Dipotassium propanedioate

Endpoint summary

Description of key information

Two in vitro skin sensitisation assays were performed on dipotassium malonate: a DPRA and a KeratinoSens assay.
The test item was found tto be negative (not sensitising) in both assessments.

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:

key study

Study period:

30.10.-02.12.2017

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))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: dist. water : acetonitrile 1:1 (v/v)
- Reason for the vehicle: The test substance was not soluble in acetonitrile, dist. water, isopropanol

CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity
of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile

PEPTIDES
- Synthetic peptides:
-- 19.43 mg cysteine peptide; amino acid sequence of Ac-RFAACAA; dissolved in 39.98 mL of phosphate buffer pH 7.5; final concentration 0.667 mM
-- 18.43 mg lysine peptide;amino acid sequence of Ac-RFAAKAA; dissolved an ammonium acetate buffer pH 10.2 (35.0 mL); final concentration of 0.667 mM

DOSE GROUPS
- Reference Control C (solvent control) undiluted
- Test Item 100 mM stock solution
- Positive Control 100 mM stock solution

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220 nm: HPLC analysis after solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h
tarted 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis22 to 26 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution 20% acetonitrile : 80% buffer ( v / v ) using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated for 24 +/- 2 hours
and visually investigated for any precipitate that may occur during the exposure period.
- Reference controls, co-elution controls as well as the positive control were set up in parallel
- Method: HPLC Agilent 1200 series
- Wavelength: 220 nm and 258 nm
- Detector: UV detector

DATA ANALYSIS
- The percent peptide depletion (PPD) was calculated according to the following formula:
PPD = [ 1 – ( Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference
Control C)] * 100

ACCEPTANCE CRITERIA
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD)
for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates
and three reference control C replicates in acetonitrile is < 15.0%.

EVALUATION RESULTS
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
- In case the mean peptide depletion cannot be determined due to invalid K-peptide depletion the
evaluation is performed as follows:
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.81%. The controls confirmed the validity of the study for both, the cysteine and lysine run.

Key result

Run / experiment:

other: Cysteine Peptide

Parameter:

other: Mean Peptide Depletion [%]

Value:

0.56

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Lysine Peptide

Parameter:

other: Mean Peptide Depletion [%]

Value:

4.89

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Cysteine Peptide and Lysine Peptide

Parameter:

other: Prediction Model 1: Mean Peptide Depletion [%]

Value:

2.72

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Cysteine Peptide

Parameter:

other: Prediction Model 2: Mean Peptide Depletion [%]

Value:

0.56

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

All acceptance criteria passed

Results of the Cysteine Peptide Depletion

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.1577	70.95	71.10	0.18	0.25
	0.1571	71.06
	0.1559	71.29
Test item	0.5346	0.02	0.56	0.47	84.89
	0.5299	0.91
	0.5307	0.75

Results of the Lysine Peptide Depletion

Sample	Peptide concentration (mM)	Peptide Depletion (%)	Mean Peptide Depletion (%)	SD of Peptide Depletion (%)	CV (%)
Positive control	0.2164	56.37	56.51	0.47	0.83
	0.2175	56.14
	0.2131	57.04
Test item	no	0.84	4.89	4.1	83.92
	0.4716	4.79
	0.4505	9.04

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered non-sensitiser .

Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

In the present study Dipotassium malonate was dissolved in dist. water : acetonitrile 1:1 (v/v) based on the results of the pre-experiments.

Based on a molecular weight of 180.24 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Samples of the positive control (excluding the co-elution of the positive control) were centrifuged prior to the HPLC analysis.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation and turbidity was observed for the samples of the positive control (including the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations, turbidity and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC dist. water : acetonitrile 1:1 (v/v).).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (2.72%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.81%.

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered non-sensitiser. The study was performed according to OECD TG guidelines and in compliance to GLP.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13.11.-04.12.2017

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)

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Preparation of the Test Item:
- All test item solutions were freshly prepared immediately prior to use
- The test item was dissolved in dist. water

Controls:
A blank, a negative control and a positive control were set up in parallel
--Blank: A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.
-- Negative Control: DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate
-- Positive Control: Cinnamic aldehyde dissolved in DMSO resulting in a concentration range of 0.4 mM – 6.4 mM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.

Cell line:
- transgenic cell line KeratinoSens™ (Givaudan, Switzerland), derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct

Maintenance Medium:
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate.
The medium was supplemented with the following components:
- 10% fetal bovine calf serum
- 1% geneticin (final concentration: 500 μg/mL)

Assay Medium:
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum

Test Item Exposure Medium:
Dulbecco’s Modified Eagle Medium (GlutaMAX™) with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum

Dose Groups:
- Negative Control: 1% (v/v) DMSO in test item exposure medium
- Positive Control: 4 μM, 8 μM, 16 μM; 32 μM; 64 μM
- Test Item: 12 concentrations of the test item

Replicates:
- Test item and the positive control were assessed in three replicates in every independent run
- The negative control was assessed using six replicates per plate in every independent run

Experimental Procedure:
- Incubation: Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2
- Luciferase activity: washed once with DPBS, 20 μL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light
- Luminescence measurement: In a plate reader, waited for 1.000 ms before assessing the luciferase activity for 2.000 ms

Cell viability:
- 27 μL MTT solution added to each individual well and incubated for 4 h at 37 °C ± 1 °C and 5% CO2
- Afterwards the medium was removed and replaced by 200 μL 10% SDS solution per well and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1) and over the weekend (experiment 2).
- After the incubation period the OD was measured at λ = 600 nm.

Data Analysis:
For each test item two independent repetitions using separately prepared test item solutions and independently harvested cells are necessary to derive a prediction. Each independent run consisted of three replicates for every concentration step of the test item and the positive control.
The following parameters were calculated:

Calculation of Cell Viability:
Cell viability [%]= (Vsample−Vblank)(Vsolvent−Vblank)×100
Vsample = MTT absorbance reading in the test chemical well
Vblank = MTT absorbance reading in the blank well containing no cells and no treatment
Vsolvent = average MTT absorbance reading in the wells containing cells and solvent (negative) control

Calculation of the Maximal Induction of the Luciferase Activity (Imax):
Fold induction = (Lsample−Lblank)(Lsolvent − Lblank)
Lsample = luminescence reading in the test chemical well
Lblank = luminescence reading in the blank well containing no cells and no treatment
Lsolvent = luminescence reading in the wells containing cells and solvent (negative) control

Calculation of the EC1.5:
The EC1.5 will be calculated by linear extrapolation and the overall EC1.5 was calculated as the geometric mean of the individual repetitions.
EC1.5=(Cb−Ca)×((1.5-Ia)/(Ib−Ia))+ Ia
Ca = lowest concentration in μM with >1.5 fold induction
Cb = highest concentration in μM with <1.5 fold induction
Ia = fold-induction measured at the lowest concentration with >1.5 fold induction (mean of three replicate wells)
Ib = fold-induction measured at the highest concentration with <1.5 fold induction (mean of three replicate wells)

Calculation of IC50 and IC30:
The IC50 and IC30 were calculated by linear extrapolation and the overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
ICx =(Cb−Ca)×((100-x)-Va)/(Vb−Va))+ Ia
X = % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca = the lowest concentration in μM with >X% reduction in cell viability
Cb = highest concentration in μM with Va = % viability at the lowest concentration with >X% reduction in cell viability
Vb = % viability at the highest concentration with
Statisitcal evaluation:
For every concentration showing >1.5 fold luciferase activity induction, statistical significance (p <0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.

Prediction Model:
The test item is considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 μM
- an apparent overall dose-response for luciferase induction
- a negative result obtained with concentrations <1000 μM is considered as inconclusive.

Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 μM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

Run / experiment:

other: Experiment 1

Parameter:

other: max luciferase activity (Imax) induction

Value:

1.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: max luciferase activity (Imax) induction

Value:

0.99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

The controls confirmed the validity of the study.

Induction of Luciferase Activity – Overall Induction

	Concentration (microM)	Fold induction
		Experiment 1	Experiment 2	Mean	SD
Solvent Control		1.00	1.00	1.00	0.00
Positive Control	4.0	1.11	1.12	1.11	0.01
	8.0	1.00	1.18	1.09	0.13
	16.0	1.28	1.43	1.36	0.10
	32.0	1.71	1.97	1.84*	0.19
	64.0	6.34	3.67	5.01	1.89
Test Item	0.98	1.20	0.98	1.09	0.16
	1.95	1.04	0.98	1.01	0.05
	3.91	1.10	0.92	1.01	0.12
	7.81	0.96	0.91	0.94	0.03
	15.63	1.16	0.93	1.05	0.16
	31.25	1.08	0.97	1.03	0.08
	62.50	1.00	0.97	0.99	0.02
	125.00	1.06	0.99	1.02	0.05
	250.00	1.02	0.93	0.97	0.06
	500.00	1.06	0.96	1.01	0.08
	1000.00	1.06	0.95	1.01	0.07
	2000.00	0.99	0.90	0.94	0.07

* = significant induction according to Student’s t-test, p<0.05

Interpretation of results:

GHS criteria not met

Conclusions:

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.
Under the conditions of this study the test item is therefore considered as non-sensitiser.

Executive summary:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. The test was performed according to OECD TG 442D and in compliance to GLP.

The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 μM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (Imax) induction of 1.20 was determined at a test item concentration of 0.98 μM. The corresponding cell viability was 105.3%. No significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, a max luciferase activity (Imax) induction of 0.99 was determined at a test item concentration of 125 μM. The corresponding cell viability was 192.8%. In the second experiment all cell viability values are in the range of 186.2% - 212.0% which can be an indicator of cell stress. However, since no induction of the luciferase was observed and the cell viability curve is similar to the one of experiment 1, an increased number of cells per well is assumed. Presumably at the seeding of the cells the double amount of cell suspension was applied. Since no significant luciferase induction >1.5 was found in the tested concentration range, this is not considered as relevant for the outcome of the study. No EC1.5 value could be calculated.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 (6.34 in experiment 1; 3.67 in experiment 2). The calculated EC1.5 was between 7 and 34 μM (24.15 in experiment 1; 18.10 in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (17.5% in experiment 1; 12.5% in experiment 2).

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the conditions of this study the test item is therefore considered as non-sensitiser.

In conclusion, for this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Two in vitro skin sensitisation assays were performed on dipotassium malonate: a DPRA and a KeratinoSens assay.
The test item was found tto be negative (not sensitising) in both assessments. These findings, when taken into a weight-of-evidence approach, can be used to conclude that dipotassium malonate does not need to be classified for skin sensitisation effects.