Potassium isobutyrate

Endpoint summary

Description of key information

An inital review of skin sensitisation potential has been performed using the following tests:

- OECD 422C

- OECD 422D

- OECD 422e

All studies were performed under the conditions of GLP.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 March 2019 - 28 March 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

adopted 04. Feb. 2015

Deviations:

yes

Remarks:

Demineralized water was used instead of HPLC grade or Millipore Milli-Q grade water. This was seen as uncritical, because the reference controls showed that the used water has no negative impact on the test system.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Study provided as part of the recommended weight of evidence approach for skin sensitsation.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
sponsor / 181101001
- Expiration date of the lot/batch:
31. Oct. 2020
- Purity test date:
16 Jan 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room Temperature (20 ± 5 °C); Keep away from humidity. The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), protected from humidity.
- Stability under test conditions:
stable
- Solubility and stability of the test substance in the solvent/vehicle:

Stability in solvents: H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:

Preparation of test item
The concentration of the test item to be used was determined based on the molecular weight (MW) 126.1955 g/mol and the purity 98.25%, by titration. Based on a non-GLP pre-test, the 100 mM test item solution was prepared by dissolving 38.5 mg and 38.7 mg test item in 3 mL water for the Cys-peptide and Lys-peptide assay, respectively. The solution was vortexed until it was dissolved completely.

Positive control results:

The mean peptide depletion with 81.70 % and a standard deviation of 0.41 % of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, respectively, for the Cys-peptide.
The mean peptide depletion with 23.91 % and a standard deviation of 3.92 % of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and < 11.6 %, respectively, for the Lys-peptide

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

4.4 %

At concentration:

5 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

11.6

At concentration:

25 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

no or minimal reactivity [in chemico]

Other effects / acceptance of results:

Acceptance criteria
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100.0 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10.0 % - 45.0 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
c) The standard deviation for the test item replicates should be < 14.9 % for the per-cent cysteine depletion and < 11.6 % for the percent lysine depletion.

Assessment
a) The mean peptide depletion with 81.70 % and a standard deviation of 0.41 % of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and < 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion with 23.91 % and a standard deviation of 3.92 % of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and < 11.6 %, respectively, for the Lys-peptide.
c) The standard deviation for the test item replicates with 0.93 % was < 14.9 % for the percent cysteine depletion for the test item.
The standard deviation for the test item replicates with 0.59 % was < 11.6 % for the percent lysine depletion for the test item.

The ten proficiency chemicals listed in the guideline were tested using the analysis method described in the report.
All ten proficiency chemicals showed the expected DPRA prediction and all ten chemi-cals showed depletion values consistent with the classification reported in the OECD guideline (LAUS in-house study).

Calculated peptide depletion values for the Cys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	81.76	81.70	0.41
Positive control Rep. 2	81.25
Positive control Rep. 3	82.07
Test item Rep. 1	3.79	4.40	0.93
Test item Rep. 2	3.95
Test item Rep. 3	5.47

 

Calculated peptide depletion values for the Lys-Peptide

Sample name	Depletion [%]
	Single	Mean	SD
Positive control Rep. 1	22.47	23.91	3.92
Positive control Rep. 2	20.91
Positive control Rep. 3	28.34
Test item Rep. 1	0 (-1.49)*	0.34	0.59
Test item Rep. 2	0 (-0.99)*
Test item Rep. 3	1.03

* Note: Negative depletion values were considered as “zero” when calculating the mean.

 

The mean peptide depletion in the Cys-peptide and Lys-peptide assay was 2.37 %, therefore the test item was classified with:

 

DPRA Prediction: Negative

Reactivity class: No or Minimal

Interpretation of results:

study cannot be used for classification

Conclusions:

The DPRA prediction for the test item Potassium isobutyrate was negative with reactivity class no or minimal according to the Cysteine 1:10/Lysine 1:50 prediction model.
It can be stated that in this study and under the experimental conditions reported, the test item Potassium isobutyrate possesses no skin sensitisation potential.
No observations arousing doubts concerning the accuracy of the results and the validity of the study were made.

Executive summary:

The study was performed in order to evaluate the reactivity of the test itemPotassium isobutyratetowards cysteine (Cys-) and lysine (Lys-) containing peptides. The test item was incubated for 22 h at 25 °C together with the Cys-peptide and 22 h 20 min with the Lys-peptide, respectively. The peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured simultaneously.

 

One experiment was performed and it was valid for the Cys- and Lys-peptide assay.

The results were evaluated according to the Cysteine 1:10/Lysine 1:50 prediction model.

The peptide depletion values after incubation are shown below:

Results

	Cys-peptide depletion [%]	Lys-peptide depletion [%]	Mean peptide depletion [%]
Experiment 1	4.40	0.34	2.37

 

In conclusion, the DPRA prediction is “negative” with no or minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. It can be stated that in this study and under the experimental conditions reported, the test item Potassium isobutyrate possesses no skin sensitisation potential.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 Jan 2019 - 14 Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

adapted: 25. June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Study provided as part of the recommended weight of evidence approach for skin sensitsation.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
sponsor / 181101001
- Expiration date of the lot/batch:
31. Oct. 2020
- Purity test date:
16 Jan 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Room Temperature (20 ± 5 °C); Keep away from humidity. The test item was stored in the test facility in a closed vessel at room temperature (20±5°C), protected from humidity.
- Stability under test conditions:
stable
- Solubility and stability of the test substance in the solvent/vehicle:

Stability in solvents: H2O: 96h; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown
Solubility: H2O: > 1 g/L; EtOH: unknown; acetone: unknown; CH3CN: unknown; DMSO: unknown

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The LuSens cell line was specially designed for this test system by the BASF SE (Lud-wigshafen, Germany). It employs the use of a luciferase reporter gene placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

Dose Selection for Experiments
In accordance with the OECD guideline 442D, the maximum final test item concentration should be 2000 µM. For a test chemical which has no defined molecular weight, the final test item concentration 2000 µg/mL can also be used. Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or poor solubility).
In the case of a cytotoxic result, the concentrations for the experiments should be deter-mined so that at least one of them is in the cytotoxic range.
Since no cytotoxic reaction was observed in the CRFT the following 12 nominal concen-trations were chosen for the experiments:
269 µM, 323 µM, 388 µM, 465 µM, 558 µM, 670 µM, 804 µM, 965 µM, 1157 µM, 1389 µM, 1667 µM, 2000 µM

In the main experiments, a reduction of the viability below 70 % is considered as cytotoxic and the concentration that is cytotoxic is not allowed to be evaluated for luciferase induc-tion.

Demonstration of proficiency
Prior to routine use, the validity of the LuSens test at LAUS GmbH was demonstrated in a proficiency study. 22 proficiency chemicals indicated by the OECD 442 D (status: 25. June 2018) and the OECD PERFORMANCE STANDARDS FOR ASSESSMENT OF PROPOSED SIMILAR OR MODIFIED IN VITRO SKIN SENSITISATION ARE-NRF2 LUCIFERASE TEST METHODS) (version: 22. May 2015) were tested. The 10 proficiency chemicals which are indicated by the current version of the OECD 442D (status: 25. June 2018) were all included in the proficiency study.
From 22 proficiency chemicals more than 80 % of the results were correctly categorized. From the 10 of the proficiency chemicals of the current OECD 442D (chemicals of the current version of the OECD 442 D (status: 25. June 2018) 80 % of the results were correctly categorized.
Therefore, the proficiency of the LuSens test was demonstrated.
For all control substances historical data are available in the test report, which demonstrates the reliability and the validity of those substances.

Positive control results:

Experiment III: Fold induction 3.4, Relative viability 80.8%
Experiment IV: Fold induction 2.6, Relative viability 80.1%

Key result

Group:

test chemical

Run / experiment:

other: Experiment III

Parameter:

other: Luciferase fold induction

Value:

0.9

Cell viability:

99.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: Experiment IV

Parameter:

other: Luciferase fold induction

Value:

0.9

Cell viability:

99.6%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Results of Experiment III

The results of experiment III are indicated in table 8-a and figures 8-a and 8-b.

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values > 80 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 0.9 fold). However, the positive control induced a clear effect with an induction value of 3.4 fold in comparison to the solvent control.

No cytotoxic effect was observed at all test item concentrations. The viability values were all ≥ 94 % and therefore analysable for luciferase induction.

In the Luciferase assay, none of the tested concentrations induced a luciferase induction above or equal 1.5 fold in comparison to the solvent control.

Table8‑a         Results of experiment III

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.05	5.04	100.0	2.60	2.60
Growth Control	-	1.0	0.08	7.80	101.1	2.14	2.12
Negative Control	5000 µM	0.9	0.04	4.08	98.2	1.97	2.01
Positive Control	60 µM	3.4	0.33	9.60	80.8	1.62	2.01
Test item	269	1.0	0.06	5.72	96.6	1.31	1.35
Test item	323	1.0	0.05	4.81	98.0	1.29	1.32
Test item	388	0.9	0.05	5.82	98.6	0.86	0.87
Test item	465	0.9	0.10	10.82	98.3	1.90	1.94
Test item	558	1.0	0.02	2.10	99.2	1.25	1.26
Test item	670	1.0	0.06	5.78	100.4	2.32	2.31
Test item	804	1.0	0.04	4.53	100.8	1.78	1.77
Test item	965	0.9	0.04	4.71	98.3	1.69	1.72
Test item	1157	0.9	0.04	3.99	95.5	2.80	2.94
Test item	1389	1.0	0.06	6.26	94.4	2.92	3.09
Test item	1667	1.0	0.04	4.09	95.9	2.25	2.35
Test item	2000	0.9	0.04	3.86	99.4	1.80	1.82

 

Results of Experiment IV

The results of experiment IV are indicated in table 8-b and figures 8-c and 8-d.

All control substances indicated the expected effect. No considerable reduction of the viability was detected (all values ≥ 80 %). Regarding the Luciferase induction, the growth control and the negative control did not exceed the threshold of 1.5 fold in comparison to the solvent control (growth control: 1.0 fold, negative control: 0.8 fold). However, the positive control induced a clear effect with an induction value of 2.6 fold in comparison to the solvent control.

No cytotoxic effect was observed at all test item concentrations. The viability values were all ≥ 96 % and therefore analysable for luciferase induction.

In the Luciferase assay, none of the tested concentrations induced a luciferase induction above or equal 1.5 fold in comparison to the solvent control.

Table 8‑b         Results of experiment IV

		Induction of Luciferase			Viability of the Cells
Parameter	Concentration	Induction	Standard Deviation	Standard Deviation	Relative Viability	Standard Deviation	Standard Deviation
	[µM]	fold		[%]	[%]		[%]
Solvent Control	-	1.0	0.08	8.21	100.0	1.72	1.72
Growth Control	-	1.0	0.09	8.85	102.2	1.68	1.64
Negative Control	5000 µM	0.8	0.07	8.44	97.1	2.28	2.35
Positive Control	60 µM	2.6*	0.10	3.82	80.1*	2.72	3.39
Test item	269	0.9	0.17	18.51	100.3	2.79	2.78
Test item	323	0.9	0.01	1.09	101.4	1.03	1.01
Test item	388	1.1	0.20	18.22	98.9	1.73	1.75
Test item	465	0.9	0.15	17.23	100.5	1.58	1.57
Test item	558	0.9	0.13	15.08	99.5	1.36	1.36
Test item	670	1.0	0.06	5.86	101.9	0.71	0.70
Test item	804	0.9	0.05	5.56	100.7	0.77	0.76
Test item	965	0.9	0.02	2.23	99.5	1.68	1.69
Test item	1157	0.8	0.11	13.67	96.3	0.92	0.96
Test item	1389	0.9	0.07	7.02	96.2	0.20	0.21
Test item	1667	0.8	0.06	6.78	96.6	0.78	0.81
Test item	2000	0.9	0.09	9.69	99.6	3.78	3.79

* = Due to one outlier in the five replicates of the positive control in the plate for evaluation of the relative viability, only four replicates were used to calculate the Viability of the Cells and the Induction of Luciferase

Interpretation of results:

study cannot be used for classification

Conclusions:

This in vitro study was performed to investigate the potential of Potassium isobutyrate to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.
In total four experiments were performed. Since in experiment I and II the relative viability of the positive control showed a cytotoxic effect and therefore one acceptance criterion was not fulfilled, they were declared as invalid. These experiments are not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. In the end two valid experiments were performed and evaluated.
12 concentrations of the test item were evaluated. The exposure time was 48 h. The fol-lowing nominal concentrations of the test item were investigated in experiment III and IV:
269 µM, 323 µM, 388 µM, 465 µM, 558 µM, 670 µM, 804 µM, 965 µM, 1157 µM, 1389 µM, 1667 µM, 2000 µM
None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration in the experiments.
p-Phenylenediamine (60 µM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected.
Experiment IV: The relative viability and the luciferase fold induction of the positive con-trol were calculated from four values. The OD value (MTT plate (Viability plate, well H7)) varied compared to the values of the other four wells. Therefore, this value was declared as an outlier and was not used to calculate the mean relative viability, which was only calculated from the four remaining values. The corresponding value from the luciferase plate (H7) was therefore not included in the calculation of the mean luciferase induction. It was calculated from the remaining four values.
The luciferase induction of the positive control of experiment III is well within the historical data range, whereas the luciferase induction of the positive control of experiment IV is slightly below the historical range. Since this value is only marginal outside the range, and the experiment is valid regarding the acceptance criteria for the positive and negative control, there is no influence on the result of the study.
DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control and well within the historical data range of the negative control (table 18-a).
The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.
Since all acceptability criteria of the assay were met, the study is valid.
No cytotoxic effect was observed in all tested test item concentrations. Therefore, all tested concentrations could be evaluated for luciferase induction.
In all tested concentrations of the test item no increase ≥ 1.5 fold in luciferase induction in comparison to the solvent control was measured. Therefore, both experiments are clearly negative.
In conclusion, it can be stated that under the experimental conditions of this study, the test item, Potassium isobutyrate, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Executive summary:

This in vitro study evaluates the potential of the test item Potassium isobutyrate to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay based on the OECD 442D Guideline with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and four independent experiments (experiment I, II, III and IV) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the experiments were determined.

Since in experiment I and II the relative viability of the positive control showed a cytotoxic effect and therefore one acceptance criterion was not fulfilled, they were declared as invalid. These experiments are not reported, all documentation is kept with the raw data and will be archived at the GLP test facility. To obtain a valid result, experiment III and IV were per-formed with an adjusted concentration of the positive control.

In the valid experiments, the highest nominal applied concentration (2000 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMEM (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 μM) was used as negative control and p-Phenylenediamine (60 μM) as positive control.

The evaluated experimental points and the results are summarised in chapter 8, page 20.

No substantial and reproducible dose-dependent increase in luciferase induction equal or above 1.5 fold was observed in both experiments up to the maximal tested concentration of the test item.

Conclusion:

Under the experimental conditions of this study, the test item, Potassium isobutyrate, was negative in the LuSens assay and is therefore considered not to have the potential to activate the Nrf2 transcription factor (no sensitizing potential).

Reference 3

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

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.

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.

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.)

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.

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”.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The weight of evidence based on 3 studies suggests that the substance should not be classified as a skin sensitiser. Further testing is not proposed.