Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 947-907-6 | CAS number: -
- 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:
- key study
- Study period:
- From May 30, 2018 to June 13, 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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
- Justification for non-LLNA method:
- OECD TG 442E cites the h-CLAT model using THP-1 cells as a validated method for skin sensitisation testing in the context of an integrated approach to testing and assessment.
Test material
- Reference substance name:
- [3-({4-carbamimidamido-1-[(1,3-dicarboxypropyl)carbamoyl]butyl}amino)-2-hydroxypropyl](dodecyl)dimethylazanium {3-[(1,3-dicarboxypropyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium {3-[(4-carbamimidamido-1-carboxybutyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium trichloride
- EC Number:
- 947-907-6
- Molecular formula:
- Molecular formula of major active constituents: C22H45Cl1N2O5 (representative for C12 alkyl chain quaternised glutamic acid) C23H50Cl1N5O3 (representative for C12 alkyl chain quaternised arginine) C28H57Cl1N6O6 (representative for C12 alkyl chain quaternised peptides of arginine and glutamic acid)
- IUPAC Name:
- [3-({4-carbamimidamido-1-[(1,3-dicarboxypropyl)carbamoyl]butyl}amino)-2-hydroxypropyl](dodecyl)dimethylazanium {3-[(1,3-dicarboxypropyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium {3-[(4-carbamimidamido-1-carboxybutyl)amino]-2-hydroxypropyl}(dodecyl)dimethylazanium trichloride
- Test material form:
- other: paste
Constituent 1
In vitro test system
- Details on the study design:
- Description of the test method
Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.
Characterisation of the test method
The h-CLAT has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Method workflow summary
Solubility was first determined for the test substance using either culture medium (RPMI 1640) or DMSO. Note that for this method, test substances with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test substances 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 72 ±2 h. Following this, the cells were dosed with the test substance 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 substance 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 for either 48 ±2 or 72 ±2 h. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test substance. 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.
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. E-mail: atcc@lgcstandards.com. Tel: +44 (0)20 8943 8489. 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 09 Oct 2017.
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 02 Mar 2017). Methodology is detailed in an XCellR8 SOP (L0094) that covers the h-CLAT. Study data is collected using XCellR8 internal protocols (IPs).
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:
- 167.6
- 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 201.12 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 201.12 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
a) Solvent Selection and CV75 Determination
Prior to the CV75 determination, the test substance was assessed for solubility and was found to be soluble in complete RPMI culture medium at 250 mg/mL. The CV75 value derived from two independent experiments was as follows:
CV75 (Rep1) |
CV75 (Rep2) |
Average CV75 (µg/mL) |
123.7 |
211.6 |
167.6 |
b) Acceptance Criteria
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. Note that the cell viability in the medium control in Run 2 for the CD54/86 measurements was a borderline value (86.61%) and was deemed acceptable even though it fell below the acceptance range for the control.
CV75 Determination |
|||
Criterion |
Run 1 |
Run 2 |
Outcome |
Cell viability must be ≥ 75% at the lowest dose. |
95.24 |
96.98 |
PASS |
The highest test substance concentration should produce cytotoxicity (< 90% cell viability) unless5mg/mL in medium, 1mg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test substance. |
51.30 |
39.17 |
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% |
97.07 |
86.61 |
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 |
N/A |
N/A |
N/A |
For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105% |
CD54: 159.29% CD86: 139.77% |
CD54: 219.67% CD86: 179.02% |
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%. |
CD54 RFI: 229 CD86 RFI: 158 CD54 Via: 90.20% CD86 Via: 90.96% |
CD54 RFI: 266 CD86 RFI: 155 CD54 Via: 72.06% CD86 Via: 67.67% |
PASS |
For each test substance, the cell viability should be greater than 50% in at least four tested concentrations in each run |
7/8 |
8/8 |
PASS |
Negative results are acceptable only for test substances 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 is used as the maximal test concentration of a test substance. |
35.8 |
90.69 (Test substance is positive) |
PASS |
RFI - Relative Fluorescence Intensity, MFI - Mean Fluorescence Intensity, DMSO - Dimethyl Sulphoxide
c) Results Summary
The CV75 dose informs the dosing range selected for the CD54/86 expression assay. The top dose for the CD54/86 expression assay (main test) is 1.2 x CV75 which was equal to 201.12 µg/mL. The following tables show the expression of CD54 and CD86 against test substance dose with concurrent cytotoxicity measurement:
Run 1 (Valid): Result = Positive
Test Substance Dose (µg/mL) |
Cell Viability (%) |
Average Cell Viability (%) |
CD54 RFI |
CD86 RFI |
||
Isotype |
CD54 |
CD86 |
||||
201.12 |
30.44 |
34.90 |
42.07 |
35.80 |
218 |
160 |
167.60 |
45.11 |
50.45 |
59.28 |
51.61 |
299 |
206 |
139.67 |
50.40 |
56.94 |
60.93 |
56.09 |
127 |
108 |
116.39 |
54.80 |
62.05 |
69.13 |
61.99 |
171 |
123 |
96.99 |
62.20 |
65.90 |
69.85 |
65.98 |
84 |
74 |
80.83 |
54.96 |
61.37 |
66.19 |
60.84 |
89 |
98 |
67.35 |
62.23 |
63.06 |
66.47 |
63.92 |
91 |
108 |
56.13 |
58.61 |
59.35 |
61.78 |
59.91 |
102 |
74 |
Run 2 (Valid): Result = Positive
Test Substance Dose (µg/mL) |
Cell Viability (%) |
Average Cell Viability (%) |
CD54 RFI |
CD86 RFI |
||
Isotype |
CD54 |
CD86 |
||||
201.12 |
90.44 |
90.59 |
91.05 |
90.69 |
1086 |
1159 |
167.60 |
75.29 |
78.95 |
75.41 |
76.55 |
74 |
130 |
139.67 |
76.50 |
71.13 |
76.90 |
74.84 |
47 |
110 |
116.39 |
68.33 |
76.45 |
74.61 |
73.13 |
105 |
99 |
96.99 |
67.61 |
73.84 |
74.95 |
72.13 |
62 |
115 |
80.83 |
78.53 |
79.11 |
72.49 |
76.71 |
83 |
130 |
67.35 |
74.22 |
76.57 |
77.31 |
76.03 |
113 |
155 |
56.13 |
80.29 |
55.51 |
80.78 |
72.19 |
71 |
110 |
As can be seen from the data, the expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2 (highlighted in green). The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2 (highlighted in gold). As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%, highlighted in pink) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result is deemed to be valid.
The EC200 and EC150 values derived for the test substance were as follows:
Marker |
EC200 (Rep1) |
EC200 (Rep2) |
Final EC200 Value (µg/mL) |
CD54 |
152 |
172 |
172 |
Marker |
EC150 (Rep1) |
EC150 (Rep2) |
Final EC150 Value (µg/mL) |
CD86 |
152 |
66 |
152 |
For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were 172 and 152 µg/mL respectively, therefore, Test substance was classified as positive as per the prediction model.
Applicant's summary and conclusion
- Conclusions:
- Under the study conditions, the test substance was concluded to be skin-sensitiser.
- Executive summary:
A study was conducted to determine the skin sensitisation potential of test substance, 'Cocodimonium hydroxypropyl hydrolysed wool (62.8% active)', 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 (a cell line that mimics DCs). 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 were pre-cultured for 72 h. Following this, the cells were dosed with the test substance over an 8 dose range (2500, 1250, 625, 312.5, 156.25, 78.13, 39.06 and 19.53 µg/mL) in RPMI culture medium 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 concentrations tested for CD54/CD86 expression assay were 201.12, 167.60, 139.67, 116.39, 96.99, 80.83, 67.35 and 56.13 µg/mL. The expression of CD54 as measured by the RFI crossed the threshold (RFI ≥200) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 µg/mL in Run 2. The expression of CD86 as measured by the RFI crossed the threshold (RFI ≥150) at the following doses; 201.12 and 167.60 µg/mL in Run 1 and at 201.12 and 67.35 µg/mL in Run 2. As the CD54/CD86 expression crossed the threshold in both runs the test substance is classified as Positive. Cell viability fell below 50% at the top concentration (201.12 µg/mL) in Run 1 (viability 35.80%) however the threshold was also crossed for both markers at 167.60 µg/mL at a non-cytotoxic concentration (51.61%), therefore, the result was considered to be valid. For test substance the dose that gave 75% cell viability was found to be 167.6 µg/mL. The EC200 and EC150 values for CD54 and CD86 expression were calculated to be 172 and 152 µg/mL respectively; therefore, the test substance was classified as positive as per the prediction model. 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 the study conditions, the test substance was concluded to be skin-sensitiser (XcellR8, 2018).
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.