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EC number: 288-327-7 | CAS number: 85711-66-6
- 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:
- 2021
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 021
- Report date:
- 2021
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))
- Version / remarks:
- Data Analysis Section 7.5.6 of the protocol describes the steps followed in typical data analysis. They include a description of the gates applied to the data to determine viability and geometric mean. The test article, MTDID 60827, however required an addition gating step which is not described in this section. The additional gating was required to obtain more accurate data due to observed precipitate in the dot plots in the Flow Cytometer. Without the additional gate, the analysis applied to cells and test article residue was skewed by the values collected for the residual test article. The h-CLAT is designed to measure the presence of CD86 and CD54 on the surface of the cells; therefore, it is important to isolate the cell population when performing the data analysis. In the case of this test article, an additional gate was necessary to eliminate the very small particles of debris (presumably residualtest article) remaining in the well at the time of data collection. Therefore, there was a significant effect on the procedure performed and those additional procedural steps (gating) resulted in a significant change in the experimental results. These changes were necessary to ensure the data analyzed was relevant to cellular expression of CD54 and CD86. There was no significant effect on the overall quality, integrity, and outcome of the study.
- Deviations:
- yes
- Remarks:
- See 'Version/remarks' field for the description of the deviations.
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- human Cell Line Activation Test (h-CLAT)
- Justification for non-LLNA method:
- The Human Cell Line Activation Test (h-CLAT) was used to assess the skin sensitization potential of the
test article by monitoring the upregulation of cell surface markers, CD54 and CD86, on the surface of
human acute monocytic leukemia cells (THP-1). The upregulation of CD54 and CD86 in response to a skin
sensitizer is correlated to dendritic cell activation, which is the third key event of the skin sensitization
pathway.
Test material
- Reference substance name:
- Resin acids and Rosin acids, esters with glycerol and diethylene glycol
- EC Number:
- 288-327-7
- EC Name:
- Resin acids and Rosin acids, esters with glycerol and diethylene glycol
- Cas Number:
- 85711-66-6
- Molecular formula:
- UVCB substance
- IUPAC Name:
- esterification product of (1R,4aR,4bR,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,4b,5,6,10,10a-decahydrophenanthrene-1-carboxylic acid; (1R,4aR,4bS,10aR)-1,4a-dimethyl-7-(propan-2-ylidene)-1,2,3,4,4a,4b,5,6,7,9,10,10a-dodecahydrophenanthrene-1-carboxylic acid; (1R,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,5,6,9,10,10a-decahydrophenanthrene-1-carboxylic acid; (1R,4aS,10aR)-1,4a-dimethyl-7-(propan-2-yl)-1,2,3,4,4a,9,10,10a-octahydrophenanthrene-1-carboxylic acid and 2,2'-oxydiethanol and propane-1,2,3-triol
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: 3M, lot: K02007
- Purity, including information on contaminants, isomers, etc.: no data.
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 30 °C (Room Temp)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: no data
- Stability in the medium, i.e. sensitivity of the test material to hydrolysis and/or photolysis: no data
- Solubility and stability of the test material in the solvent/vehicle and the exposure medium: The top stock dilution of the test article was found to be non-soluble at 100 mg/mL in Saline or at 500 mg/mL in DMSO. Therefore, the test article final stock was attempted to be solubilized at a concentration at 10 mg/mL directly in THP-1 Culture Medium
- Reactivity of the test material with the incubation material used (e.g. plastic ware): no data
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing (e.g. warming, grinding): Grinding into poweder, vortexing and sonicating.
- Preliminary purification step (if any):
- Final concentration of a dissolved solid, stock liquid or gel: 39 to 5000 µg/mL
- Final preparation of a solid (e.g. stock crystals ground to fine powder using a mortar and pestle): no data.
FORM AS APPLIED IN THE TEST (if different from that of starting material)
- Specify the relevant form characteristics if different from those in the starting material, such as state of aggregation, shape of particles or particle size distribution: The test article stock and working dilutions were observed to be cloudy pink with white particles non-viscous suspension.
OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added:
In vitro test system
- Details of test system:
- THP-1 cell line [442E]
- Details on the study design:
- 442E
PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution
: test article will be dissolved to the maximum appropriate concentration determined in the solubility test, or up to a maximum contration of 100 mg/ml in saline, or a maximum concentration of 500 mg/ml in DMSO.
- Preparation of the test chemical serial dilutions
: seven serial doses using a typical dilution factor of 1.2 to 1.5 will be prepared such that 8 doses will be tested in the definitive assay.
- Preparation of the positive controls
: 2,4-Dinitrochlorobenzene at stock concentration of 2 mg/ml
- Preparation of the solvent, vehicle and negative controls : the solvent control will be culture medium for test articles diluted in saline or culture medium. The solvent control will be DMSO in culture medium for test articles diluted in DMSO. A single concentration of the solvent control(s) will be prepared in culture medium and dosed on the cells in the same manner as the test article(s) so the final concentration of saline on the cells is 1% and DMSO is 0.2%.
- Stable dispersion obtained
DOSE RANGE FINDING ASSAY:
- Highest concentration used
: 500 mg/ml
- Solubility in solvents
: The top stock dilution of the test article was found to be non-soluble at 100 mg/mL in
Saline or at 500 mg/mL in DMSO. Therefore, the test article final stock was attempted to be solubilized
at a concentration at 10 mg/mL directly in THP-1 Culture Medium, after grinding the test article into a
powder, and then vortexing and sonicating. Using this strategy, the final concentration of the test article
on the cells ranged from 39 to 5000 µg/mL.
- Cytotoxicity assessment performed
A preliminary (dose range finding assay) was performed to determine the viability of the THP-1 cells after
24 ± 0.5 hour exposure to 8 test article concentrations. The CV75, which is the calculated test article
concentration leading to 75% cell viability resulted in 2230 µg/mL.
APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 8
SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density)
: cells resuspended in fresh medium to a density of 2.0 x 10^6 cells/ml and 500 microliters of the cell supsension will be seeded into the appropriate wells. Resulting in 1.0x10^6 cells/well.
- Incubation conditions
: 37 degree in a humidified atmosphere of 5 (+/-) 1% CO2 in air.
- Washing conditions
: Centrifugation 200 to 300 g for 5 minutes at 4 degrees C.
MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used : MACSQuant Analyzer (Miltenyi) with three laser system capable of both FITC and PI acquisition
- Plate used : 24 well plate
- Propidium iodide staining/cytotoxicity measurements : After 24 hours of exposure and cell rinsing, Propidium Iodide was added to the samples to a final concentratino of 0.625 ug/mL.
- Preparation for CD54 and/or CD86 expression measurements/cell staining : PI uptake was analyzed via flow cytometry. Cells stained with PI represent the non-viable cell population and will be gated out to identify the viable populations. - Vehicle / solvent control:
- cell culture medium
- Negative control:
- not applicable
- Positive control:
- dinitrochlorobenzene (DNCB) [442E]
Results and discussion
In vitro / in chemico
Resultsopen allclose all
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- EC200, CD54 [442E]
- Value:
- 1 239 µg/mL
- Cell viability:
- Cell viability was approximately 93%
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- EC200, CD54 [442E]
- Value:
- 822 µg/mL
- Cell viability:
- Cell viability was approximately 94%
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 1
- Parameter:
- other: CD 86 RFI > 150
- Remarks:
- CD 86 RFI > 150. The lowest concentration tested (754 ug/mL) elicited an RFI of 169.
- Value:
- 754 µg/mL
- Cell viability:
- 94.45%
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Group:
- test chemical
- Run / experiment:
- run/experiment 2
- Parameter:
- EC150, CD86 [442E]
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- not determinable
- Remarks:
- The highest concentration (2250 ug/mL) elicted an RFI of 80.59 so an EC150 could not be calculated for this run.
- Group:
- test chemical
- Run / experiment:
- mean
- Parameter:
- CV75 [442D and 442E]
- Value:
- 2 230 µg/mL
- Outcome of the prediction model:
- positive [in vitro/in chemico]
Applicant's summary and conclusion
- Interpretation of results:
- other: See 'Remarks'
- Remarks:
- The test article is predicted to have skin sensitization potential within Key Event 3 (KE3) of the skin sensitization adverse outcome pathway (AOP).
- Conclusions:
- Based on the results of the study (EC200 = 822 ug/mL and EC150 <750 ug/mL), rosin acid derivative is predicted to have skin sensitization potential within Key Event 3 (KE3) of the skin sensitization adverse outcome pathway (AOP).
- Executive summary:
The skin sensitization potential of rosin acid derivatives was evaluated in the h-CLAT assay. The study was conducted according to OECD 422E in compliance with OECD GLP. The sensitization potential of rosin acid derivatives was evaluated by measuring the changes in the expression of cell surface markers CD54 and CD86 associated with the process of dendritic cell activation in the human leukemia cell line, THP-1, following exposure to a test article. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescently labelled antibodies for CD54 and CD86. The upregulation of CD54 and CD86 in response to a skin sensitizer is correlated to dendritic cell activation, which is the third key event of the skin sensitization pathway. The test article was solubilized directly in THP-1 culture medium, after grinding the test article into a powder, then vortexing and sonicating. The final concentration of the test article on the cells ranged from 39 to 5000 ug/mL. THP-1 cells were seeded at a density of 2.0 x 10^6 cells/mL in culture medium in 24 -well plate format. Cells were exposed to rosin acid derivative at 754, 904, 1085, 1302, 1563, 1875, 2250, and 2700 ug/mL, the positive control (2,4-Dinitrochlorobenzene at 2 mg/ml) or negative control (culture media). Following a 24-hour exposure, cytotoxicity was assessed via Propidium Iodine (PI) staining. Cells were incubated with CD54 and CD86 antibodies for 30 minutes. Following incubation, CD54 and CD86 expression as well as PI uptake (cytotoxicity measurement) was measured via flow cytrometry. The h-CLAT is designed to measure the presence of CD86 and CD54 on the surface of the cells; therefore, it is important to isolate the cell population when performing the data analysis. In the case of this test article, an additional gate was necessary to eliminate the very small particles of debris (presumably residual test article) remaining in the well at the time of data collection. Therefore, there was a significant effect on the procedure performed and those additional procedural steps (gating) resulted in a significant change in the experimental results. These changes were necessary to ensure the data analyzed was relevant to cellular expression of CD54 and CD86. There was no significant effect on the overall quality, integrity, and outcome of the study.
The results from the first assay performed were deemed invalid due to the positive control values did not meet validation criteria. Two definitive assays were performed following the first assay and only results from the two valid assays have been reported. Based on the results of the assays, rosin acid derivative had CD54 EC200 values of 1239 and 822 ug/mL and CD86 EC150 values of <750 and >1875 ug/mL in the first and second experiments, respectively. Based on the results of the study (EC200 = 822 ug/mL and EC150 <750 ug/mL), rosin acid derivative is predicted to have skin sensitization potential within Key Event 3 (KE3) of the skin sensitization adverse outcome pathway (AOP).
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