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EC number: 233-588-4 | CAS number: 10250-45-0
- 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:
- 25 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:
- 2 018
- 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:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 2012
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- 3-methyl-1-isobutylbutyl acetate
- EC Number:
- 233-588-4
- EC Name:
- 3-methyl-1-isobutylbutyl acetate
- Cas Number:
- 10250-45-0
- Molecular formula:
- C11H22O2
- IUPAC Name:
- 3-methyl-1-isobutylbutyl acetate
- Test material form:
- liquid
- Details on test material:
- - Physical Description: Clear colourless liquid
- Storage Conditions: At room temperature
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 Small Model™ (EPISKIN-SM™, 0.38 cm^2)
- Tissue batch number(s): 18 EKIN 039
- This model is a three-dimensional human epidermis model, which consists of adult human derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
TEST FOR THE INTERFERENCE OF THE TEST MATERIAL WITH THE MTT ENDPOINT
- A test material may interfere with the MTT endpoint if it is coloured and/or it is able to directly reduce MTT. The cell viability measurement is affected only if the test material is present on the tissues when the MTT viability test is performed.
- The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model. Because solutions did not turn blue / purple and a blue / purple precipitate was not observed it was concluded that the test material did not interfere with the MTT endpoint.
APPLICATION/ TREATMENT OF THE TEST MATERIAL
- Twenty-five μL of the undiluted test material was added into 12-well plates on top of the skin tissues. Three tissues were treated with 25 μL PBS (negative control) and 3 tissues with 25 μL 5 % SDS (positive control) respectively. The positive control was re-spread after 7 minutes contact time.
REMOVAL OF TEST MATERIAL AND CONTROLS
- After the exposure period of 15 ± 0.5 minutes at room temperature, the tissues were washed with phosphate buffered saline to remove residual test material. After rinsing, the cell culture inserts were each dried carefully and moved to a new well on 2 mL pre-warmed maintenance medium until all tissues were dosed and rinsed. Subsequently the skin tissues were incubated for 42 hours at 37 °C.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- After incubation, cell culture inserts were dried carefully to remove excess medium and were transferred into a 12-wells plate prefilled with 2 mL MTT-solution (0.3 mg/mL in PBS). The tissues were incubated for 3 hours at 37 °C. After incubation the tissues were placed on blotting paper to dry the tissues. Total biopsy was made by using a biopsy punch. Epidermis was separated from the collagen matrix and both parts were placed in pre-labeled microtubes and extracted with 500 μL isopropanol (Merck, Darmstadt, Germany). Tubes were stored refrigerated and protected from light for 69 hours. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
NUMBER OF REPLICATE TISSUES:
- The test was performed on a total of 3 tissues per test material together with negative and positive controls.
INTERPRETATION
- A test material is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is ≤ 50 % of the mean viability of the negative controls. The prediction to be considered is Category 2.
- A test material is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test material and 42 hours of post incubation is > 50 % of the mean viability of the negative controls. No category is required.
ANALYSIS
- Calculation of Cell Viability
Optical Density readings were transferred into Microsoft Excel to allow further calculations to be performed.
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 positive control is calculated as follows:
%Viability = (ODc/mean ODlt_u+MTT) * 100 - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied: 25 µL
- Concentration: Undiluted
NEGATIVE CONTROL
- Amount(s) applied: 25 µL
POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration: 5 %: - Duration of treatment / exposure:
- 15 ± 0.5 minutes
- Duration of post-treatment incubation (if applicable):
- 42 hours followed by 3 hours with MTT
- Number of replicates:
- The test was performed on a total of 3 tissues per test material together with negative and positive controls.
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- Mean
- Value:
- 76
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- - The test material was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20151202). Because no colour changes were observed it was concluded that the test material did not interact with the MTT endpoint.
- Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 76 %. Since the mean relative tissue viability for the test material was above 50 % it is considered to be non-irritant.
- The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 32 %. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 14 %, indicating that the test system functioned properly.
Any other information on results incl. tables
Table 1: Mean absorption in the in vitro skin irritation test
|
A (OD570) |
B (OD570) |
C (OD570) |
Mean (OD570) ± SD |
Negative control |
0.963 |
0.925 |
1.034 |
0.974 ± 0.055 |
Test material |
0.775 |
0.702 |
0.737 |
0.738 ± 0.037 |
Positive control |
0.184 |
0.310 |
0.449 |
0.314 ± 0.132 |
OD = Optical density
SD = Standard deviation
Triplicate exposures are indicated by A, B and C.
In this table the values are corrected for background absorption (0.043). Isopropanol was used to measure the background absorption.
Table 2: Mean tissue viability in the in vitro skin irritation test
|
Mean tissue viability (percentage of control) |
Standard deviation (percentage) |
Negative control |
100 |
5.7 |
Test material |
76 |
3.8 |
Positive control |
32 |
14 |
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 is non-irritant in the in vitro skin irritation test.
- 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 objective of this study was to evaluate the test material for its ability to induce skin irritation on a human three dimensional epidermal model (EPISKIN Small model (EPISKIN-SM™)). The possible skin irritation potential of the test material was tested through topical application for 15 minutes.
The test material was applied undiluted (25 μL) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-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 MTT at the end of the treatment. Skin irritation is expressed as the remaining cell viability after exposure to the test material.
The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with the test material compared to the negative control tissues was 76 %. Since the mean relative tissue viability for the test material was above 50 % after 15 ± 0.5 minutes treatment the test material is considered to be non-irritant.
The positive control had a mean cell viability of 32 % after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 14 %, indicating that the test system functioned properly.
Under the conditions of this study, the test material is non-irritant in the in vitro skin irritation test.
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