N,N'-[(methylimino)bis(trimethylene)]bis(stearamide)

Endpoint summary

Description of key information

N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was proven to be not irritating in an in vitro skin irritation study, according to OECD Guideline 439.

In an in vitro eye irritation BCOP assay using fresh bovine corneae according to OECD guideline 437, N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was proven to be not irritating to the eye as well.

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:

2017-04-06 to 2017-05-22

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)

Version / remarks:

2015-07-28

Deviations:

no

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:

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

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

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).

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).

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:

2017-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2013-07-26

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

2010-12-08

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Source OF fresh bovine corneae are animals is cattle (species Bos primigenius Taurus).
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. The eyes were transported within 1 hour to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a cooled container. Then, the corneae were dissected and incubated in medium at 32 ± 1 °C in an incubation chamber for 1 h.

PREPARATION
Clean and sterile cornea holders were kept in the in-cubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bi-carbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.

After the arrival of the corneae, they were examined and only corneas which were free from damages were used. The corneae were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneae showed tissue damage; therefore, all corneae were used. For each treatment group (negative control solution, test item and positive con-trol solution), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative control solution, positive control solution and a defined amount of test item were applied to each replicate.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The test item was applied onto the epithelium so that the cornea was completely and homogeneously covered.
Replicate Amount of neat pestled test item
1 535.8 mg
2 563.3 mg
3 560.6 mg

Duration of treatment / exposure:

Exposure time of the test item and the controls on the corneas was 4 hours at 32 ± 1 °C.

Observation period (in vivo):

Exposure time of the test item and the controls on the corneas was 4 hours at 32 ± 1°C.

Number of animals or in vitro replicates:

3 corneae per group (test item, negative control, positive control)

Details on study design:

REMOVAL OF TEST ITEM
After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.
The cMEM without phenol red was then removed from the front chamber, and 1 mL so-dium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the perme-ability of the cornea was measured as optical density of the liquid with a spectrophotome-ter at 492 nm.

SCORING SYSTEM
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference (opacity of treated corneae - opacity of negative control) + (15 x corrected OD492 value)

SPECIFIC EQUIPMENT
Opacitometer BASF OP 3.0 (BASF)
Cornea holders (Duratec Analysentechnik GmbH)

VALIDITY
According to the OECD guideline 437 (26 July 2013), the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The negative control has to show an IVIS ≤ 3.

Irritation parameter:

in vitro irritation score

Value:

0.7

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:

VALIDITY
Parameter Criterion Found Assessment
IVIS of negative control ≤ 3 0.55 OK
HBSS-solution
IVIS of positive control 2.75 - 166.63 108.80 OK
20% imidazole solution

Values for negative and positive controls were within the range of historical data of the test facility.
Therefore, the test system was acceptable and valid.

Results afer 4 h incubation

Test group	Opacity difference corrected	Permeability at 492 nm*	In vitro Irritation Score	Mean in vitro Irritation Score	Relative SD of IVIS
Negative control	-	-	0.58	0.55	25.02 %
	-	-	0.40
	-	-	0.68
Positive control	91.71	2.3362	126.75	108.80	16.75 %
	68.35	1.4646	90.32
	75.20	2.2746	109.32
Test substance	0.15	0.0021	0.12	0.70	76.33 %
	0.39	0.0286	0.82
	0.23	0.0626	1.17

- The mean opacity difference of the negative control was 0.35.

- For the blank, the mean optical density at 492 nm was 0.038.

- The values for Permeability at 492 nm were all corrected by subtracting the mean blank value.

- For the negative control, the mean Permeability at 492 nm was 0.0138.

- For the positive control, the all values for the Permeability at 492 nm were obtained by measurement of a fivefold diluted solution and multiplication of the absorbances with factor 5.

- The high relative standard deviation of the IVIS of test item is due to mathematical reasons, as the respective means are very small.

COMPARISON WITH HISTORICAL DATA

In the following table, the means of the negative control and positive control of all experiments which were performed at the test facility up to 11. May 2017 are stated and compared with the values which were found in this study.

	Negative Control	Positive Control
Mean IVIS	1.99	119.69
Standard Deviation IVIS	1.03	23.47
Range of IVIS (validity)	≤ 3	72.75 – 166.63
Study 17031603G850	0.55	108.80
Mean Opacity	1.73	82.38
Standard Deviation Opacity	1.01	18.45
Range of Opacity	-1.86 – 4.08	42.92 – 133.11
Study 17031603G850	0.35	78.42
Mean Permeability	0.02	2.52
Standard Deviation Permeability	0.02	1.02
Range of Permeability	-0.01 – 0.10	0.75 – 5.89
Study 17031603G850	0.01	2.03

Interpretation of results:

GHS criteria not met

Conclusions:

In the Bovine Corneal Opacity and Permeability Test, the test item N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) has an IVIS of 0.7.
This result lies within the range of IVIS ≤ 3 and, according to OECD Guideline 437 (26 July 2013), this substance requires no classification for eye irritation or serious eye damage.

Executive summary:

An in-vitro eye irritation study in accordance with OECD Guideline 437, with N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was performed using the Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular corrosives and Severe Irritants.

Bovine corneas, collected from slaughtered cattle which were between 12 and 60 months old, were used for the test.

The test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) was tested pure. It was pestled and brought onto the cornea of a bovine eye, which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for4 hours at 32 ± 1 °C. After removal of the test item opacity and permeability values were measured.

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 0.55.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean.

The calculated IVIS (in vitro irritancy score) is 108.80.

 

Under the conditions of this study, the test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0.70.

Since the IVIS of N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) has a value of ≤ 3, according to OECD Guideline 437 (26 July 2013), this substance requires no classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

An in-vitro skin irritation study in accordance with OECD Guideline 439, with N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was performed using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.

In a preliminary test the ability of the test item to reducing MTT per se and ability of colouring water per se was investigated. No interaction of the test substance with MTT neither acolouringpotential was recorded.

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).

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).

Eye irritation:

An in-vitro eye irritation study in accordance with OECD Guideline 437, with N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) was performed using the Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular corrosives and Severe Irritants.

Bovine corneas, collected from slaughtered cattle which were between 12 and 60 months old, were used for the test. The test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) was tested pure. It was pestled and brought onto the cornea of a bovine eye, which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for4 hours at 32 ± 1 °C. After removal of the test item opacity and permeability values were measured.

HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 0.55.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and falls within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 108.80.

Under the conditions of this study, the test item N,N'-[(methylimino)bis(trimethylene)]bis(stearamide) showed no effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 0.70.

Since the IVIS of N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) has a value of ≤ 3, according to OECD Guideline 437 (26 July 2013), this substance requires no classification for eye irritation or serious eye damage.

Conclusion:

N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not need to be classified for skin and eye irritation according to the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008)

 

 

Justification for classification or non-classification

N,N’-[(methylimino)bis-(trimethylene)]bis-(stearamide) does not need to be classified for skin and eye irritation according to the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008)