N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

Endpoint summary

Description of key information

OECD 431 (2017): Non- Corrosive to the skin

OECD 439 (2017): Non-irritant to Skin.

OECD 437 (2017): Non- Corrosive to the eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2017 - 27 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (in the dark*)
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Not applicable - test item applied undiluted.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, applied as supplied.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: n/a

Test system:

human skin model

Source species:

human

Cell type:

other: epidermal keratinocytes

Cell source:

other: MatTek Corporation, Bratislava, Slovakia

Source strain:

other: N/A

Details on animal used as source of test system:

N/A

Justification for test system used:

N/A

Vehicle:

unchanged (no vehicle)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): approximately 25 mg (~ 39 mg/cm2 according to guideline) of the test item were applied to the tissues, wetted with 25 μL DPBS prior to application.
- Concentration (if solution): See above.

VEHICLE
- Amount(s) applied (volume or weight with unit): See above.
- Concentration (if solution): See above.
- Lot/batch no. (if required): 062717
- Purity: Not reported.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5 % w/v

Duration of treatment / exposure:

60 mins

Duration of post-treatment incubation (if applicable):

42 hours and 20 minutes

Number of replicates:

3

Type of coverage:

other: Not applicable

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 86.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 2. Results after treatment with

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

and the controls.

Dose Group	Tissue No.	Absor-bance 570 nm Well 1	Absor-bance 570 nm Well 2	Absor-bance 570 nm Well 3	Mean Absor-bance of 3 Wells	Mean Absor-bance of three wells blank corrected	Mean Absor-bance of 3 tissues after blank correction	Rel. Absor-bance [%] Tissue 1, 2 + 3*	Relative Standard Deviation [%]	Mean Rel. Absorbance [%]**
Blank		0.039	0.038	0.044	0.040	0.000
Negative Control	1	1.396	1.399	1.398	1.398	1.357	1.495	90.8	8.1	100.0
	2	1.580	1.593	1.581	1.584	1.544		103.3
	3	1.631	1.626	1.612	1.623	1.583		105.9
Positive Control	1	0.103	0.105	0.104	0.104	0.064	0.063	4.3	3.6	4.2
	2	0.104	0.105	0.105	0.104	0.064		4.3
	3	0.100	0.101	0.100	0.100	0.060		4.0
Test Item	1	1.383	1.397	1.384	1.388	1.348	1.288	90.2	5.1	86.2
	2	1.255	1.273	1.249	1.259	1.219		81.5
	3	1.348	1.327	1.342	1.339	1.299		86.9

OD= Optical Density

SD= Standard deviation

*= The mean viability of the negative control tissues is set at 100 %

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study the test substance is not considered to be irritant to the skin and does not meet the criteria for classification in accordance with UN GHS and EU CLP regulation.

Executive summary:

OECD 439 (2017) -The skin irritation potential of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP.

Triplicate tissues were exposed to the test item for 60 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a spectrophotometer.

Mean viability of tissues exposed to the test substance after 60 minutes were 86.2 %. It was considered unnecessary to perform IL-1a analysis as the results of the MTT test were unequivocal. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance,

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

is not irritant to skin according to UN GHS and EU CLP regulation.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 June - 22 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with GLP. All guideline validity criteria were met.

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no

Test system:

human skin model

Source species:

human

Cell type:

other: Reconstructed human epidermis

Cell source:

other: MatTek model kit - MatTek Corporation (Bratislava, Slovakia).

Justification for test system used:

Guideline specific test system.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: MatTek
- Tissue batch number(s): 25828
- Production date: not reported
- Shipping date: not reported
- Delivery date: 04 July 2017
- Date of initiation of testing: not reported

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by gently shaking the tissue insert and blotting the bottom of the tissue insert with blotting paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: No
- Modifications to validated SOP: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)
- Wavelength: 570 nm
- Filter: Not reported
- Filter bandwidth: Not reported
- Linear OD range of spectrophotometer: Not reported

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: A comparison of laboratory historical data for negative and positive controls was made to verify the functioning of the test system.
- Barrier function: Not reported
- Morphology: Not reported
- Contamination: Not reported
- Reproducibility: Coefficient of Variation between tissue replicates was ≤ 30%.

NUMBER OF REPLICATE TISSUES: 2

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item did not reduce MTT.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 μL of deionised water and 25 ± 2mg (39.7 mg/cm2) of the test item were applied onto the surface of duplicate EpiDermTM tissue.
- Concentration (if solution): unchanged - see above

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): n/a

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3 and 60 mins

Duration of post-treatment incubation (if applicable):

3 h MTT incubation followed by overnight isopropanol extraction

Number of replicates:

2 per test group

Type of coverage:

other: Not applicable

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

98.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes exposure

Value:

98

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:
- Visible damage on test system: No
- Direct-MTT reduction: No
- Colour interference with MTT: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD562 for the negative control treated tissues was 1.658 for the 3-Minute exposure period and 1.586 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.5% relative to the negative control following the 60-Minute exposure period. Thus, confirming the validity of the test system and the specific batch of tissue models.
- Acceptance criteria met for variability between replicate measurements: In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: All values for the positive and negative control were within the historical ranges achieved by the testing facility in the previous twelve months, thus confirming the acceptable functioning of the test system.

Table 1. Results after treatment with

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamid

and the controls.

Dose Group	Exposure Interval	Absorbance Well 1 (Tissue 1/2)	Absorbance Well 2 (Tissue 1/2)	Absorbance Well 3 (Tissue 1/2)	Mean Absorbance (Tissue 1/2)	Mean Absorbance (OD) of 3 Wells minus Blank	Mean Absorbance (OD) of 2 Tissues	Rel. Absorbance [%]*	CV [%]	Mean Rel. Absorbance [%]**
Blank	3 minutes	0.037	0.037	0.037	0.037	0.000
Negative Control		1.746	1.675	1.716	1.712	1.675	1.658	101.0	1.5	100.0
		1.683	1.673	1.677	1.678	1.641		99.0
Positive Control		0.477	0.465	0.453	0.465	0.428	0.430	25.8	0.6	25.9
		0.474	0.464	0.467	0.468	0.431		26.0
Test Item		1.636	1.641	1.647	1.641	1.605	1.641	96.8	3.0	98.9
		1.706	1.716	1.713	1.712	1.676		101.1
Blank	1 Hour	0.036	0.036	0.036	0.036	0.000
Negative Control		1.672	1.581	1.633	1.629	1.593	1.586	100.4	0.6	100.0
		1.603	1.607	1.638	1.616	1.580		99.6
Positive Control		0.109	0.111	0.111	0.111	0.075	0.087	4.7	20.6	5.5
		0.147	0.136	0.125	0.136	0.100		6.3
Test Item		1.575	1.569	1.567	1.570	1.536	1.555	96.8	1.7	98.0
		1.600	1.628	1.597	1.608	1.574		99.2

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin under the conditions of the test.

Executive summary:

OECD 431 (2017) - The skin corrosivity potential of reaction mass of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP.

 

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a Versamax microplate reader.

Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 98.9 and 98.0 %, respectively. The quality criteria required for acceptance of the results was met.

Under the conditions of this study the test substance is not considered to be corrosive to the skin.

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:

28 July 2017 - 31 January 2018

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

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (in the dark*)
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
- Preliminary purification step (if any): no
- Final dilution of a dissolved solid, stock liquid or gel: undiluted
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: no

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Freshly isolated bovine cornea obtained from AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Number of animals: Not reported
- Characteristics of donor animals (e.g. age, sex, weight): At least 9-month-old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyes were removed after slaughter, the isolated eyes were stored in HBSS containing 1% (v/v) Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) in the cooled slaughter-house until transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: Same day
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: Penicillin/Streptomycin (100 units/mL penicillin and 100 µg/mL streptomycin) added to Styrofoam box during transportation.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL (of suspension)
- Concentration (if solution): The test item was tested as a 20% suspension (w/v) in saline

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

Duration of treatment / exposure:

240 minutes

Observation period (in vivo):

n/a

Duration of post- treatment incubation (in vitro):

90 minutes

Number of animals or in vitro replicates:

3

Details on study design:

APPLICATION DOSE AND EXPOSURE TIME

0.75 mL applied to each cornea and incubated at 32 ± 1 °C in the water-bath for 240 minutes.

TREATMENT METHOD

Closed chamber

POST-INCUBATION PERIOD

Following rinsing, the corneas were incubated (horizontally) for 240 minutes after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the Corneae were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red
- POST-EXPOSURE INCUBATION: Following rinsing, the corneas were incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated again in a horizontal position for 90 minutes in a water-bath at 32 ± 1 °C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacitometer
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of spectrophotometer (OD490)
- Others (e.g, pertinent visual observations, histopathology): (please specify) N/A

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: As described in OECD 437.

Irritation parameter:

in vitro irritation score

Value:

ca. 0.48

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 1. Results after 240 Minutes Treatment Time

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposedin vitroIrritancy Score
		Mean		Mean
Negative Control	0	0.33	0.065	0.070	0.98	1.38	Not categorized
	0		0.068		1.02
	1		0.077		2.16
Positive Control	133.67*		0.006*		133.76	127.81	Category 1
	122.67*		0.011*		122.83
	126.67*		0.011*		126.83
Test Item	-0.33*		0.041*		0.28	0.48	Not categorized
	0.67*		0.033*		1.16
	-0.33*		0.022*		0.00

*corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that under the condition of this study, the test item, produced an IVIS score of 0.48 and therefore, the test item is not categorised according to UN GHS/EU CLP regulation.

Executive summary:

OECD 437 (2017) -The Bovine Corneal Opacity and Permeability (BCOP) test was conducted using  

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

 in accordance with 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" (2013).

Undiluted test item of 0.75 mL volume was applied evenly to the surface of three corneas before being washed off with media solution after 10 minute test item contact time. A negative and positive control group, each containing 3 corneas, were also prepared using a similar concentration and volume of the test item .

Measurements for corneal opacity were made after 240 minutes incubation in the horizontal position with fresh media. Measurements for corneal permeability were made following 90  min incubation in the vertical position with sodium fluorescein.

Corneal opacity and corneal permeability media solutions were analysed by a spectrophotometer at 490 nanometers (OD490). The mean corrected opacity reading and permeability readings for the test item were 0.44 and 0.032, resulting in a mean In Vitro Irritation Score (IVIS) of 0.48.

It was concluded that under the condition of this study, the test item, produced an IVIS score of 0.48 and therefore, the test item is not categorised according to UN GHS/EU CLP regulation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation

OECD 431 (2017) - The skin corrosivity potential of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 431 and GLP. Duplicate tissues were treated with the test item for exposure periods of 3 and 60 mins. At the end of the exposure period the test item was rinsed from each tissue before being loaded with MTT. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction. After extraction, each tissue was pierced and the extraction solution aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a Versamax microplate reader. Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 98.9 and 98.0%, respectively. The quality criteria required for acceptance of the results was met. Under the conditions of this study the test substance is not considered to be corrosive to the skin.

OECD 439 (2018) - The skin irritation potential of

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

was assessed using an EpiDerm Reconstructed Human Epidermis (RHE) Model Kit in accordance with OECD guidance 439 and GLP. Triplicate tissues were exposed to the test item for 15 minutes. At the end of the exposure period the test item was rinsed and the tissues incubated for a further 42 h in the presence of maintenance solution which would be used for possible inflammatory mediator determination. Each tissue was then loaded with MTT. After incubation and extraction, the solutions were aliquoted for absorbance measurements. Absorbency at 570 nm of each well was measured using a Versamax microplate reader. Mean viability of tissues exposed to the test substance after 15 minutes were 86.2 %. The quality criteria required for acceptance of the results was met. Under the conditions of this study the test substance is not considered to be irritant to the skin.

As both the in vitro skin corrosion and skin irritation studies produced negative results, no further testing is required in accordance with Annex VII of REACH. The subtance can be concluded as non-corrosive and non-irritating to skin.

Eye Irritation

OECD 437 (2018) - The study was performed to evaluate the possible corrosivity or severe irritancy potential of the test item,

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

as measured by its ability to induce toxicity in fresh bovine corneae. The anterior compartment received the test item suspension or the negative or positive controls at a volume of 0.75 mL each on the surface of the corneae, respectively. The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. After 240 mins incubation (post rinsing) the opacity measurements permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. All control showed expected results within the historical control data. The calculated mean in vitro irritancy score was 0.48 and was concluded that the test item

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

did not cause an increase of the corneal opacity or permeability. Under condition of this study, the test item is not categorized according to UN GHS/EU CLP regulation.

Based on the results of the in vitro eye corrosion/irritation study conducted according to OECD 437, no local toxicity to the eye can be concluded in accordance with Annex VII of REACH.

Justification for classification or non-classification

The substance does not meet the criteria for classification under local toxicity in accordance with GHS and Regulation (EC) No 1272/2008 (CLP).