Potassium isobutyrate

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:

2020

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

Test material information

Constituent 1

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

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

Results

Results 1

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

Results 2

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

Results 3

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.