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EC number: 273-159-9 | CAS number: 68951-62-2
- 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
Skin sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- From August 04, 2017 to August 15, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
- Version / remarks:
- OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT)
- Version / remarks:
- EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 01 Jul 2015)
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol
- EC Number:
- 273-159-9
- EC Name:
- Octadecanoic acid, reaction products with 2-amino-2-methyl-1-propanol
- Cas Number:
- 68951-62-2
- Molecular formula:
- C20H41NO2 (C16 chain representative) & C22H45NO2 (C18 chain representative)
- IUPAC Name:
- N-(1-hydroxy-2-methylpropan-2-yl)hexadecanamide; N-(1-hydroxy-2-methylpropan-2-yl)octadecanamide
- Test material form:
- solid
- Details on test material:
- Appearance: Off white cream
Constituent 1
In vitro test system
- Details on the study design:
- Test System Material Source
The human monocytic leukaemia cell line, THP-1, (Cat# TIB-202) were obtained from American Type Culture Collection (ATCC) via their UK subsidiary LGC standards; LGC Standards, Queens Road, Teddington, Middlesex TW11 0LY, UK. Two initial vials of cells were received in April 2016 and data to verify the referenced sources was archived.
OECD Test Guideline relating to the test method
OECD TG 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted 29 Jul 2016.
XCellR8 SOP
The study was performed using the h-CLAT method as detailed in OECD TG 442E and also in EURL ECVAM DB-ALM Protocol No. 158 (Issued 01 Jul 2015). Methodology is detailed in an XCellR8 SOP (L0094) that covers the h-CLAT. Study data was collected using XCellR8 internal protocols (IPs).
Method workflow summary
Solubility was first determined for the test item using either culture medium (RPMI 1640) or DMSO. Note that for this method, test items with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test items with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid. THP-1 cells were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test item over an 8 dose range and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test item that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs. THP-1 cells were pre-cultured again for either 48 or 72 h. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test item. This dilution range was used to dose the cells again for 24 ±0.5 h. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: Average of two run
- Parameter:
- other: CV75 (µg/mL)
- Remarks:
- A concentration showing 75% of THP-1 cell survival (25% cytotoxicity)
- Value:
- 250
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Run / experiment:
- other: Average of two representatives
- Parameter:
- other: CD54 expression measured as RFI (up to 300 µg/mL, top dose concentration)
- Value:
- 200
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Run / experiment:
- other: Average of two representatives
- Parameter:
- other: CD86 expression measured as RFI (up to 300 µg/mL, top dose concentration)
- Value:
- 150
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Other effects / acceptance of results:
- Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.
Any other information on results incl. tables
Results
Solvent Selection and CV75 Determination
Prior to the CV75 determination, the test substance was assessed for solubility and was found to be soluble in Isopropanol at 25 mg/mL.
The CV75 value derived from two independent experiments was as follows:
CV75 (Rep1) |
CV75 (Rep2) |
Average CV75 |
>250 |
77.1 |
163.55 µg/mL |
The final CV75 was taken as the top dose (250 µg/mL) even though the CV75 was lower in Rep2, as the average of the viabilities across the 2 replicates at the top dose was 64.59% and the top dose produced cytotoxicity in both replicates. In the second lowest dose cytotoxicity was not observed in Rep1. The graph for the two replicates has been included for clarity, note that the second lowest dose for Rep1 was considered to be an outlier datapoint.
Acceptance Criteria
Acceptance criteria for all controls and the test item were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.
CV75 Determination |
|||
Criterion |
Run 1 |
Run 2 |
Outcome |
Cell viability must be ≥ 75% at the lowest dose. |
96.89% |
92.55% |
PASS |
The highest test substance concentration should produce cytotoxicity (< 90% cell viability) unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance. |
86.13% |
43.04% |
PASS |
Measurement of CD54 and CD86 Expression |
|||
Criterion |
Run 1 |
Run 2 |
Outcome |
Cell viabilities of medium and solvent controls should be higher than 90% |
Medium: 98.02% Isopropanol: 95.75% |
Medium: 97.49% Isopropanol: 94.98% |
PASS |
In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) compared to the medium control |
CD54 = 118 CD86 = 122 |
CD54 = 98 CD86 = 124 |
PASS |
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105% |
Medium CD54: 140.50 CD86: 127.20 Isopropanol CD54: 142.24 CD86: 129.13 |
Medium CD54: 148.44 CD86: 127.21 Isopropanol CD54: 142.69 CD86: 130.43 |
PASS |
In the positive control (Nickel Sulphate), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be greater than 50%.
|
RFI CD54: 384 CD86: 165 Viability (%) CD54: 91.02 CD86: 90.33 |
RFI CD54: 280 CD86: 153 Viability (%) CD54: 89.43 CD86: 91.20 |
PASS |
For each test substance, the cell viability should be greater than 50% in at least four tested concentrations in each run
|
6/8 |
8/8 |
PASS |
Negative results are acceptable only for test items exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration in solvent is used as the maximal test concentration of a test substance. |
N/A as test item positive |
N/A as test item positive |
PASS |
RFI - Relative Fluorescence Intensity, MFI - Mean Fluorescence Intensity.
Applicant's summary and conclusion
- Interpretation of results:
- other: Category 1 (skin sensitising) based on EU CLP criteria
- Conclusions:
- Under study conditions, the test substance was concluded to be a skin sensitiser.
- Executive summary:
A study was conducted to determine the skin sensitisation potential of test substance, C16-18 AMP, using the in vitro human Cell Line Activation Test (h-CLAT) method, according to OECD 442E, in compliance of GLP. The h-CLAT assay measures markers of dendritic cell (DC) activation in the human leukaemia cell line THP-1. The ability of a chemical to induce activation of CD86 and CD54 in THP-1 cells was used as an assessment of the chemical’s skin sensitisation potential. Activation of CD54 and CD86 protein markers was measured by flow cytometry following 24 h exposure to the test substance. The concentration of the test substance used was selected on the basis of a pre-determined CV75 value (i.e. the estimated concentration yielding 75% cell viability). For the CV75 determination, THP-1 cells (a cell line that mimics DCs) were pre-cultured for either 48 or 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (300, 250, 208.33, 173.61, 144.68, 120.56, 100.47, 83.72 µg/mL) and incubated for 24 ±0.5 h. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The CV75 was found to be 250 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were found to be 12 µg/mL and 11 µg/mL respectively. As the CD54/CD86 expression crossed the sensitisation threshold the test substance was classified as a skin sensitiser. Cell viability only fell marginally below 50% at the 120.56 µg/mL test substance concentration in Run 1. All other concentrations that induced sensitisation had a cell viability of >50% and therefore the final result were deemed to be valid. An inverse relationship of dose to viability was observed in the main test, however this has not affected the end result, as all test concentrations (with the exception noted above) had cell viabilities above 50% and the RFI data were conclusive and consistent in both runs. Acceptance criteria for all controls and the test substance were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression. Under study conditions, the test substance was concluded to be a skin sensitiser (XcellR8, 2017).
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