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EC number: 208-793-7 | CAS number: 541-85-5
- 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:
- 27.03.2013-22.04.2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Fully documented study run under GLP and according to international guidelines
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 013
- Report date:
- 2013
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Qualifier:
- according to guideline
- Guideline:
- other: • ECVAM international validation study on in vitro tests for acute skin irritation: Report on the validity of the EPISKIN and EpiDerm assays and on the Skin Integrity Func-tion Test (Altern Lab Anim. 2007 Dec; 35 (6): 559-601).
- Qualifier:
- according to guideline
- Guideline:
- other: • Protocol for IN VITRO EpiDermTM SKIN IRRITATION TEST (EPI-200-SIT), Rev. 3/23/2009, MatTek Corporation, Ashland, MA 01721, USA
- Principles of method if other than guideline:
- This in-vitro study was performed in order to evaluate the potential of Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) to evoke skin irritation in a human-skin-model.
The test consists of a topical exposure of the neat test item to a human reconstructed epidermis model followed by a cell viability test. Cell viability is measured by dehydrogenase conversion of MTT (3-[4,5-dimethyl thiazole 2-yl] 2,5-diphenyl-tetrazoliumbromide), present in cell mitochondria, into a blue formazan salt that is quantitatively measured after extraction from tissues. The percent reduction of cell viability in comparison of untreated negative controls is used to predict skin irritation potential. - GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- 5-methylheptan-3-one
- EC Number:
- 208-793-7
- EC Name:
- 5-methylheptan-3-one
- Cas Number:
- 541-85-5
- Molecular formula:
- C8H16O
- IUPAC Name:
- 5-methylheptan-3-one
- Details on test material:
- -Name of the test material: Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5)
-Batch no./Ref. No.: 12023305
-Expiration date: 31. May 2013
-Storage: store in a tightly closed container, cool and dry, in a well ventilated place
Constituent 1
Test animals
- Species:
- other: human-derived epidermal keratinocytes (EPI-200 tissues)
- Strain:
- other: Commercially available Epi-200-SIT-Kit
- Details on test animals or test system and environmental conditions:
- The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi-layered, 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 EpiDermTM tissues are cultured on specially prepared cell culture inserts.
Epi-200 tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava (Batch: 18311).
Preincubation of tissues:
Eight 6-well-plates were prepared with 0.9 mL assay medium in three of the six wells (up-per row). The tissues were inspected for viability. Viable tissues were transferred (three per plate) in the wells with the medium using sterile forceps under the laminar air flow bank and placed into the incubator at 37 °C and 5 % CO2 for one hour.
After the pre-incubation (one hour), the other three wells of each plate (lower row) were filled with fresh assay medium (0.9 mL). Every tissue was transferred into a well of the lower row. All 6-well-plates were set into the incubator at 37 °C and 5 % CO2 for 18 hours.
Test system
- Type of coverage:
- other: nylon mesh
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent vehicle
- Amount / concentration applied:
- Applied volume: 30 uL of test item
- Duration of treatment / exposure:
- 60 minutes
- Details on study design:
- Pre-Tests:
First, it was tested whether the test item develops a colour without MTT addition. 30 µL were given in a test tube with 0.3 mL H2O demin. and incubated at 37 °C and 5 % CO2 for 60 minutes. The resulting solution was colourless, therefore no binding capacity had to be tested.
Then, the test item Ethylamylketon was tested for the ability of direct formazan reduction. To test for this ability, 30 µL were added to 1 mL of MTT solution and the mixture was in-cubated in the dark at 37 °C and 5 % CO2 for 60 minutes. Untreated MTT solution was used as control. The MTT solution didn’t change its colour within one hour, therefore, di-rect MTT reduction had not taken place, and no data correction was necessary.
Finally, Ethylamylketon was tested for possible reaction with the nylon mesh which is used to ensure sufficient contact with the tissue surface. 30 µL Ethylamylketon were brought onto a nylon mesh on a microscope slide. No reaction with the mesh was visible after 1 h incubation at 37 °C.
Treatment
The pre-incubated tissues were placed into fresh 6-well-plates containing 0.9 mL assay medium per well, using the upper row only.
One plate (three tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer. One plate was used as positive control; each tissue was treated with 30 µL SDS-solution. One plate was used for treatment with the test item. 30 µL test item were applied, and a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
Tissues were dosed in one minute intervals. After dosing the last tissue, all plates are transferred into the incubator for 35 minutes. 60 minutes after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in one-minute-intervals.
Dosing of first tissue: 11:00 h
Dosing of last tissue: 11:15 h
Start of incubation at 37 °C: 11:25 h
End of incubation at 37 °C: 12:00 h
Rinsing of first tissue: 12:00 h
Rinsing of last tissue: 12:15 h
After rinsing, each tissue was dried with a sterile cotton tip and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The tissue surfaces were evaluated visually under the stereo microscope, excess test item was removed, where necessary.
Then the tissues were set in the incubator for 24 hours.
Medium Renewal
For three incubated tissues, a new 6-well-plate with 0.9 mL assay medium in the upper row was prepared. The tissues were removed from the incubator and shaken for ten min (500 rpm). Then the inserts were transferred into the new 6-well-plate and set into the in-cubator for 18 ± 2 hours for post-incubation.
The medium from the “old” 6-well-plates was collected in the labelled 24-well-plate. It can be stored for 12 months at – 20 °C for possible interleukin analysis. As the result of the test was unambiguous, the samples were destroyed after finalisation of the final report.
MTT Assay
After a total incubation time of 42 hours, a 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissues were blotted on the bottom and then transferred into the 24-well-plate. Then the 24-well-plate was set into the incubator for 3 hours ± 5 min.
After this time, the MTT reagent was aspirated and replaced by PBS buffer. This was then aspirated, too, and replaced several times. At last, each insert was thoroughly dried and set into the empty, pre-warmed 24-well-plate. Into each well, 2 mL isopropanol were pipet-ted, taking care to reach the upper rim of the insert. The plate was then shaked for two hours room temperature.
After two hours, the inserts in which formazan had been produced were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate which was read in a plate spectral photometer at 570 nm.
Calculations
The photometric absorption of the negative controls is considered as 100 %. For each replicate of test item and positive control, formazan production is calculated as % photometric absorption compared with the mean of the negative controls:
%Formazan production=(OD1replicate test item resp.positve control/ ODmean of negative controls)*100
1OD= Optical Density
Validation criteria:
Skin irritation potential of the test item is assessed as given in the following table:
Table8.2-a Assessment of Irritation Potential
% Formazan production
Assessment
=50 % of negative control
Irritant
> 50 % of negative control
Non-irritant
Results and discussion
In vitro
Results
- Irritation / corrosion parameter:
- other: other: % Formazan production
- Value:
- 6
- Remarks on result:
- other:
- Remarks:
- Basis: mean. Max. score: 100.0. Reversibility: not reversible. (migrated information)
Any other information on results incl. tables
Measured Values
As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:
Table1.1-a Absorption values blank isopropanol (OD at 570 nm)
Replicate |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
Mean |
Absorption |
0.040 |
0.056 |
0.043 |
0.037 |
0.039 |
0.042 |
0.038 |
0.034 |
0.041 |
The absorption values of negative control, test item and positive control are given in the following table:
Table1.1-b Absorption values negative control, test item and positive control (OD at 570 nm)
Designation |
Measurement |
Negative Control |
Ethylamylketon |
Positive Control |
Tissue 1 |
1 |
2.292 |
0.178 |
0.100 |
2 |
2.245 |
0.182 |
0.102 |
|
Tissue 2 |
1 |
2.179 |
0.180 |
0.120 |
2 |
2.219 |
0.179 |
0.119 |
|
Tissue 3 |
1 |
2.055 |
0.151 |
0.116 |
2 |
2.081 |
0.148 |
0.115 |
From the measured absorptions, the mean of each tissue was calculated, subtracting the mean absorption of isopropanol as given in table 1.1-a. Mean and relative standard deviation (comparison of the three tissues) were also calculated.
Table1.1-c Mean Absorption Values
Designation |
Negative Control |
Ethylamylketon |
Positive Control |
Mean – blank (Tissue 1) |
2.228 |
0.139 |
0.060 |
Mean – blank (Tissue 2) |
2.158 |
0.139 |
0.079 |
Mean – blank (Tissue 3) |
2.027 |
0.109 |
0.075 |
Mean of the three Tissues |
2.138 |
0.129 |
0.071 |
Relative Standard Deviation |
4.8 % |
13.4 % |
14.1 % |
Comparison of Formazan Production
For the test item and the positive control, the following percentage values of formazan production were calculated in comparison to the negative control:
Table1.2-a % Formazan Production
Designation |
Ethylamylketon |
Positive Control |
% Formazan production (Tissue 1) |
6.5 % |
2.8 % |
% Formazan production (Tissue 2) |
6.5 % |
3.7 % |
% Formazan production (Tissue 3) |
5.1 % |
3.5 % |
% Formazan production Mean |
6.0 % |
3.3 % |
Applicant's summary and conclusion
- Interpretation of results:
- irritating
- Remarks:
- Migrated information Criteria used for interpretation of results: EU
- Conclusions:
- The test item Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) is considered irritant.
- Executive summary:
Three tissues of the human skin model EpiDermTMwere treated with Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) for 60 minutes.
30 µL of the liquid test item (using a nylon mesh) were applied to each tissue and spread to match the tissue size (0.63 cm2; as indicated by supplier). DPBS-buffer was used as negative control, 5 % SDS-solution was used as positive control.
After treatment with the negative control, the absorbance values were within the required acceptability criterion of 1.0 < mean OD < 2.5. The positive control showed clear irritating effects. Variation within tissues was acceptable (< 18 %). After the treatment with the test item, the relative absorbance values were reduced to 6.0 %. This value is well below the threshold for irritation potential (50 %).
Therefore, Ethylamylketon (5-methylheptan-3-one, CASNr: 541-85-5) is considered as irritant in the Human Skin Model Test.
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