N-(2-hydroxyethyl)dodecanamide

Endpoint summary

Description of key information

In vitro skin corrosion: Non-corrosive; OECD 431; C Spohr. (2017)

In vitro skin irritation: Not irritating; OECD 439;  C Spohr. (2018)

In vitro eye damage: No prediction could be made; OECD 437;  C Spohr. (2018)

In vitro eye irritation:  Irritating; OECD 492;  C Spohr. (2018)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 August 2017 - 01 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 28 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

23 July 2009

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
- 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 (82105 Bratislava, Slovakia)

Source strain:

other:

Details on animal used as source of test system:

n/a

Justification for test system used:

n/a

Vehicle:

unchanged (no vehicle)

Details on test system:

n/a

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, and spread to match the surface of the tissue for a complete treatment time of 60 minutes.
- 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): Not reported.
- 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 minutes.

Duration of post-treatment incubation (if applicable):

42 hours and 20 minutes

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Value:

ca. 101.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Table 2. Results after treatment with N-(2-hydroxyethyl) dodecanamide  and the controls.

Dose Group	Tissue No.	Absorbance 570 nm Well 1	Absorbance 570 nm Well 2	Absorbance 570 nm Well 3	Mean Absorbance of 3 Wells	Mean Absorbance of three wells blank corrected	Mean Absorbance of 3 tissues after blank correction	Rel. Absorbance [%] Tissue 1, 2 + 3	Relative Standard Deviation [%]	Mean Rel. Absorbance [%]
Blank		0.038	0.039	0.037	0.038	0.000
Negative Control	1	1.240	1.333	1.344	1.306	1.268	1.277	99.3	3.3	100.0
	2	1.357	1.369	1.359	1.361	1.323		103.6
	3	1.278	1.273	1.283	1.278	1.240		97.1
Positive Control	1	0.109	0.109	0.100	0.106	0.068	0.064	5.3	0.3	5.0
	2	0.110	0.100	0.098	0.103	0.065		5.1
	3	0.100	0.099	0.096	0.098	0.060		4.7
Test Item	1	1.212	1.300	1.315	1.276	1.238	1.301	96.9	4.9	101.9
	2	1.398	1.395	1.406	1.400	1.362		106.6
	3	1.326	1.355	1.347	1.343	1.305		102.2

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-hydroxyethyl) dodecanamide  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 101.9 %. 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-hydroxyethyl) dodecanamide  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:

02 June 2017 - 07 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

July 29 2016

Deviations:

yes

Remarks:

Instead of at room temperature, the formazan salt was extracted overnight in the refrigerator

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

30 May 2008

Deviations:

yes

Remarks:

Instead of at room temperature, the formazan salt was extracted overnight in the refrigerator

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.
- 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 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 ± 2 mg (39.7 mg/cm2 according to guideline) of the test item were applied onto the surface of duplicate EpiDermTM tissue. The test item was wetted with 25 μL of deionised water.
- Concentration (if solution): unchanged - See above.

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

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 isopropnaol extraction

Number of replicates:

2 per test group

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

ca. 96.6

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:

ca. 83.1

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.

Exposure Period	Percentage Viability^*
	Negative Control^§	Positive Control	Test Item
3 minutes	100	25.9	96.6
60 minutes	100	5.50	83.1

*mean of 2 replicates

§negative controls set to 100 %

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 N-(2-hydroxyethyl)dodecanamide 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 96.6 and 83.1 %, 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

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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:

9th October 2017

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:

9.12.2010

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.
- 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

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 Schlachth of 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 (as a 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): See above
- Concentration (if solution): 0.9% NaCl in deionised water
- Lot/batch no. (if required): Not reported.
- Purity: Not reported.

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

Run / experiment:

240 minutes

Value:

ca. 7.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not classified as serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for hazard classification cannot be made.

Executive summary:

In an OCED 437 (2018),  fresh bovine corneae were exposed to the test item, N-(2-hydroxyethyl)dodecanamide  for a duration of up 240 minutes. The damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability. 


The study was performed three times since the negative control did not meet the acceptance criteria in the first run (was above the historical established boundaries) and in the second experiment, the positive control did not meet the acceptance criteria since the IVIS was higher than two Standard Deviations of the Mean IVIS of the positive control of historical established boundaries. Both experiments were declared as invalid and will not be reported. A third experiment was performed. This report reflects the data of the third experiment.

After a first opacity measurement of the fresh bovine corneae (t₀), the 20% (w/v) suspension in saline of the test item N-(2-hydroxyethyl)dodecanamide, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t₂₄₀).

After 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.

For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.24).

The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 107.58) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)).

Relative to the negative control, the test item N-(2-hydroxyethyl)dodecanamide caused an increase of the corneal opacity. The calculated mean IVIS was 7.70 (threshold for serious eye damage: IVIS> 55).According to OECD 437, no prediction for the damage hazard of the test item to the eye can be made.

In conclusion, according to the current study and under the experimental conditions reported, the test item is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for hazard classification cannot be made.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

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)

Version / remarks:

9th October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

ADIOLABELLING 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.
- 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

Species:

human

Details on test animals or tissues and environmental conditions:

The EpiOcular™ tissue consists of normal, human-derived epidermal 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 which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) were cultured on specially prepared cell culture inserts (MILLICELL, 10 mm ).
EpiOcular™ tissues were shipped at 2 - 8 °C on medium-supplemented agarose gels in a 24-well plate. On day of receipt (27 February 2018) of the EpiOcular™ tissues, the equilibration step (15 minutes at room temperature in the 24-well shipping container) started. 1.0 mL of the medium was aliquoted into the appropriate wells of pre-labelled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with 70% isopropanol- or ethanol-soaked tissue paper. The sterile gauze was removed and each tissue was inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping containers using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for one hour in the Assay Medium. After one hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (about 16 - 24 hours).

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg
- Concentration (if solution): undiluted

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

Irritation parameter:

other: cornea viability

Run / experiment:

6 hours incubation

Value:

ca. 43.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Table 1.       Results after treatment for 6 hours with the test item and the controls.

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]
Blank		0.037	0.036	0.036
Negative Control	1	1.895	1.833	1.864	1.828	1.859	98.3	3.3	100.0
	2	1.965	1.887	1.926	1.890		101.7
Positive Control	1	0.715	0.683	0.699	0.662	0.718	35.6	6.0	38.6
	2	0.828	0.794	0.811	0.774		41.7
Test Item	1	0.838	0.802	0.820	0.784	0.824	42.2	4.3	43.1
	2	0.905	0.896	0.900	0.864		46.5
Blank		0.037	0.036	0.036
Negative Control Freeze killed Tissues	1	0.076	0.074	0.075	0.038	0.038	2.1	0.0	2.0
	2	0.074	0.074	0.074	0.038		2.0
Test Item Freeze killed Tissues	1	0.095	0.093	0.094	0.057	0.060	3.1	0.3	3.2
	2	0.100	0.098	0.099	0.063		3.4

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

Executive summary:

In an OECD 492 (2018) study, the test item, N-(2-hydroxyethyl)dodecanamide applied to human Cornea in order to assess it irritation potential.

The test item’s intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with viable tissues (without MTT addition) did not have to be performed.

The test item proved to be an MTT reducer in the MTT pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.

Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues.

Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.

The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

A reduction in tissue viability was observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (determined value for the test item 43.1%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, thet test item possesses an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin Irritation/Corrosion

OECD 431 (2017) - The skin corrosivity potential of N-(2-hydroxyethyl)dodecanamide 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 Versmax microplate reader. Mean viability of tissues exposed to the test substance after 3 and 60 minutes were 96.6 and 83.1 %, 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 (2017) - The skin irritation potential of N-(2-hydroxyethyl) dodecanamide 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 101.9 %. 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-hydroxyethyl) dodecanamide  is not irritant to skin according to UN GHS and EU CLP regulation.

Eye Irritation

OCED 437 (2018) - Fresh bovine corneae were exposed to the test item, N-(2-hydroxyethyl)dodecanamide  for a duration of up to 240 minutes. The damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability. 
 The study was performed three times since the negative control did not meet the acceptance criteria in the first run (was above the historical established boundaries) and in the second experiment, the positive control did not meet the acceptance criteria since the IVIS was higher than two Standard Deviations of the Mean IVIS of the positive control of historical established boundaries. Both experiments were declared as invalid and will not be reported. A third experiment was performed. This report reflects the data of the third experiment. After a first opacity measurement of the fresh bovine corneae (t0), the 20% (w/v) suspension in saline of the test item N-(2-hydroxyethyl)dodecanamide, the positive, and the negative controls were applied to the different corneae and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneae and opacity was measured again (t₂₄₀). After 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. For the negative control (saline) an increase of neither opacity nor permeability of the corneae could be observed (mean IVIS = 1.24). The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneae (mean IVIS = 107.58) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item N-(2-hydroxyethyl)dodecanamide caused an increase of the corneal opacity. The calculated mean IVIS was 7.70 (threshold for serious eye damage: IVIS> 55). According to OECD 437, no prediction for the hazard classification of the test item to the eye can be made. In conclusion, according to the current study and under the experimental conditions reported, N-(2-hydroxyethyl)dodecanamide is not serious eye damaging (CLP/EPA/GHS (Cat 1) but a prediction for the damage hazard cannot be made (GHS).

OECD 492 (2018) - The test item, N-(2-hydroxyethyl)dodecanamide applied to human Cornea in order to assess it irritation potential. The test item’s intrinsic colour was not intensive and it did not prove to dye water or isopropanol in the colour interference pre-test. Therefore, additional tests with viable tissues (without MTT addition) did not have to be performed. The test item proved to be an MTT reducer in the MTT pre-test. Therefore, additional tests with freeze-killed tissues had to be performed. The viability values resulted in these additional tests were used to correct the values gained in the normal tests.  Each 50 mg of the test item, were applied to each of duplicate tissue for 6 hours. Each 50 µL of the negative control (deionised water) and of the positive control (methyl acetate) were also applied to duplicate tissues each. After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system. The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues). A reduction in tissue viability was observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased below 60% (determined value for the test item 43.1%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

Whilst the OECD 437 (BCOP) and OECD 492 (EpiOcular) methodology for eye irritation does not permit discrimination between serious eye damage and eye irritation in accordance with CLP, a conclusion on classification and labelling is reached for this substance based on a weight-of-evidence approach, based on the integrated testing approach conducted in accordance with ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a. (v6.0 - July 2017). Both assays conducted indicate with certainty that the substance should not be classified for serious eye damage (Eye Dam. 1), yet both assays confirm that the substance should not have no classification with respect to eye irritation and is considered to have eye irritation potential. Therefore, the proposed classification and labelling scheme for this substance under CLP is category 2 (H319: causes serious eye irritation).

Justification for classification or non-classification

The substance meets the criteria for classification as an eye irritant (Eye Irrit. 2, H319: causes serious eye irritation) in accordance with Regulation (EC) No 1272/2008 (CLP).