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EC number: 205-560-1 | CAS number: 142-78-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
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
- 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
- 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.
- 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. - 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.
Referenceopen allclose all
Table 2. Results after treatment with N-(2-hydroxyethyl) dodecanamide and the controls.
Dose Group |
Tissue No. |
Absorbance 570 nm |
Absorbance 570 nm |
Absorbance 570 nm |
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 |
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 %
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- 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.
- 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
- 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.
Referenceopen allclose all
Table 1. Results after treatment for 6 hours with the test item and the controls.
Treatment Group |
Tissue No. |
OD 570 nm |
OD 570 nm |
Mean OD of 2 Wells |
Mean OD of 2 Wells blank corrected |
Mean OD of Treatment Group blank corrected |
Rel. Viability [%] Tissue |
Absolute Value of the Difference of Rel. Viability |
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 |
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 |
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).
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