Registration Dossier
Registration Dossier
Data platform availability banner - registered substances factsheets
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Diss Factsheets
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 214-262-0 | CAS number: 1118-39-4
- 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 irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- Experimental start date 24 May 2016, experimental completion date 30 May 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- 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
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Myrcenyl acetate
- EC Number:
- 214-262-0
- EC Name:
- Myrcenyl acetate
- Cas Number:
- 1118-39-4
- Molecular formula:
- C12H20O2
- IUPAC Name:
- myrcenyl acetate
Constituent 1
In vitro test system
- Test system:
- other: EPISKIN reconstructed human epidermis model
- Source species:
- human
- Cell type:
- other: adult human-derived epidermal keratinocytes
- Source strain:
- other: Not applicable
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- TEST SYSTEM:
EPISKIN™ Reconstructed Human Epidermis Model Kit:
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 24 May 2016
EpiSkinTM Tissues (0.38cm2) lot number: 16-EKIN-021
Maintenance Medium lot number: 16-MAIN3-035
Assay Medium lot number: 16-ESSC-021
The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
PRE-TEST PROCEDURE:
Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay:
The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt by mitochondrial succinate dehydrogenase in viable cells.
One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.
Test for Direct MTT Reduction:
As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure:
10 µL of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control.
If the MTT solution containing the test item turns blue or purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.
Assessment of Color Interference with the MTT endpoint:
A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed.
10 µL of test item was added to 90 µL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the color was made.
PRE-INCUBATION (Day 0: TISSUE ARRIVAL):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
MAIN TEST:
Application of Test Item and Rinsing (Day 1):
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12 well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 10 µL (26.3 µL/cm2) of the test item was applied to the epidermis surface. Triplicate tissues treated with 10 µL of DPBS served as the negative controls and triplicate tissues treated with 10 µL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
MTT Loading/Formazan Extraction (Day 3):
Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination.
2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 µL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Absorbance/Optical Density Measurements (Day 6):
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution.
For each tissue, duplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 µL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 562 nm (without a reference filter) using the Anthos 2001 microplate reader.
DATA EVALUATION:
Quantitative MTT Assessment (Percentage Tissue Viability):
For the test item the mean tissue viability obtained after the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period were compared to the mean of the negative control treated tissues (n=3) to generate the relative mean tissue viability. The relative mean viability was calculated in the following way:
Relative mean viability (%) = ((mean OD562 of test item) / (mean OD562 of negative control)) * 100
Classification of irritation potential is based upon relative mean tissue viability following the 15-Minute exposure period followed by the 42-Hour post-exposure incubation period according to the following table:
Criteria for in vitro interpretation: Prediction
Relative mean tissue viability is ≤50%: Irritant
Relative mean tissue viability is >50%: Non-irritant
Quality Criteria:
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.
Major Computerized Systems:
The following computerized system was used in the study:
Delta Building Monitoring System - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- 10 µL
- Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- other: Relative mean viability (%)
- Value:
- 16.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- Direct MTT Reduction:
The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
Assessment of Color Interference with the MTT endpoint:
The solution (10 µL of test item in 90 µL of sterile water) containing the test item was colorless. It was therefore unnecessary to run color correction tissues.
Test Item, Positive Control Item and Negative Control Item:
The individual and mean OD562 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given under "Any other information on results incl. tables". The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given under "Any other information on results incl. tables".
The relative mean viability of the test item treated tissues was 16.2% after a 15-Minute exposure period and 42-Hour post-exposure incubation period.
It was considered unnecessary to perform IL-1alpha analysis as the results of the MTT test were unequivocal.
Quality Criteria:
The relative mean tissue viability for the positive control treated tissues was 4.9% relative to the negative control treated tissues and the standard deviation value of the viability was 0.9%. The positive control acceptance criteria were therefore satisfied.
The mean OD562 for the negative control treated tissues was 1.293 and the standard deviation value of the viability was 3.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 11.9%. The test item acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item
Item |
OD562 of tissues |
Mean OD562 of triplicate tissues |
+/- SD of OD562 |
Relative individual tissue viability (%) |
Relative mean viability (%) |
+/- SD of Relative mean viability (%) |
Negative Control Item |
1.265 |
1.293 |
0.045 |
97.8 |
100* |
3.5 |
1.345 |
104.0 |
|||||
1.269 |
98.1 |
|||||
Positive Control Item |
0.076 |
0.063 |
0.011 |
5.9 |
4.9 |
0.9 |
0.058 |
4.5 |
|||||
0.055 |
4.3 |
|||||
Test Item |
0.387 |
0.210 |
0.154 |
29.9 |
16.2 |
11.9 |
0.124 |
9.6 |
|||||
0.118 |
9.1 |
OD = Optical Density
SD = Standard deviation
* = The mean viability of the negative control tissues is set at 100%
Applicant's summary and conclusion
- Interpretation of results:
- other: Skin irritant Category 2
- Remarks:
- according to EU CLP criteria and its amendments (1272/2008)
- Conclusions:
- The relative mean tissue viability after 15 minutes treatment with the substance compared to the negative control tissues was 16.2%. Since the mean relative tissue viability for the substance was below 50%, the substance is considered to be a skin irritant Category 2 according to GHS.
- Executive summary:
The possible skin irritation potential of the substance was evaluated using the EPISKIN reconstructed human epidermis model in compliance with OECD TG 439 and GLP principles. Triplicate skin tissues were treated by application of 10 µL undiluted test substance for an exposure period of 15 minutes. After 42 hours post-exposure incubation period, determination of the cytotoxic (irritancy) effect was performed. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test substance treated tissues relative to the negative controls. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissues was 16.2%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritating to the skin. The positive control had a relative mean tissue viability of 4.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, for the substance, the positive and the negative control, indicating that the test system functioned properly. Based on the result of this study the substance is considered to be irritating to the skin.
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.