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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
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 07-25 February 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- The tester strain WP2uvrA was accidentally additionally tested in the dose range finding test.
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- yes
- Remarks:
- The tester strain WP2uvrA was accidentally additionally tested in the dose range finding test.
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- bacterial reverse mutation assay
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: refrigerated
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: water solubility 5.35 mg/L at 25°C
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: diluted in Milli-Q water, the stock solution was treated with ultrasonic waves to obtain a homogeneous suspension
- 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
Method
- Target gene:
- his
Species / strainopen allclose all
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Species / strain / cell type:
- E. coli WP2 uvr A
- Remarks:
- Tested only in direct plate assay
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9
- Test concentrations with justification for top dose:
- 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (TA 100, WP2uvrA; direct plate assay, dose-range finding)
52, 164, 512, 1600 and 5000 µg/plate (TA 98, TA 102, TA1535, TA 1537; direct plate assay)
52, 164, 512, 1600 and 5000 µg/plate (TA 98, TA 100, TA 102, TA1535, TA 1537; pre-incubation assay) - Vehicle / solvent:
- Milli-Q water
Controls
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- methylmethanesulfonate
- other: ICR-191 (-S9 mix), tert-butyl hyperoxide (-S9 mix), 2-aminoanthracene (+S9 mix)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: agar plates, 1st experiment: direct plate assay, 2nd experiment: pre-incubation assay
DURATION
- Preincubation period: 30 ± 2 minutes at 70 rpm at 37 ± 1°C (2nd experiment)
- Exposure duration: 48 ± 4 h at 37 ± 1°C (1st and 2nd experiment)
NUMBER OF REPLICATIONS: triplicates
DETERMINATION OF CYTOTOXICITY
- Method: visual inspection - Rationale for test conditions:
- In accodrance with the OECD Testing Guideline 471.
- Evaluation criteria:
- A test item is considered negative (not mutagenic) in the test if:
1) The total number of revertants in the tester strains TA100, TA102 and WP2uvrA is not greater than two times the concurrent control, and the total number of revertants in tester strain TA1535, TA1537 and TA98 is not greater than three times the concurrent control.
2) The negative response should be reproducible in at least one independently repeated experiment.
A test item is considered positive (mutagenic) in the test if:
1) The total number of revertants in the tester strains TA100, TA102 and WP2uvrA is greater than two times the concurrent control, or the total number of revertants in tester strain TA1535, TA1537 and TA98, is greater than three times the concurrent control.
2) In case a second experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Remarks:
- Only direct plate assay
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
Applicant's summary and conclusion
- Conclusions:
- In a Bacterial Reverse Mutation assay, conducted according to OECD test guideline 471 and to GLP, Alfalfa, ext. was concluded to have negative mutagenic potential.
- Executive summary:
Bacterial Reverse Mutation assay conducted according to OECD test guideline 471 and to GLP was performed to determine mutagenic potential of Alfalfa, ext.
In the dose-range finding study, the test item was initially tested up to concentrations of 5000 µg/plate in the strains TA100 and WP2uvrA in the direct plate assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose-range finding test were reported as part of the first mutation assay.
In the first mutation experiment, the test item was tested at a concentration range of 52 to 5000 µg/plate in the strains TA1535, TA1537, TA98 and TA102. The test item did not precipitate on the plates at these dose levels. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In the second mutation experiment, the test item was tested at a concentration range of 52 to 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and TA102 in the pre-incubation assay. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.
In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.
The test item did not induce any increase in the number of revertant (His+) colonies in any of the five Salmonella tester strains and in the number of revertant (Trp+) colonies in E. coli tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.
In conclusion, based on the results of this study it is concluded that Alfalfa ext. is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
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