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

Endpoint summary

Description of key information

N,N’-[(methylimino)bis(trimethylene)]bis(oleamide) is not irritating to skin and the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-09-03 to 2019-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular™ Eye Irritation Test (EIT) predicts the acute eye hazard potential of chemicals by measurement of its tissue damage caused by cytotoxic effects in the reconstructed human cornea-like tissue model.
Within a tired testing strategy, the EpiOcular™ EIT is used as areplacement of the in vivo Draize Eye Irritation Test.
It is utilized for the classification and labelling of ehernieals concerning their eye hazard potential.
The EpiOcular™ EIT can be used to identify ehernieals that do not require classification for eye irritation or serious eye damage according to the UN GHS classification system.
A limitation of this guideline is that it neither allows discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1), nor between eye irritants (optional Category 2A) and mild eye irritants (optional Category 2B). For these purposes, further testing with other suitable test methods is required.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
The EpiOcular™ tissue consists of normal, human-derived keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea.
It consists of highly organized basal cells. These cells are not transformed or transfected with genes to induce an extended life span.
The EpiOcular™ tissues are cultured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells.
Certificate of analysis is available for tissues, confirming cell source, absence of biological contaminants and analysis of quality and functionality.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test substance:
50 mg
Positive and negative control: 50 µl for each

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

duplicate

Details on study design:

Details of the test procedure used

- RhCE tissue construct used, including batch number
EplOcular™Tissue, MatTek Lot: 27029

- Doses of test chemical and control substances used : 50 mg of test substance, each 50µL of negative and positive control

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable)
After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and ~ 95 % relative humidity.
At the end of exposure time, the inserts were removed from the plates in one-minuteintervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS.
Then, the tissues were immediately transferred into 5 mL of assay medium in pre-Iabelled 12-well plate for 25 minutes post soak at room temperature.
For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % C02 and ~ 95 % relative humidity.

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable)
The test item was tested for the ability of direct MTT reduction.
The colour of the MTT turned blue/purple, therefore the test item is presumed to have reduced the MTT.

- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable)
duplicate tissues were used

- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer)
The wavelength used in the test is 570 nm.
Quarterly, a calibration of the microplate reader by using a calibration plate provided by the manufacturer is performed in order to ensure the linearity of photometric measurements.
The plate photometer has an indication range for optical density (OD) between 0.0 and 3.3 and a linear range of 0.1 - 2.5. Only measurements made within the linear range are used for the quantitative calculations.

- Description of the method used to quantify MTT formazan
Microtiter plate photometer, Anthos Reader 2010 Flexi, Anthos Microsysteme GmbH

- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria
Values for negative control and for positive control were within the range of historical data of the test facility.
All validity criteria were met, therefore the experiment is considered valid.

- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals
The validity of the EpiOcular™ test at test lab was demonstrated in a proficiency study.
For this purpose 15 proficiency ehernieals (indicated by the OECD 492 guideline) were tested. All of the 15 proficiency ehernieals were correctly categorized.

- Positive and negative control means and acceptance ranges based on historical data
% mean relative viability of positive control: < 50 % of negative control

- Acceptable variability between tissue replicates for positive and negative controls
< 20 % (test: 0.3% (negative control); 3.3% (positive control)

- Acceptable variability between tissue replicates for the test chemical
< 20 % (test: 1.1 % (test item)

Irritation parameter:

other: % Tissue Viability Mean

Run / experiment:

Experiment 1

Value:

98.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item did not show any colour development in isopropanol.
The result of this pre-test was that the colour of the MTT turned blue/purple, therefore the test item has shown its ability to reduce MTT.
Therefore, it was necessary to perform an additional test with freeze killed tissues that possess no metabolic activity but absorb and bind the test item like viable tissues.
Results showed that the MTT reduction by the test item did not influence the result of the main test.

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 2.0 (> 0.8 and < 2.5).
The positive control induced a decrease in tissue viability as compared to the negative control to 28.0%.
The variation within the replicates of the controls and the test item was acceptable (< 20%).
For these reasons, the result of the test is considered valid.

Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

 

	Measurement	Negative Control	Positive Control	Test substance
Tissue 1	1	1.989	0.545	2.005
	2	2.044	0.572	1.935
Tissue 2	1	2.025	0.623	1.987
	2	1.997	0.624	1.998

 

 

Absorbance Values of Negative Control and Test Item with Freeze-killed Tissues (OD at 570 nm)

 

	Measurement	Negative Control	Test substance
Tissue 1	1	0.078	0.066
	2	0.076	0.063
Tissue 2	1	-	0.073
	2	-	0.073

 

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test, N,N'-[(methylimino)bis(trimethylene)]bis(oleamide) is considered non-eye irritant in the EpiOcular™ Eye Irritation Test.

Executive summary:

An in-vitro eye irritation study was conducted using the EpiOcular™ Reconstructed human Cornea-like Epithelium (RhCE) test method according to OECD guideline 492 with the test substance N,N'-[(methylimino)bis(trimethylene)]bis(oleamide).

 

Since in the pre-test, potential MTT reduction by the test item was observed, an additional test was performed to correct photometrical measurement values due to direct MTT reduction by the test item. The results of the additional test showed that the MTT reduction by the test item did not influence the result of the main test.

 

The test item N,N'-[(methylimino)bis(trimethylene)]bis(oleamide) was applied to a three dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

After treatment, the test item was rinsed off the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

 

Demineralised water was used as negative control and methyl acetate was used as positive control.

After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD > 0.8 and <2.5, the OD was 2.0.

The positive control showed clear eye irritating effects, the mean value of the relative tissue viability was 28.0% (< 50%).

The variation within tissue replicates of the controls and the test item was acceptable(< 20%).

 

After treatment with the test item, the mean value of relative tissue viability was 98.3%. This value is well above the threshold for eye irritation potential (≤ 60%).

Test items that induce values above the threshold are considered non-eye irritant.

Under the conditions of the test, N,N'[(methylimino)bis(trimethylene)]bis(oleamide) is considered non-eye irritant in the EpiOcular™ Eye Irritation Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin:

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 did not induce cell death in any replicate, the mean cell viability after the blank subtraction was 92% when compared to the negative control.

The validity criteria were fulfilled, the assay was regarded as valid.

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).

Eye:

Data from an OECD 437 guideline study withN,N’-[(methylimino)bis(trimethylene)]bis(oleamide) are available. Under the conditions of this study, the test item N,N'-[(methylimino)bis(trimethylene)]bis(oleamide) showed effects on the cornea of the bovine eye.

The calculated IVIS (in vitro irritancy score) is 15.70.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category.

Thus, no prediction can be made based on study results.

Therefore an in-vitro eye irritation study was conducted using the EpiOcular™ Reconstructed human Cornea-like Epithelium (RhCE) test method according to OECD guideline 492.

After treatment with the test item, the mean value of relative tissue viability was 98.3%. This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

The validity criteria were fulfilled, the assay was regarded as valid.

Under the conditions of the test, N,N'[(methylimino)bis(trimethylene)]bis(oleamide) is considered non-eye irritant in the EpiOcular™ Eye Irritation Test.

Justification for classification or non-classification

N,N’-[(methylimino)bis(trimethylene)]bis(oleamide) does not need to be classified for skin or eye irritation according to the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008), based on data from a study according to OECD Guideline 439 for skin irritation and data from a study according to OECD Guideline 492 for eye irritation.