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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 695-735-2 | CAS number: 68489-14-5
- 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:
- May 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Principles of method if other than guideline:
- not applicable
- GLP compliance:
- yes
Test material
- Reference substance name:
- ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate
- EC Number:
- 695-735-2
- Cas Number:
- 68489-14-5
- Molecular formula:
- C15H27NO3
- IUPAC Name:
- ethyl 2-{[(1R,2S,5R)-5-methyl-2-(propan-2-yl)cyclohexyl]formamido}acetate
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Symrise, WS-5, 83100003
- Expiration date of the lot/batch: 09 December 2018
- Purity test date: 99.76%
- Physical state/Appearance: White solid
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: three-dimensional reconstructed human epidermis model (EPISKINTM model)
- Cell source:
- other: adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen.
- Details on animal used as source of test system:
- not applicable
- Justification for test system used:
- EpiSkinTM reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.
The procedure followed is based on the recommended EpiSkin™ SOP, Version 1.8 (February 2009), ECVAM Skin Irritation Validation Study and acceptable to the current OECD guideline. - Vehicle:
- unchanged (no vehicle)
- Remarks:
- neat test item WS-5
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKINTM reconstructed human epidermis model
- Supplier: SkinEthic Laboratories, Lyon, France
- EpiSkinTM Tissues (0.38cm2) lot number: 17-EKIN-028
- Date received: 11 July 2017
Maintenance Medium lot number: 17-MAIN3-028
Assay Medium lot number :17-ESSC-027
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): at 37 C, 5% CO2 in air
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: not specified
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours at 37 °C, 5% CO2 in air
- Spectrophotometer: Labtech LT-4500 microplate reader, (LT-4500 IFU DOC Revision No.1.0)
- Wavelength: 570 nm (without a reference filter).
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
To identify the possible interference with the MTT endpoint, WS-5 was checked for the ability to directly reduce MTT according to the following procedure:
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 mg 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.
PREDICTION MODEL / DECISION CRITERIA
The results of the assay are considered acceptable if the following assay acceptance criteria (EpiskinTM SOP February 2009 Version 108 ECVAM Skin Irritation validation Study) 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 OD570 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%. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: Approximately 10 mg (26.3 mg/cm2) of WS-5.
The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis.
NEGATIVE CONTROL
- Concentration (if solution): 10 µL of DPBS.
POSITIVE CONTROL
- Concentration (if solution): 10 µL of SDS 5% w/v
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. - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours at 37C, 5% CO2 in air
- Number of replicates:
- 3
Test system
- Details on study design:
- TEST SITE
- Area of exposure: Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.
The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 µL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis.
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes
- Time after start of exposure: at the end of the exposure period (after 15 min)
SCORING SYSTEM:
- Method of calculation:
For the test item the relative mean tissue viabilities 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). The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD570of test item / OD570of negative control) X 100
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 2
- Value:
- ca. 63.8
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100%
- Positive controls validity:
- valid
- Remarks:
- 22.7%
- Remarks on result:
- no indication of irritation
- Irritation / corrosion parameter:
- other: Mean OD570
- Remarks:
- triplicate tissues
- Run / experiment:
- 2
- Value:
- ca. 0.396
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 0.621
- Positive controls validity:
- valid
- Remarks:
- 0.141
- Remarks on result:
- no indication of irritation
Any other information on results incl. tables
Table 1. 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:
Criteria for in vitro interpretation |
Prediction |
EU CLP |
UN GHS |
Relative mean tissue viability is ≤50% |
Irritant |
H315 |
H315 |
Relative mean tissue viability is >50% |
Non-irritant |
Not classified for irritation |
Not classified or UN GHS Category 3 can not be determined |
Table 2. Mean OD570Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item
Item |
OD570of tissues |
Mean OD570of triplicate tissues |
±SDof OD570 |
Relative individual tissue viability (%) |
Relative mean viability (%) |
± SD of Relative mean viability (%) |
Negative Control Item DPBS |
0.581 |
0.621 |
0.051 |
93.6 |
100* |
8.2 |
0.604 |
97.3 |
|||||
0.679 |
109.3 |
|||||
Positive Control Item SDS |
0.212 |
0.141 |
0.069 |
34.1 |
22.7 |
11.0 |
0.137 |
22.1 |
|||||
0.075 |
12.1 |
|||||
Test Item WS-5 |
0.334 |
0.396 |
0.070 |
53.8 |
63.8 |
11.2 |
0.382 |
61.5 |
|||||
0.471 |
75.8 |
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: not classified for Irritation according to EU CLP
- Conclusions:
- Based on available data on skin irritaiton performed according the OECD TG 439, WS-5 is not classified for skin irritation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.
- Executive summary:
The purpose of this study performed according to OECD Test Guideline 439 was to evaluate the skin irritation potential of the test item, WS-5 using the EPISKIN TM reconstructed human epidermis model.
In the direct MTT Reduction assay, WS-5 did not turn blue or purple which indicated that the test item did not directly reduce MTT. The solution containing WS-5 produced no colour. It was therefore unnecessary to run colour correction tissues.
Triplicate human skin tissues were treated with the neat test item, WS-5 for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing 500 µL acidified isopropanol for extraction of formazan crystals out of the MTT‑loaded tissues.
At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µL samples were transferred to the appropriate wells of a pre‑labeled 96‑well plate. The optical density was measured at 570 nm.
The relative mean viability of WS-5 treated tissues was 63.8% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. It was therefore considered unnecessary to perform IL-1 alpha analysis as the results of the MTT test were unequivocal.
Conclusions:
Based on the above results, the test item, WS-5 was classified as non-irritant according to OECD Test Guideline 439.
The following classification criteria apply:
EU DSD and CLP - not classified for Irritation.
UN GHS - not classified for Irritation (category 3 can not be determined).
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.