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EC number: 943-175-7 | 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:
- weight of evidence
- Study period:
- 2015-02-18 to 2015-02-27
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 015
- Report date:
- 2015
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: U-SENS (former MUSST)
- Version / remarks:
- MUSST SOP ECVAM version 03/26/2013
- Deviations:
- not applicable
- Principles of method if other than guideline:
- - Principle of test:
Cell activation by contact sensitizers is determined by the overexpression of CD86 on U937 cell surface, which is a ligand required for T-cell activation. CD86 expression is measured using fluorescent labeled anti-CD86 monoclonal antibody after a 45 hours treatment by flow cytometry.
Cell culture
U937, a human myelomonocytic cell line, was obtained from the America, Type Culture Collection (ATCC, Ref: CRL-1593.2). On the day of experiment, cells were seeded in 96-well culture plates at a density of 0.5 x 106 cells/mL with or without a test items (doses ranging from 1 to 200 μg/mL) for 45±3 hours to determine a dose-response.
Cell treatment
Treatments were performed at the following concentration for the first run: 1, 10,20,50, 100 and 200 μg/mL. For the next runs, the adjustment of the dose could be necessary to determine a sub-toxic dose-response of the test item and the calculation of the EC150. Test item is tested up to five validated runs. The sensitizing potential of the test item is determined after a minimum of two concordant runs.
Determination of CD86 Expression
Cell staining was performed using mouse mAb FITC-anti-human-CD86. Briefly, cells were washed in cold PBS (containing 5% FBS), stained with antibodies in the dark at 4°C for 30 min, washed twice with cold PBS (containing 5% FBS) and once with PBS without FBS and resuspended in PBS. Cells were then analyzed using FACS DIVA Software based on a collection of 10,000 cells with a FACS CANTO II flow cytometer. Necrotic cells were gated out after staining with 50 μg/mL Propidium Iodide solution. The percentage of CD86 positive cells was determined according to the IgGl isotype control (% cCD86).
Automatized test system using MAIA (Methode Alternative In vitro Automatisée) was used for cell treatment and cell staining. - GLP compliance:
- no
- Remarks:
- adequate for C&L; sufficient documentation is provided to assess the adequacy of the study; the data are valid for the endpoint investigated and the study is performed using an acceptable level of quality assurance
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria
- EC Number:
- 943-175-7
- IUPAC Name:
- Rhamnolipids: fermentation products of glucose with Pseudomonas bacteria
- Test material form:
- solid: particulate/powder
Constituent 1
In vitro test system
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
No solubility assessment was performed because the solvent RPMI was weil known for this test item
Lactic acid (LA 200 μg/mL as known non sensitizer) treated cells were used as negative control 2,4,6- Trinitrobenzenesulfonic acid (TNBS 50 μg/mL, as well known skin sensitizer) treated cells were used as positive control.
A set of control wells corresponding to untreated (RPMI control) and treated with either the vehicle (DMSO), negative and positive controls were also added in each plate to guarantee the validity of each run.
Cell culture
U937, a human myelomonocytic cell line, was obtained from the ATCC (Ref: CRL-1593.2). On the day of experiment, cells were seeded in 96-well culture plates at a density of 0.5E06 cells/mL with or without a test items (doses ranging from 1 to 200 μg/mL) for 45±3 hours to determine a dose-response.
Cell treatment
Test item was tested in two validated runs.
Concentration for the first run: 1, 10, 20, 50, 100 and 200 μg/mL.
Concentration for the second run: 10, 20, 50, 100, 160 and 200 μg/mL.
Determination of CD86 Expression
Cell staining was performed using mouse mAb FITC-anti-human-CD86. Briefly, cells were washed in cold PBS (containing 5% FBS), stained with antibodies in the dark at 4°C for 30 min, washed twice with cold PBS (containing 5% FBS) and once with PBS without FBS and resuspended in PBS. Cells were then analyzed using FACS DIVA Software based on a collection of 10,000 cells with a FACS CANTO II flow cytometer. Necrotic cells were gated out after staining with 50 μg/mL Propidium Iodide solution. The percentage of CD86 positive cells was determined according to the IgG1 isotype control (%cCD86).
Results and discussion
- Positive control results:
- 2,4,6- Trinitrobenzenesulfonic acid (TNBS 50 μg/mL, as well known skin sensitizer) treated cells were used as positive control. In both runs, all positive control wells gave positive results (CD86 SI ≥ 150).
In vitro / in chemico
Resultsopen allclose all
- Parameter:
- other: EC150 [µg/ml]
- Value:
- 200
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Parameter:
- other: CV70 [µg/mL]
- Value:
- 200
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Other effects / acceptance of results:
- DEMONSTRATION OF TECHNICAL PROFICIENCY:
If one or more of the following acceptance criteria is not met, the run is invalidated and had to be repeated:
- At least 2 RPMI controls out of 3 presents a % IgG1 between 0.6% and 1.5%.
- The mean viability of RPMI cells have to be >90%.
- The mean corrected %CD86+ values of RPMI control wells have to be ≥ 2 and ≤ 25%.
- At least 2 out of 3 wells of the positive control TNBS have to be positives (CD86 SI ≥ 150).
- At least 2 out of 3 wells of the negative control LA have to be negatives (CD86 SI ≤ 150).
- The mean viability of the vehicle control DMSO has to be ≥ 90%.The mean CD86 SI of the vehicle control DMSO has to be ≤ 250. If not a marginal value can be discarded.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Other acceptance criteria met: yes
Any other information on results incl. tables
Summary results
|
Dose |
Individual runs |
Mean and SD |
FL3 (lgG1) |
FL1 (lgG1) |
||||||||
|
testsubstance |
% vialbility (mean) |
CD86-lgG1(S.I.) |
Viability |
CD86-lgG1 |
X Geo |
Mean S.I. |
X Geo |
Mean S.I. |
||||
|
|
A |
B |
A |
B |
Mean |
SD |
Mean |
SD |
A |
B |
A |
B |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
1.00 |
95 |
|
76 |
|
95 |
0 |
76 |
0 |
165 |
|
108 |
|
|
10.00 |
96 |
96 |
66 |
70 |
96 |
0 |
68 |
2 |
110 |
93 |
96 |
95 |
|
20.00 |
96 |
96 |
61 |
70 |
96 |
0 |
65 |
4 |
96 |
97 |
96 |
94 |
|
50.00 |
95 |
96 |
77 |
61 |
96 |
1 |
69 |
8 |
92 |
95 |
95 |
95 |
|
100.00 |
95 |
96 |
90 |
72 |
95 |
0 |
81 |
9 |
105 |
87 |
101 |
99 |
|
160.00 |
|
93 |
|
78 |
93 |
0 |
78 |
0 |
|
82 |
|
109 |
|
200.00 |
67 |
76 |
|
99 |
72 |
5 |
99 |
0 |
|
84 |
|
95 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
CV70 / EC 150 |
|
189 |
NA |
NA |
NA |
NA |
|
NA |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Run conclusion |
|
|
|
NS |
NS |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
|
NA: Not applicable NS: Non-Sensitizer
EC150: calculated value corresponding to the concentration beyond which the RM leads 150% of expression of CD86 with regard to the control.
CV70: calculated value corresponding to the concentration beyond which the moleeule is considered as being cytotoxic.
Applicant's summary and conclusion
- Interpretation of results:
- other: not sensitising
- Remarks:
- expert statement
- Conclusions:
- Under the experimental conditions of this study, the test item, Rhamnolipids is predicted to be a non sensitizer (no EC150) in the MUSST assay without cytotoxicity.
- Executive summary:
The purpose of this study was to evaluate the capability of Rhamnolipids (79% a.i.) to induce the surface marker CD86 expression in the human myeloid U937 cell line. This study was conducted in compliance with the MUSST SOP ECVAM version 03/26/2013.
Cell activation by contact sensitizers is determined by the overexpression of CD86 on U937 cell surface, which is a ligand required for T-cell activation. CD86 expression is measured using fluorescent labeled anti-CD86 monoclonal antibody after a 45 hours treatment by flow cytometry.
All validity criteria were fulfilled. An EC150 could not be determined up to the highest tested concentration of 200 µg/L. Rhamnolipids is predicted a non-sensitizer in the MUSST assay without cytotoxicity.
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