover the epidermis surface while avoiding an infinite dose (as indicated in OECD 439, § 24).
Results obtained demonstrated that test method has been successfully established.

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

N,N'-[(methylimino)bis(trimethylene)] bis(oleamide) was not irritating in the in vitro skin irritation test.

Executive summary:

The potential of the test item N,N’-[(methylimino)bis(trimethylene)]bis(oleamide) 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(oleamide) (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. Purple precipitate was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se. A colourless, opaque solution was observed, indicating that the test item has no potential interfering ability. Based on these results, an additional control for non specific MTT reduction (NSMTT) was added in the Main Assay, using two killed tissues and compared with negative control performed with alive tissues.

 

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

The positive control caused the expected cell death (5% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.8). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid.

The NSMTT value was 1%, thus only the OD-blank background subtraction was performed. The test item did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 92% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability value equal to 1.7 (lower than 18, as stated in the Study Protocol).

 

Based on the results obtained, the test item N,N’-[(methylimino)bis(trimethylene)] bis(oleamide) is classified as non-irritant to the skin (UN GHS No Category).