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EC number: 281-984-0 | CAS number: 84082-36-0 Extractives and their physically modified derivatives such as tinctures, concretes, absolutes, essential oils, oleoresins, terpenes, terpene-free fractions, distillates, residues, etc., obtained from Medicago sativa, Leguminosae.
- 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
- Remarks:
- OECD 442D
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From 28 March to 6 April 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
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of keratinocytes
Test material
- Reference substance name:
- Alfalfa, ext.
- EC Number:
- 281-984-0
- EC Name:
- Alfalfa, ext.
- Cas Number:
- 84082-36-0
- IUPAC Name:
- Alfalfa, ext.
- Test material form:
- liquid: viscous
- Remarks:
- Alfalfa, ext was observed to be a very thick dark coloured paste at room temperature.
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: AL01
- Expiration date of the lot/batch: 10.09.2019
- Purity test date: not available
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: refrigerator (4°C)
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: 20 mg/ml in DMSO
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: none
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable
OTHER SPECIFICS: none
In vitro test system
- Details on the study design:
- DESCRIPTION OF THE TEST SYSTEM
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection using a BMG Optima FLUOstar) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.
METHOD OF ADMINISTRATION OF TEST ITEM
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.
METHOD OF ADMINISTRATION OF REFERENCE ITEMS
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
EXPOSURE TIMES OF TEST ITEMS AND REFERENCE ITEMS
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.
NUMBER OF REPETITIONS
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT.
NEGATIVE CONTROL
DMSO, 99.7%
POSITIVE CONTROL
Cinnamic aldehyde, 99%
DATA ANALYSIS
The following parameters were calculated in the KeratinoSens test method:
- The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration of the test item and positive control.
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity).
- For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
-The percentage of viability as compared to the negative control.
ASSAY ACCEPTANCE CRITERIA
Test results are acceptable if:
- The positive control (cinnamic aldehyde) produces positive results, i.e., the luciferase gene induction by this control is statistically above the threshold of 1.5 in at least one of the tested concentrations.
-The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet the following targets:
* Average induction in the three replicates for cinnamic aldehyde at 32 µM is between the XCellR8 historical range (currently 1.6 and 3)
* EC1.5 value for cinnamic aldehyde is between the XCellR8 historical range (currently 6 µM and 39 µM).
Note: At least one of these criteria must be met, otherwise the run is discarded. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
- CV% of blank values < 20%
INTERPRETATION OF RESULTS AND SKIN SENSITISATION PREDICTION MODEL
A test item is considered a sensitiser if the following conditions are met in 2 of 3 repetitions:
- The IMAX is higher than 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test).
- The cellular viability is higher than 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
- The EC1.5 value is < 1000 µM or < 200 µg/mL for test chemicals with no defined MW.
- There is an apparent overall dose-response for luciferase induction (or a biphasic response).
Results and discussion
- Positive control results:
- Induction (=1.5-fold):
1.518 at 8 µM
2.664 at 16 µM
5.019 at 32 µM
11.866 at 64 µM
20.861 at 128 µM
In vitro / in chemico
Results
- Key result
- Positive controls validity:
- valid
- Remarks on result:
- other: Negative
Any other information on results incl. tables
See attached additional information on results.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In an in vitro KeratinoSens test, conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative.
- Executive summary:
The in vitro KeratinoSens test, conducted according to OECD test guideline 442D and to GLP, was performed to determine skin sensitisation potential of Alfalfa, ext. by assessing keratinocytes activation.
After 48h exposure of cells to 12 concentrations of Alfalfa, ext. luciferase measurements and MTT viability test were performed.
The sensitisation potential of Alfalfa ext. was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value.
Alfalfa, ext. only caused luciferase induction above 1.5 in repetition 2, with an EC1.5 value below 0.098 µg/ml. Since no clear dose response was observed, this was classified as an inconclusive result for this repetition. In repetitions 1 and 3, the threshold for induction was not exceeded at any of the concentrations tested and, therefore, based on 2 out of 3 concordant results from independent experiments, the result for Alfalfa, ext. in this KeratinoSens test was negative.
The maximum induction for repetition 1 was observed at 200 µg/ml Alfalfa, ext. (1.238). For repetitions 2 and 3 the IMAX values of 3.139 and 0.969, respectively, were observed at 0.391 µg/ml.
All of the formal acceptance criteria of the tests were met except for acceptance criterion 2 (average induction of positive control at 32 µM). However, as all other acceptance criteria were met, and there was a dose-dependent increase of induction with the positive control, results are considered as valid.
Therefore, in the in vitro test, conducted according to OECD test guideline 442D and to GLP, the skin sensitisation potential of Alfalfa, ext. was determined negative for the second key event of the skin sensitisation Adverse Outcome Pathway.
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