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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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: 202-235-6 | CAS number: 93-28-7
- 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:
- 04 July to 29 September 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted in compliance with OECD Guideline No. 439 without any deviation.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
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:
- 23 July 2009
- Deviations:
- no
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz (inspected on July 13-16, 2015 / signed on September 14, 2015)
Test material
- Reference substance name:
- 4-allyl-2-methoxyphenyl acetate
- EC Number:
- 202-235-6
- EC Name:
- 4-allyl-2-methoxyphenyl acetate
- Cas Number:
- 93-28-7
- Molecular formula:
- C12H14O3
- IUPAC Name:
- 4-allyl-2-methoxyphenyl acetate
- Test material form:
- liquid
- Details on test material:
- - Physical state/Appearance: Colourless to pale yellow liquid, slightly viscous.
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Stable under normal temperatures and pressure
Constituent 1
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: human-derived epidermal keratinocytes
- Justification for test system used:
- Following the REACH top-down strategy, the EPISKIN™ Reconstructed Human Epidermis Model method was used to assess skin irritation as recommended in the OECD test guideline No. 439.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin™ kits are purchased from SkinEthic Laboratories (69007 Lyon, France).
- Tissue batch number(s): 17-EKIN-030
- Expiry date: 31 July 2017
- Date of initiation of testing: 04 July 2017
The EpiSkin™ tissue consists of NHEK, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate and reached Envigo CRS GmbH on 25 July 2017. EpiSkin™ tissues were transferred to 12-well plates with maintenance medium and the pre-incubation phase of the EpiSkin™ tissues started.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: After the end of the treatment interval the inserts were removed immediately from the 12-well plate. The tissues were be gently rinsed with PBS to remove any residual test material. Excess PBS were removed by gently shaking the inserts and blotting the bottom with blotting paper. The inserts were placed in the plates with 2 mL maintenance medium. Tissues were incubated for 42 ± 1 hour at 37 ± 1.5 °C, 5 ± 0.5% CO2.Number of washing steps not reported.
- Observable damage in the tissue due to washing: none reported
- Modifications to validated SOP: none
IL-1 ALPHA IMMUNOASSAY
If the results obtained by means of the MTT assay are unclear or borderline, additionally the IL-1 α concentration in the medium after 42 hours incubation can be determined by order of the Sponsor. For this purpose, samples of all treatment groups are taken from the wells. The plates are shaken for approximately 15 minutes to homogenise the released mediators in the medium before sampling. At least 1.6 mL medium from each well is taken and is stored in the freezer at ≤ -18 °C until analysis.
Following the instruction from the QuantikineTM kit the amount of released IL-1 α will be determined. Therefore 200 μL of each sample and the standard solutions is mixed with 50 μL Assay Diluent RD1C in the wells of the IL-1 α Microplate. After 2 hours incubation and washing steps 200 μL IL-1 α Conjugate is added to each well. Meanwhile the substrate solution is prepared by mixing Colour Reagent A and B in equal volumes. After 1 hour incubation and washing steps 200 μL of the substrate solution is added to each well. The plates are covered with the adhesive strips and incubated for further 20 min under light protection. After 20 min 50 μL of the stop solution is added to each well. The plates are read at 450 nm in a photometer (Versamax® Molecular Devices, software version 4.7.1) within 30 minutes. Each sample is tested in duplicate.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, software version 4.7.1
- Wavelength: 570 nm
- Filter: with 570 nm filter
- Linear OD range of spectrophotometer: not reported
FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: 2.1 mg/L (1.5 mg/L < IC50 < 3;0 mg/L)
- Morphology: well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: on blood of the same donors: absence of HIV1 and 2 antibodies, of hepatitis C antibodies, and hepatitis B antigen HBs. On epidermal cells of the same donors: absence of bacteria, fungus and mycoplasma.
- Reproducibility: the results for the positive and negative controls are within the historical ranges obtained by Envigo CRS GmbH in the previous eleven months (means. rel. standard deviation. and ranges) of Envigo CRS GmbH
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
Not needed
NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤ 50% after 15 minutes of exposure.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is > 50% after 15 minutes of exposure.
- For the current test, an irritation potential of a test item is predicted if the release of IL-1 α is above the threshold of 60 pg/mL. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- The test item was used as supplied (undiluted).
- Amount(s) applied (volume or weight with unit): approximately 10 μL (26.3 μL/cm² according to guideline)
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL (in DPBS)
POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 μL
- Concentration (if solution): Sodium Dodecyl Sulphate (SDS) at a 5% (w/v) aqueous solution - Duration of treatment / exposure:
- 15 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours
- Number of replicates:
- Triplicate tissues for test substance, negative and positive controls
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1
- Value:
- 52.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 100%
- Positive controls validity:
- valid
- Remarks:
- 6.9%
- Remarks on result:
- no indication of irritation
- Remarks:
- > 50 %
- Irritation / corrosion parameter:
- other: IL-alpha concentration (pg/L)
- Run / experiment:
- 1
- Value:
- 26.15
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Remarks:
- 3.14
- Positive controls validity:
- valid
- Remarks:
- 129.21
- Remarks on result:
- no indication of irritation
- Remarks:
- < 50 pg/mL
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: no
- Colour interference with MTT: no
DEMONSTRATION OF TECHNICAL PROFICIENCY: yes
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.672 and the standard deviation value of the viability was 2.8%.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 6.9% relative to the negative control treated tissues and the standard deviation value of the viability was 11.9%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 5.6%. - Reference to historical values: All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.
Any other information on results incl. tables
Table 7.3.1/1: Results of the MTT assay
Test Group |
Absor-bance 570 nm |
Absor-bance 570 nm |
Mean Absor-bance 570 nm |
Mean Absor-bance 570 nm |
Mean Absor-bance of 3 Tissues |
Relative Viability [%] Tissue |
Relative Standard Deviation [%] |
Mean Rel. viability [%]** |
|
Blank |
0.038 |
0.039 |
0.038 |
|
|
||||
Negative Control |
0.718 |
0.684 |
0.701 |
0.663 |
0.672
|
98.7 |
2.8
|
100.0 |
|
0.759 |
0.705 |
0.732 |
0.694 |
103.2 |
|||||
0.717 |
0.677 |
0.697 |
0.659 |
98.1 |
|||||
Positive Control |
0.089 |
0.087 |
0.088 |
0.050 |
0.047
|
7.4 |
11.9
|
6.9 |
|
0.086 |
0.090 |
0.088 |
0.050 |
7.4 |
|||||
0.075 |
0.082 |
0.078 |
0.040 |
6.0 |
|||||
Test Item |
0.406 |
0.406 |
0.406 |
0.368 |
0.351 |
54.8 |
5.6 |
52.2 |
|
0.395 |
0.392 |
0.394 |
0.355 |
52.9 |
|||||
0.376 |
0.360 |
0.368 |
0.329 |
49.0 |
* Relative absorbance [rounded values]
** Mean relative absorbance [rounded values]
The mean relative absorbance value of the test item, corresponding to the cell viability, decreased to 52.2% (threshold for irritancy: ≤ 50%), consequently the test item was not irritant to skin.
This value is very close to the threshold for irritancy (≤ 50%), consequently, the results of the MTT test were considered borderline. It was considered necessary to perform IL-1α analysis.
Table 7.3.1/2: Results of the IL1 ELISA
Abs. 1 |
Abs. 2 |
Mean |
IL1αConc. |
|
Medium |
0.020 |
0.017 |
0.019 |
0.903 |
42h Negative Ctrl, Tissue 1 |
0.060 |
0.078 |
0.069 |
4.507 |
42h Negative Ctrl, Tissue 2 |
0.059 |
0.055 |
0.057 |
3.641 |
42h Negative Ctrl, Tissue 3 |
0.105 |
0.115 |
0.110 |
7.510 |
42h Positive Ctrl, Tissue 1 |
1.447 |
1.554 |
1.501 |
150.458 |
42h Positive Ctrl, Tissue 2 |
1.051 |
1.360 |
1.206 |
113.458 |
42h Positive Ctrl, Tissue 3 |
1.189 |
1.482 |
1.336 |
129.320 |
42h Test Item, Tissue 1 |
0.264 |
0.236 |
0.250 |
18.287 |
42h Test Item, Tissue 2 |
0.503 |
0.451 |
0.477 |
37.482 |
42h Test Item, Tissue 3 |
0.406 |
0.270 |
0.338 |
25.476 |
*the parallel
entrainment of a definite IL1α
standard led to a curve with the following formula:
y = 20,654x2+ 69,547x -
0,3903
The
IL1α concentration of the samples (y) was calculated by setting the value
Mean Abs. 1-2 (OD) as x.
Applicant's summary and conclusion
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the experimental conditions of this study, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
- Executive summary:
An in vitro skin irritation study was performed according to the OECD Guideline 439 and in compliance with GLP, using the EPISKINTM reconstructed human epidermis model.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. The test item was found to directly reduce MTT and therefore additional non-viable tissues were incorporated into the testing for correction purposes. 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‑labeled 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‑labeled 96‑well plate. The optical density was measured at 570 nm.
The relative mean viability of the test item treated tissues was 52.2% after the 15‑Minute exposure period and 42‑Hours post‑exposure incubation period. The amount of mean IL1 -alpha release is 26.15 pg/mL.
The relative mean tissue viability for the positive control treated tissues was 6.9% relative to the negative control treated tissues and the standard deviation value of the viability was 11.9%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.672 and the standard deviation value of the viability was 2.8%. 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 5.6%. The test item acceptance criterion was therefore satisfied.
All of the values for the negative and positive control groups fell within the historical ranges of the testing laboratory obtained in the previous six months. This was taken to show the correct functioning of the test system.
Under the experimental conditions of this study, the viability being above 50% and the IL1 -alpha release below 50 pg/mL, the test substance is not classified for skin irritation according to Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
This study is considered as acceptable and satisfies the requirement for skin irritation endpoint.
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.