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EC number: 947-375-5 | 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
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Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20-23 March 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study performed according to OECD Guideline 439 without any deviation.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- adopted 28 July 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- dated 23 July 2009
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- 27 April 2017
Test material
- Test material form:
- solid
- Details on test material:
- Name: cocoa oleoresine F9202
Batch No: K17 017-1
Appearance: dark brown sticky solid
CAS No: 84649-99-0
Production date: 17 January 2017
Expiry date: 16 January 2019
Storage condition of test material: room temperature
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No. of test material: K17 017-1
- Date received: 21 February 2017
- Manufacturing date: 17 January 2017
- Expiry date: 16 January 2019
- Purity test date: January 2017
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Used as supplied
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Cell source:
- other: foreskin
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- 0.50 cm² reconstructed epidermis (Episkin SA, RHE/S/17 Batch No. 17-RHE-035) were received on 21 March 2017.
- On the same day, the insert (filter + epidermis) was gently removed from the agarose while avoiding leaving agarose on the polycarbonate filter. The inserts were placed in 6 wells culture plate which had been previously filled with 1 mL of growth medium (Episkin SA) during 2 hours and 35 minutes. Then just before treatment, the inserts were placed in 24 wells culture plate which had been previously filled with 300 µL of maintenance medium (Episkin SA).
TREATMENT
- The test item was applied, as supplied, on a nylon mesh, at the approximate dose of 16 mg, then applied on the epidermal surface of 3 living human skin models during 42 minutes at room temperature.
- In the same experimental conditions, a positive control (5% SDS) and a negative control (DPBS) were carried out. The 5% SDS solution was prepared by weighing 0.5 g of SDS in a 10 mL volumetric flask qsp 10 mL of distilled water. Then, the preparation was magnetically stirred, just before the treatment. To ensure a good contact with the epidermises, during all the treatment period, the test item was recovered with a nylon mesh provided by Episkin SA.
REMOVAL OF TEST MATERIAL AND CONTROLS
- 42 minutes after the test item application, the nylon mesh was removed and the human epidermises were washed with 25 x 1 mL of DPBS. The rinsed tissues were checked for any coloration and noted to be slightly brown instead of being whitish as for the coloration of the negative control tissues. Residual test item with brown coloration was noted on all Reconstructed Human epidermis after the rinse. They were incubated for a 41 hours and 35 minutes post-treatment incubation period in fresh medium at 37°C, 5% CO2. Then, the epidermises were put in contact with the MTT solution.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- The cell viability was quantified by the measurement of the cell succinate dehydrogenase activity. This enzyme was responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS No. 298-93-1)] reduction into blue formazan crystal that is quantitatively measured by Optical Density (OD) after extraction from tissues. The measured OD were proportional to the number of living cells.
- The skin samples were placed in 300 µL of a MTT solution at 1.0 mg/mL for 3 hours at 37°C, 5% CO2. The precipitated blue formazan product was then extracted using isopropanol during 2 hours under gentle agitation in the dark, and the concentration of formazan was measured by determining the OD (Optical Density) at 570 nm, just after dilution of the extracts (1:2 in isopropanol).
- The OD of MTT extract was measured in triplicate. The measurement of OD was performed using the ELx800 absorbance microplate reader (controlled every year and calibrated if necessary) supplied by BioTek and the validated software Gen5 ELISA V1.05.11 supplied by BioTek.
VIABILITY CALCULATION:
- The results were expressed as a viability percentage compared with the negative control: viability % = (mean OD test item / mean OD negative control) * 100
- Data from individual replicate tissues (OD values and calculated percent tissue viability data for the test item and controls), mean percent tissue viability and standard deviation for each individual test item and control were reported in Table 7.3.1/1.
PREDICTION MODEL / DECISION CRITERIA
The OD values obtained for each test sample were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%. The cut-off values for the prediction of irritation associated with the RHE models were as follows:
- The test item is considered to be non-irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is >50%.
- The test item is considered to be irritant to skin if the tissue viability after 42 minutes of exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is "non-corrosive". In accordance with Regulation EC No. 1272/2008, the test item has to be classified in Category 2 “Irritant”. The corresponding hazard statement is “H315: Causes skin irritation” with the signal word “Warning”.
- The test item is considered to be irritant or corrosive to skin if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤50% and in absence of information on a skin corrosion test. In accordance with the Regulation (CE) No.1272/2008 and in absence of information on a skin corrosion test, the item has to be classified in Category 2 "Irritant" or in Category 1 "Corrosive". The corresponding hazard statement is respectively, "H315: Causes skin irritation" with the signal word "Warning" or "H314: Causes severe skin burns and eye damage" with the signal word "Danger". - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg
- Concentration (if solution): Undiluted - Duration of treatment / exposure:
- 42 minutes at room temperature
- Duration of post-treatment incubation (if applicable):
- 41 hours and 35 minutes post-incubation period at 37°C, 5% CO2
- Number of replicates:
- 3 living human skin models
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- mean
- Run / experiment:
- main test (duration of exposure: 42 minutes)
- Value:
- 105.6
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- PRELIMINARY TESTS
- Test for direct interaction with MTT:
A yellow solution with test item at the bottom of the well was observed after 3 hours of incubation between 36.1°C and 37.0°C, 5% CO2. Therefore, there was no direct interaction between the test item and MTT.
- Spectral analvsis of the test item in isopropanol:
A yellow solution was obtained after 2 hours of incubation at ambient temperature with gentle shaking. The mean of the corrected OD (blank subtracted) was 0.025 which was less than 0.08 (value corresponding to approximately twice the OD of the extracting solvent). Therefore the test item was considered not to interfere with the MTT assay and there was no need to add non-specific coloration controls to the study.
MTT VIABILITY ASSAY RESULTS
- The mean percent viability of the treated tissues was 105.6% (considered as 100%), versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate).
- The mean percent tissue viabilities obtained with the negative controls and positive controls were within the range of historical data and therefore validated the experiment.
Any other information on results incl. tables
Table 7.3.1/1: Main test - Individual and mean OD values and tissue viabilities for the test item, the negative and positive controls
Skin |
OD |
Mean OD / disc (#) |
Mean OD / product |
Viability % |
Mean viability % |
Standard deviation (SD) |
|
Negative control |
1 |
0.731 |
0.768 |
0.868 |
88.5 |
100.0 |
11.2 |
0.784 |
|||||||
0.791 |
|||||||
2 |
0.876 |
0.872 |
100.5 |
||||
0.880 |
|||||||
0.861 |
|||||||
3 |
0.970 |
0.963 |
111.0 |
||||
0.856 |
|||||||
1.063 |
|||||||
Positive control |
1 |
0.012 |
0.012 |
0.012 |
1.4 |
1.4 |
0.2 |
0.012 |
|||||||
0.012 |
|||||||
2 |
0.012 |
0.011 |
1.3 |
||||
0.011 |
|||||||
0.010 |
|||||||
3 |
0.015 |
0.014 |
1.6 |
||||
0.014 |
|||||||
0.014 |
|||||||
Test item |
1 |
0.894 |
0.937 |
0.916 |
108.0 |
105.6 |
10.2 |
0.960 |
|||||||
0.957 |
|||||||
2 |
0.989 |
0.992 |
114.3 |
||||
0.981 |
|||||||
1.007 |
|||||||
3 |
0.808 |
0.819 |
94.4 |
||||
0.809 |
|||||||
0.841 |
#: mean of 3 values (triplicate of the same extract)
OD: optical density
Acceptability criteria:
- SD ≤ 18%;
- Negative control: OD value of the 3 replicates in the range ≥0.8 and ≤3.0.
OD was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range >=0.4 and =<1.5 for the negative control.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
- Executive summary:
An in vitro skin irritation test using the Reconstructed Human Epidermis (SkinEthic RHE® model) was performed according to the OECD Guideline 439 and in compliance with GLP to predict the acute skin irritation potential of the test item.
As the test item was pasty, it was applied, as supplied, on a nylon mesh then on epidermis, so that to cover the entire surface of the epidermis (corresponding approximatively to 16 mg). The test substance was applied on 3 living Reconstructed Human epidermis (SkinEthic RHE® model) during 42 minutes, followed by a rinse with 25 mL of DPBS and a 41-hour and 35-minute post-incubation period at 37°C, 5% CO2. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.
In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. In the main test, the mean percent viability of the treated tissues was 105.6% (considered as 100%), versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate). The mean percent tissue viabilities obtained with the positive control and negative controls were within the range of historical data and therefore validated the experiment.
Under the test conditions and in accordance with Regulation EC No. 1272/2008, the test item was considered as non-irritant to skin. It corresponds to UN GHS No Category. No hazard statement or signal word is required.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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