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EC number: 944-753-1 | 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
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- Flash point
- Auto flammability
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- Oxidation reduction potential
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- Endpoint summary
- Stability
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- 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
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- Sediment toxicity
- Terrestrial toxicity
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- 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:
- 18 September 2017 - 16 October 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
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:
- 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:
- 20 July 2012
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- 4-methyl-1-oxaspiro[5.5]undec-4-ene
- Cas Number:
- 62062-89-9
- Molecular formula:
- C11-H18O
- IUPAC Name:
- 4-methyl-1-oxaspiro[5.5]undec-4-ene
- Reference substance name:
- 4-methyl-1-oxaspiro[5.5]undec-3-ene
- Cas Number:
- 62062-94-6
- Molecular formula:
- C11H18O
- IUPAC Name:
- 4-methyl-1-oxaspiro[5.5]undec-3-ene
- Reference substance name:
- 4-methylidene-1-oxaspiro[5.5]undecane
- Cas Number:
- 62062-84-4
- Molecular formula:
- C11H18O
- IUPAC Name:
- 4-methylidene-1-oxaspiro[5.5]undecane
Constituent 1
Constituent 2
Constituent 3
In vitro test system
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- other: epidermal keratinocytes
- Cell source:
- other: SkinEthic Laboratories, Lyon, France.
- Source strain:
- other: Not applicable
- Justification for test system used:
- In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- TEST SYSTEM
- EPISKIN Small Model (TM) (EPISKIN-SM (TM), 0.38 cm^2, Lot no.: 17-EKIN-041); a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded in 12-well plates on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen and cultured for 13 days.
On the day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for 2.5 hours at 37°C.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 36.6 - 37.1 °C
- Humidity (%): 65 - 85
REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: the tissues were washed once with phosphate buffered saline
- Observable damage in the tissue due to washing: no
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
NUMBER OF REPLICATE TISSUES: 3
CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, 25 μL sterile Milli-Q water was tested concurrently. At the end of the incubation period a color check was performed.
PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.
- Cell viability: The corrected OD (ODc) for each sample or control was calculated by subtracting the value of the blank mean (ODbl) from each reading (ODraw): ODc = ODraw – ODbl
The OD value representing 100% cell viability is the average OD of the negative controls (ODlt_u +MTT).
The % viability for each sample and the positive control is calculated as follows: %Viability = (ODc/mean ODlt_u+MTT) * 100
- Skin irritation is expressed as the remaining cell viability after exposure to the test item.
ACCEPTABILITY CRITERIA:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤ 18.
b) The mean relative tissue viability of the positive control should be ≤ 40% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤ 18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤ 18. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST ITEM:
25 μL directly on top of the tissue
NEGATIVE CONTOL:
- Amount applied: 25 μL Phosphate buffered saline
POSITIVE CONTROL
- Amount applied: 25 μL
- Concentration: 5% (aq) Sodium dodecyl sulphate
- Re-spread after 7 minutes contact time - Duration of treatment / exposure:
- 15 ± 0.5 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours and 3 hours with MTT
- Number of replicates:
- 3 for the test item, the negative and the positive control, each.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Value:
- 12
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks:
- Cell viability: 10%
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the absolute mean OD570 of the three tissues of the negative control were within the laboratory historical control data range (i.e., a mean of 1.115 ± 0.051) and the SD of the % viability was <18% (i.e., 4.5%)
- Acceptance criteria met for positive control: yes, the mean relative tissue viability of the positive control was <50% (i.e., 10%) and the SD of the % viability was <18% (i.e., 0.8%).
- Acceptance criteria met for variability between replicate measurements: yes, the SD calculated from individual % tissue viabilities of the three identically treated replicates was <18% (i.e., 5%).
- Since the mean relative tissue viability for the substance was below 50%, it is considered to be irritant.
Any other information on results incl. tables
Table Individual OD measurements (570 nm)
|
A (OD570) |
B (OD570) |
C (OD570) |
Negative control OD570measurement 1 OD570measurement 2 |
1.2414 1.1881 |
1.2934 0.9755 |
1.2003 1.0412 |
Test item OD570measurement 1 OD570measurement 2 |
0.2161 0.2166 |
0.2142 0.1402 |
0.1344 0.1054 |
Positive control OD570measurement 1 OD570measurement 2 |
0.1637 0.1397 |
0.1728 0.1625 |
0.1501 0.1536 |
OD = Optical density
Triplicate exposures are indicated by A, B and C.
Applicant's summary and conclusion
- Interpretation of results:
- other: Skin irritant Cat. 2
- Remarks:
- according to Regulation (EC) No. 1272/2008 and its amendments.
- Conclusions:
- An in vitro skin irritation test with the substance was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the substance is irritating in the in vitro skin irritation test.
- Executive summary:
The possible skin irritation potential of the substance was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. All validity criteria were met and the study was considered to be valid. The positive control had a mean cell viability of 10% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 12%. Since the mean relative tissue viability for the substance was below 50% after 15 minutes treatment the substance is considered to be irritant to the skin.
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