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EC number: 270-151-7 | CAS number: 68411-85-8
- 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:
- Human Cell Line Activation Test
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 01 Oct 2017 - 16 Feb 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:
- other: OECD 442E In-Vitro Skin Sensitization: Human Cell Line Activation Test (hCLAT)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- Principles of method if other than guideline:
- - Principle of test: The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. 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 marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.
- GLP compliance:
- yes
- Type of study:
- activation of dendritic cells
Test material
- Reference substance name:
- Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone
- EC Number:
- 270-151-7
- EC Name:
- Hydrazinecarboximidamide, 2-[(2-hydroxyphenyl)methylene]-, reaction products with 2-undecanone
- Cas Number:
- 68411-85-8
- Molecular formula:
- C19H32N4O2
- IUPAC Name:
- 2-{[(6-oxocyclohexa-2,4-dien-1-ylidene)methyl]amino}guanidine; undecan-2-one
- Test material form:
- liquid: viscous
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: BASF (Puebla, Mexico) batch# 6191104
- Expiration date of the lot/batch: August 2018
- Purity test date: 08 March 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at room temperature
- Stability under test conditions: stable under conditions of assay
- Solubility and stability of the test substance in the solvent/vehicle: soluble in vehicle based on initial solubility screen
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: used as received
In vitro test system
- Details on the study design:
- The h-CLAT method is an in vitro assay that quantifies changes of cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukemia cell line, THP-1 cells, following 24 hours exposure to the test chemical. These surface molecules are typical markers of monocytic THP-1 activation and may mimic DC activation, which plays a critical role in T-cell priming. 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 marker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers
THP-1 cells are cultured, at 37°C under 5% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 μg/mL streptomycin. The positive controls for this assay are 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity) and the negative control, is lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity).
A dose finding assay is performed to determine the CV75, being the test chemical concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value is used to determine the concentration of test chemicals for the main assay involving CD86/CD54 expression measurement. The test substance working solutions are mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well or 96-well flat-bottom plate. The treated plates are then incubated for 24±0.5 hours at 37°C under 5% CO2. After 24±0.5 hours of exposure, CD54/86 expression is measured by FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies at 4°C for 30 min. Cell viability was measured by Propidium Iodine staining.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Cell viabilities of medium and solvent/vehicle controls should be > 90%. The RFI values of both CD86 and CD54 should not exceed positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). The MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
-Acceptance criteria for positive control: RFI values of both CD86 and CD54 should meet the positive criteria CD86 RFI ≥ 150% and CD54 RFI ≥ 200% and cell viability should be more than 50%.
-Acceptance criteria for test chemicals: Cell viability should be more than 50% in at least four tested concentrations. Negative results are acceptable only for test chemicals exhibiting a cell viability of less than 90% at the highest concentration tested, unless 5000 ug/mL is used as the maximal test concentration of a test chemical than a negative result is acceptable even if the cell viability is above 90%.
Results and discussion
- Positive control results:
- The positive control 2,4-dinitrochlorobenzene (DNCB) produced positive results (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in three out of five replicates. The positive control nickel sulfate (NiSO4) produced positive results (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in all five replicates.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Run / experiment:
- other: mean of three replicates
- Parameter:
- other: CV75 (ug/mL)
- Remarks:
- estimated concentration causing 75% viability
- Value:
- 56.2
- Run / experiment:
- other: CD54 Main Assay #1
- Parameter:
- other: EC200 (ug/mL)
- Value:
- 32.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- not valid
- Run / experiment:
- other: CD86 Main Assay #1
- Parameter:
- other: EC150 (ug/mL)
- Value:
- 67.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- not valid
- Run / experiment:
- other: CD54 Main Assay #2
- Parameter:
- other: EC200 (ug/mL)
- Value:
- 32.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- not valid
- Run / experiment:
- other: CD86 Main Assay #2
- Parameter:
- other: EC150 (ug/mL)
- Value:
- 67.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- not valid
- Key result
- Run / experiment:
- other: CD54 Main Assay #3
- Parameter:
- other: EC200 (ug/mL)
- Remarks:
- concentration showing an RFI value >200 in CD54
- Value:
- 56.9
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Run / experiment:
- other: CD86 Main Assay #3
- Parameter:
- other: EC150 (ug/mL)
- Remarks:
- concentration showing an RFI value >150 in CD86
- Value:
- 67.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: CD54 Main Assay #4
- Parameter:
- other: EC200 (ug/mL)
- Remarks:
- concentration showing an RFI value >200 in CD54
- Value:
- 67.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: CD86 Main Assay #4
- Parameter:
- other: EC150 (ug/mL)
- Remarks:
- concentration showing an RFI value >150 in CD86
- Value:
- 67.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Run / experiment:
- other: CD54 Main Assay #5
- Parameter:
- other: EC200 (ug/mL)
- Remarks:
- concentration showing an RFI value >200 in CD54
- Value:
- 27.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Run / experiment:
- other: CD86 Main Assay #5
- Parameter:
- other: EC150 (ug/mL)
- Remarks:
- concentration showing an RFI value >150 in CD86
- Value:
- 67.5
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of skin sensitisation
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The RFI values of both CD86 and CD54 did not exceed positive crtieria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) for the negative control Lactic Acid in any of five replicates.
-Acceptance criteria for positive control: The positive control 2,4-dinitrochlorobenzene (DNCB) did not meet the crtieria for acceptance (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%) in the first two replicates, tehrefore the results of the test substance were considered invalid. The assay was repeated with 3 additional replicates, and DNCB met the acceptance criteria for these additional replicates. The positive control nickel sulfate (NiSO4) met the acceptance crtieria in all 5 replicates.
Applicant's summary and conclusion
- Interpretation of results:
- other: Sensitizing
- Conclusions:
- The in-vitro Human Cell Line Activation Assay (hCLAT) was used to assess Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone for its potential to activate dendritic cells through measurement of markers CD86 and CD54. A positive result was obtained for CD54 (RFI≥ 200%) in two out of three replicates which resulted in an overall positive h-CLAT prediction. However, this study only gives information on one key event of the skin sensitization AOP and should be assessed with other information within a defined approach or weight of evidence.
- Executive summary:
In an OECD TG 442E hCLAT assay, Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone was exposed to THP-1 cells at 67.5, 56.2, 46.7, 38.8, 32.3, 27.0, 22.5, and 18.8 ug/ml based on the initial cytotoxicity screens. A positive result was obtained for CD54 (RFI≥ 200%) in two out of three replicates which resulted in an overall positive h-CLAT prediction. Based on these results, Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone would be predicted as capable of activating dendritic cells (key event #3). Therefore the results of this assay are considered positive and should be used within a weight of evidence for concluding on the skin sensitization potential of Hydrazine Carboximidamide, 2-((2-hydroxyphenyl)methylene)-, reaction products with 2 undecanone under the Regulation (EC) 1272/2008 on classification, labeling, and packaging of substances and mixtures (CLP).
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