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EC number: 243-077-8 | CAS number: 19455-20-0
- 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 sensitisation
Administrative data
- Endpoint:
- skin sensitisation: in vitro
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2 Dec 2019 - 10 Dec 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 020
- Report date:
- 2020
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline for the Testing of Chemicals, Part 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT) (adapted 25. June 2018)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- activation of dendritic cells
- Justification for non-LLNA method:
- Study provided as part of the recommended weight of evidence approach for skin sensitsation.
Test material
- Reference substance name:
- Potassium isobutyrate
- EC Number:
- 243-077-8
- EC Name:
- Potassium isobutyrate
- Cas Number:
- 19455-20-0
- Molecular formula:
- C4H8O2.K
- IUPAC Name:
- potassium 2-methylpropanoate
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Sponsor / 18110908G
- Expiration date of the lot/batch: 31 Oct. 2020
- Purity test date: 09 Nov 2018
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
The solubility of the test item was determined in a non-GLP pre-test in RPMI 1640 and
DMSO.
The test item was soluble in RPMI 1640 at the concentrations 100 mg/mL and 500 mg/mL.
As RPMI 1640 is the preferred solvent by the guideline OECD 442E, it was used as solvent
in the test.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid:
Since the test item is hygroscopic, it was dried over night at 145 °C before use for the stock
solution. Afterwards the test item was kept in an exsiccator under argon until it was cooled
down. Then the test item was used for preparing the stock solutions.
First, a stock solution (first pre-test: 100 mg/mL of the test item, second pre-test and both
runs: 500mg/mL of the test item) in RPMI 1640 was prepared and used to prepare a geometric
series of solutions (factor 2 for pre-tests; factor 1.2 for main experiment). Afterwards
all concentrations were further diluted (1:50 fold) in complete culture medium (working solutions).
Another 1:2 dilution was achieved by adding 1 part of each test item concentration
and 1 part of the cell suspension to the treatment plate. In the end, the total dilution factor
was 1:100. The stock solutions as well as the dilutions were freshly prepared on the day of
treatment.
OTHER SPECIFICS
A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluorescence Detector and an excitation wavelength of 488 ± 5 nm. No emission was detected between 500 - 700 nm. Therefore, it is assumed that the test item has no influence on the result of the assay due to autofluorescence.
In vitro test system
- Details on the study design:
- Skin sensitisation (In vitro test system) - Details on study design:
Test System
Reasons for the Choice of the THP-1 Cell Line
The OECD 442E indicates that the human monocytic leukaemia cell line, THP-1 should be
used for the h-CLAT. The cells were purchased by CLS (Eppelheim, Germany).
Cell Cultures
THP-1 cells are stored in liquid nitrogen in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees similar parameters of the experiment and reproducible characteristics of the cells.
The THP-1 cells are routinely seeded every 2-3 days at the density of 0.1 – 0.2 * 106
cells/mL. They were maintained at densities from 0.1 to 1.0 * 106 cells/mL. Prior to using
them for testing, the cells were qualified by conducting a reactivity check.
For the pre-tests cells of passage 18 and 19 were used. For the main experiment cells of
passage 21 were used. After thawing the cells were cultivated in RPMI 1640 complete culture medium in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 % CO2.
Reactivity Check
Three weeks after thawing, a reactivity check of the cells was performed. For that, the two
positive controls 2,4-dinitrochlorobenzene (DNCB) (CAS n. 97-00-7, ≥ 99% purity, test concentration: 4 μg/mL) and nickel sulfate (NiSO4) (CAS n. 10101-97-0, ≥ 99% purity, test concentration: 200 μg/mL) as well as the negative control, lactic acid (LA) (CAS n. 50-21-5, ≥ 85% purity, test concentration: 1000 μg/mL) were used. These substances as well as all
additional information are given by the OECD 442E. The experimental procedure was identical to the runs in this study.
The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers.
Therefore, the cells were found to be suitable for the runs.
For the pre-test as well as the experiment only cells which have successfully passed the
reactivity check were used.
Test Vessels
All vessels used were made of glass or sterilizable plastic. In case of non-sterilization by the manufacturer, they were sterilized before usage in a heating chamber or autoclave. The test was performed on 96- and 24-well plates. For the transfer of the culture medium, pipettes were used. Glass measuring flasks and cylinders with conformity sign and standard laboratory material were also used.
Demonstration of proficiency
Prior to routine use, the validity of the h-CLAT test at LAUS GmbH was demonstrated in a
non-GLP proficiency study. 12 proficiency substances were selected to represent the range of responses for skin sensitisation hazards. The expected h-CLAT prediction as well as the reference range were correctly obtained for 10 substances. All values fell within the respective reference range (CV75, EC150, EC200). Therefore, the proficiency of the test system was demonstrated. For all control substances historical data are available, which
demonstrate the reliability and the validity of those substances.
Prior to use in the pre-test and the experiment, the proficiency of the cells was demonstrated in a reactivity check. Only the cells which passed the reactivity check were used for the pre-test and the experiment. The runs for Experiment I were performed on the same day, provided that for each run: a) independent fresh stock solutions and working
solutions of the test item and antibody solutions were prepared and b) independently
harvested cells were used (cells came from the same passage and were collected from
different culture flasks.)
Results and discussion
- Positive control results:
- The two positive controls produced a clearly positive result of the two surface markers CD86 and CD54. The negative control produced a clearly negative result of both surface markers. Therefore, the cells were found to be suitable for the runs.
In vitro / in chemico
Resultsopen allclose all
- Key result
- Group:
- test chemical
- Run / experiment:
- other: 1
- Parameter:
- RFI CD54>150 [442E]
- Value:
- 200
- Cell viability:
- >85%
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Group:
- test chemical
- Run / experiment:
- other: 2
- Parameter:
- RFI CD54>150 [442E]
- Value:
- 200
- Cell viability:
- >85%
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Group:
- test chemical
- Run / experiment:
- other: both runs and for all tested concentrations of run II except the lowest concentration of 1395.41 μg/mL.
- Parameter:
- RFI CD86>200 [442E]
- Value:
- 200
- Cell viability:
- >85%
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of skin sensitisation
- Outcome of the prediction model:
- positive [in vitro/in chemico]
- Other effects / acceptance of results:
- Cell viability in all runs was > 85%
Since the majority result of the two individual runs is positive, the test item is considered as “positive”.
Any other information on results incl. tables
Table 1 Results from experiment I run I
|
Concentration [µg/mL] |
Events (living cells) |
Viability [%] |
Antibodies |
MFI Value |
MFI ratioto Isotype [%] |
RFI Value [%] |
Medium |
- |
10358 |
97.80 |
CD86 |
1993 |
215 |
|
10314 |
97.78 |
CD54 |
1290 |
139 |
|
||
10257 |
98.17 |
ISO |
925 |
|
|
||
RPMI 1640 |
- |
10254 |
97.56 |
CD86 |
1775 |
196 |
81 |
10276 |
97.76 |
CD54 |
1442 |
159 |
147 |
||
10267 |
97.60 |
ISO |
907 |
|
|
||
DMSO |
- |
10266 |
97.86 |
CD86 |
1954 |
212 |
97 |
10276 |
98.11 |
CD54 |
1326 |
144 |
110 |
||
10230 |
98.41 |
ISO |
923 |
|
|
||
DNCB |
4.0 |
11376 |
88.41 |
CD86 |
6133 |
|
490 |
11508 |
86.06 |
CD54 |
2513 |
|
355 |
||
11385 |
84.77 |
ISO |
1082 |
|
|
||
Test Item |
1395.41 |
10656 |
95.84 |
CD86 |
2441 |
|
181 |
10541 |
96.96 |
CD54 |
1514 |
|
120 |
||
10506 |
97.06 |
ISO |
874 |
|
|
||
Test Item |
1674.49 |
10921 |
96.49 |
CD86 |
2708 |
|
211 |
10821 |
96.32 |
CD54 |
1691 |
|
152 |
||
10770 |
96.51 |
ISO |
877 |
|
|
||
Test Item |
2009.39 |
10759 |
96.05 |
CD86 |
2710 |
|
205 |
10851 |
96.07 |
CD54 |
1806 |
|
155 |
||
10849 |
95.80 |
ISO |
933 |
|
|
||
Test Item |
2411.27 |
10918 |
95.44 |
CD86 |
2974 |
|
225 |
10781 |
95.54 |
CD54 |
1850 |
|
155 |
||
10873 |
95.31 |
ISO |
1019 |
|
|
||
Test Item |
2893.52 |
11270 |
94.99 |
CD86 |
3344 |
|
272 |
11534 |
94.73 |
CD54 |
1761 |
|
145 |
||
11523 |
94.41 |
ISO |
983 |
|
|
||
Test Item |
3472.22 |
13000 |
92.21 |
CD86 |
4578 |
|
414 |
12271 |
92.79 |
CD54 |
1837 |
|
159 |
||
12797 |
92.42 |
ISO |
986 |
|
|
||
Test Item |
4166.67 |
12265 |
91.36 |
CD86 |
3970 |
|
338 |
12716 |
90.56 |
CD54 |
1970 |
|
174 |
||
12658 |
90.84 |
ISO |
1037 |
|
|
||
Test Item |
5000.00 |
13379 |
86.18 |
CD86 |
4700 |
|
416 |
12720 |
85.93 |
CD54 |
2000 |
|
171 |
||
12534 |
86.90 |
ISO |
1086 |
|
|
A calculation of the EC150 and/or EC200 was not possible since none of the RFI values was below 150 (for CD86) and /or above 200 (for CD54) at any of the tested concentrations.
Table2 Results from experiment I run II
|
Concentration [µg/mL] |
Events (living cells) |
Viability [%] |
Antibodies |
MFI Value |
MFI ratioto Isotype [%] |
RFI Value [%] |
Medium |
- |
10310 |
97.66 |
CD86 |
2123 |
221 |
|
10275 |
97.60 |
CD54 |
1285 |
134 |
|
||
10285 |
97.83 |
ISO |
961 |
|
|
||
RPMI 1640 |
- |
10285 |
97.34 |
CD86 |
2038 |
211 |
92 |
10260 |
97.36 |
CD54 |
1314 |
136 |
108 |
||
10241 |
97.25 |
ISO |
965 |
|
|
||
DMSO |
- |
10189 |
97.31 |
CD86 |
2294 |
250 |
118 |
10210 |
97.78 |
CD54 |
1330 |
145 |
127 |
||
10207 |
97.40 |
ISO |
919 |
|
|
||
DNCB |
4.0 |
11448 |
86.73 |
CD86 |
5956 |
|
352 |
11409 |
85.05 |
CD54 |
2583 |
|
357 |
||
11434 |
83.72 |
ISO |
1116 |
|
|
||
Test Item |
1395.41 |
10700 |
96.52 |
CD86 |
2765 |
|
165 |
10581 |
96.88 |
CD54 |
1547 |
|
158 |
||
10581 |
96.80 |
ISO |
996 |
|
|
||
Test Item |
1674.49 |
10742 |
96.46 |
CD86 |
2447 |
|
135 |
10762 |
96.89 |
CD54 |
1834 |
|
240 |
||
10726 |
96.88 |
ISO |
995 |
|
|
||
Test Item |
2009.39 |
10995 |
95.66 |
CD86 |
2798 |
|
171 |
10958 |
95.91 |
CD54 |
1901 |
|
268 |
||
10930 |
95.23 |
ISO |
965 |
|
|
||
Test Item |
2411.27 |
11094 |
95.02 |
CD86 |
2903 |
|
180 |
10930 |
95.43 |
CD54 |
1737 |
|
218 |
||
10940 |
95.69 |
ISO |
975 |
|
|
||
Test Item |
2893.52 |
11511 |
94.54 |
CD86 |
3756 |
|
256 |
11746 |
95.33 |
CD54 |
1838 |
|
236 |
||
11508 |
95.72 |
ISO |
1013 |
|
|
||
Test Item |
3472.22 |
12273 |
93.64 |
CD86 |
4152 |
|
291 |
12296 |
94.01 |
CD54 |
1863 |
|
239 |
||
11996 |
93.69 |
ISO |
1030 |
|
|
||
Test Item |
4166.67 |
12658 |
92.13 |
CD86 |
3818 |
|
257 |
12385 |
91.36 |
CD54 |
1878 |
|
233 |
||
12809 |
90.48 |
ISO |
1064 |
|
|
||
Test Item |
5000.00 |
13814 |
86.79 |
CD86 |
4179 |
|
287 |
13770 |
87.46 |
CD54 |
1886 |
|
226 |
||
12789 |
89.05 |
ISO |
1098 |
|
|
A calculation of the EC150 and/or EC200 was not possible since no dose response for the RFI values for CD86 and for CD54 was observed.
Applicant's summary and conclusion
- Interpretation of results:
- study cannot be used for classification
- Conclusions:
- Under the experimental conditions of this study, the test item, Potassium isobutyrate, was positive in the h-CLAT and is therefore considered to have the potential to activate dendritic cells and therefore to up-regulate the cell surface marker (CD86 and CD54) expression of THP-1 cells.
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