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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 809-934-0 | CAS number: 627034-93-9
- 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:
- 26 September 2018 to 01 October 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
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:
- updated 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:
- 23 July 2009
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 2-ethoxyethyl ethyl carbonic acid ester
- EC Number:
- 809-934-0
- Cas Number:
- 627034-93-9
- Molecular formula:
- C7H14O4
- IUPAC Name:
- 2-ethoxyethyl ethyl carbonic acid ester
- Test material form:
- liquid
- Details on test material:
- - Appearance: clear, colourless liquid
- Storage conditions: room temperature, in the dark
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- Recommended in international guidelines
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Source: EpiSkin Laboratories, Lyon, France
- Tissue lot number: 18-EKIN-039
- Delivery date: 25 September 2018
TEST FOR DIRECT MTT REDUCTION
- A test material 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 material is checked for the ability to directly reduce MTT according to the following procedure: 10 μL of the test material 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 material turns blue/purple, the test material 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 COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test material is present in the tissues when the MTT viability assay is performed.
- 10 μL of test material was added to 90 μL of sterile water. After mixing for 15 minutes on a plate shaker a visual assessment of the colour 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 Colour Satisfactory: Yes
Agar Medium Colour Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 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 material and each control material. The tissues were incubated at 37 °C, 5 % CO2 in air overnight.
MAIN TEST
- Application of Test Material (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 material for an exposure period of 15 minutes. The test material was applied topically to the corresponding tissues ensuring uniform covering. 10 μL (26.3 μL/cm²) of the test material 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 material the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the centre). 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 material). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
REMOVAL OF TEST MATERIAL AND CONTROLS
- 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 material. 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.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37 °C
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 homogenise the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C 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 labelled 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 coloured solution.
- For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
NUMBER OF REPLICATE TISSUES: 3
DATA EVALUATION
- Quantitative MTT Assessment (Percentage Tissue Viability): For the test material 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 OD570 of the test material / mean OD570 of the negative control) x 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:
Relative mean tissue viability is ≤ 50 %: Irritant (H314 or H315 Category 1 or 2)
Relative mean tissue viability is > 50 %: Non-irritant (Not classified for irritation) - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 10 µL (26.3 μL/cm²)
NEGATIVE CONTROL
- Amount(s) applied: 10 µL
POSITIVE CONTROL
- Amount(s) applied: 10 µL
- Concentration: 5 % w/v - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours followed by 3 hours with MTT
- Number of replicates:
- 3
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Mean
- Value:
- 97.3
- 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 material did not turn blue or purple which indicated that the test material did not directly reduce MTT.
ASSESSMENT OF THE COLOUR INTERFERENCE WITH THE MTT ENDPOINT
- The solution containing the test material was colourless. It was therefore unnecessary to run colour correction tissues.
MAIN TEST RESULTS
- The relative mean viability of the test material treated tissues was 97.3 % after a 15-minute exposure period and 42-hour post-exposure incubation period.
- It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.
QUALITY CRITERIA
- The relative mean tissue viability for the positive control treated tissues was 5.2 % relative to the negative control treated tissues and the standard deviation value of the viability was 2.7 %. The positive control acceptance criteria were therefore satisfied.
- The mean OD570 for the negative control treated tissues was 0.706 and the standard deviation value of the viability was 8.3 %. The negative control acceptance criteria were therefore satisfied.
- The standard deviation calculated from individual tissue viabilities of the three identically test material treated tissues was 6.8 %. The test material acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Table 1: Mean OD570 Values and Viabilities for the Negative Control, Positive Control and Test Material
Treatment |
OD570 of Tissues |
Mean OD570 of Triplicate Tissues |
± SD of OD570 |
Relative Individual Tissue Viability (%) |
Relative Mean Viability (%) |
± SD of Relative Mean Viability (%) |
Negative Control |
0.642 |
0.706 |
0.059 |
90.9 |
100* |
8.3 |
0.720 |
102.0 |
|||||
0.757 |
107.2 |
|||||
Positive Control |
0.056 |
0.037 |
0.019 |
7.9 |
5.2 |
2.7 |
0.036 |
5.1 |
|||||
0.018 |
2.5 |
|||||
Test Material |
0.742 |
0.687 |
0.048 |
105.1 |
97.3 |
6.8 |
0.656 |
92.9 |
|||||
0.663 |
93.9 |
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 in accordance with EU criteria
- Conclusions:
- Under the conditions of this study, the test material was not irritating.
- Executive summary:
The skin irritation potential of the test material was investigated in accordance with the standardised guidelines OECD 439 and EU Method B.46, under GLP conditions.
The purpose of this test was to evaluate the skin irritation potential of the test material using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test material by means of the colorimetric MTT reduction assay. Cell viability is 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 material treated tissues relative to the negative controls.
Triplicate tissues were treated with the test material 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. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing 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-labelled 96-well plate. The optical density was measured at 570 nm.
The relative mean viability of the test material treated tissues was 97.3 % after the 15-minute exposure period and 42-hours post-exposure incubation period. The quality criteria required for acceptance of results in the test were satisfied.
Under the conditions of this study, the test material was not irritating.
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.