Dipotassium propanedioate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Dipotassium propanedioate

EC Number:

820-064-0

Cas Number:

13095-67-5

Molecular formula:

C3H2K2O4

IUPAC Name:

Dipotassium propanedioate

In chemico test system

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.

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.