Dipotassium methanedisulphonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 November 2017 - 18 November 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))

Deviations:

no

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 methanedisulphonate

EC Number:

228-543-0

EC Name:

Dipotassium methanedisulphonate

Cas Number:

6291-65-2

Molecular formula:

CH4O6S2.2K

IUPAC Name:

dipotassium methanedisulphonate

Test material form:

solid

In chemico test system

Details on the study design:

TEST ITEM SOLVENT
Initial election of test item solvent: solubility of the test item in an appropriate solvent was assessed before performing the assay. The chosen solvent was Milli-Q water .

TEST SYSTEM
Cysteine peptide (supplier RS Synthesis, Louisville, KY 40270, USA)
Lysine peptide (supplier RS Synthesis, Louisville, KY 40270, USA)

CONTROLS
Positive control: cinnamaldehyde (CAS 104-55-2; batch no MKBT8955V; purity 99.1%)
Co-elution control: test item in phosphate buffer (750 µL of phosphate buffer (pH 7.5) + 200 µL of acetonitrile + 50 µL test item) for cysteine peptide and ammonium acetate buffer (750 µL of ammonium acetate buffer (pH 10.2) + 250 µL of test item) for lysine peptide.
Reference control A: prepared with acetonitrile in order to check the calibration curve accuracy (750 µL Cysteine/Lysine peptide (0.667 mM) + 250 µL of acetonitrile).
Reference control B: prepared with acetonitrile and included at the beginning and at the end of the sequence in order to check the stability of peptide over time (same as preparation for reference control A, but run before and after 24 ± 2 hours incubation at 25 ± 2.5ºC in dark).
Reference control C: prepared with Milli-Q water, the test item and the positive control solvent, in order to check its influence on the peptide stability: 750 µL of Cysteine peptide stock + 200 µL of acetonitrile + 50 µL Milli Q water (for Cysteine), 750 µL of Lysine peptide stock + 250 µL of Milli Q water (for Lysine).

PREPARATION OF THE TEST ITEM AND POSITIVE CONTROL SOLUTIONS
Test item solution: 47.2 mg was pre-weighted in a glass vial in order to prepare, right for use, 3 ml of a limpid 100 mM solution with acetonitrile.
Positive control solution: 38.2 µl of the positive control were distributed in a 5 ml glass vial in order to prepare, right before use, 3 ml of a limpid 100 mM solution with acetonitrile.

PREPARATION OF THE PEPTIDE SOLUTIONS
Peptide solutions were prepared at 0.667 mM:
Cysteine solution: 9.27 mg of cysteine peptide were pre-weighted then dissolved, right before the incubation, in 18.503 ml of phosphate buffer (pH 7.5).
Lysine solution: 9.784 mg of lysine peptide were pre-weighted then dissolved, right before the incubation, in 18.888 ml of ammonium acetate buffer (pH 10.2).

TEST SOLUTIONS
Peptides were incubated with each sample (test item and positive control) at 1:10 and 1:50 ratio for cysteine and lysine respectively.
1 ml of each solution was prepared according to the following quantities:
-Cysteine test solution: 750 µl of cysteine peptide solution (buffer only to check co-elution) + 50 µl of sample or solvent for Reference controls + 200 µl of acetonitrile.
-Lysine test solution: 750 µl of lysine peptide solution (buffer only to check co-elution) + 250 µl of sample or solvent for Reference controls.

Vials were caped, vortexed and were placed in the HPLC auto sampler at 25 ± 2.5ºC in dark for 24 ± 2 hours. HPLC analysis of the batch of samples was started 24 ± 2 hours after the test item was added to the peptide solution.

Replicates: Samples were prepared in triplicate for both peptides.

HPLC ANALYSIS
-Apparatus: Shimadzu with UV Detector. Column: ZorbaX SB-C18 (2.1 mm x 100 mm x 3.5 micron) with Guard Column Phenomenex Security Guard C18 (4 mm x 2 mm)
-Calibration curve: Six peptides standards solutions between 0.534 mM and 0.0167 mM (dilution factor 2) were prepared in 20% acetonitrile in phosphate buffer for cysteine peptide and ammonium acetate for lysine peptide. The dilution buffer was also included as blank in the standard calibration curve.
-Volume injected: 5 µl of each sample
-Run time: 20 min
-Sequence (gradient): see table 1 on “Any other information on materials and methods incl. tables”
-Analysis sequence:
The analysis was programmed according to the following principles:
The reference controls B were placed at the beginning and at the end of the analysis (3 repetitions).
The reference controls C were placed at the beginning of each repetition.
The standards of the calibration curve and the reference controls A were placed in order to be analyzed progressively throughout the sequence.
Lysine and cysteine analysis were conducted on separate day. Samples were visually inspected for precipitation prior to HPLC analysis. Precipitation was not observed with test item and positive control.

DEVIATIONS FROM OECD GUIDELINE: No.

Results and discussion

Positive control results:

The depletion mean rate was 78% for cysteine peptide and 61% for lysine peptide.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: mean of 3 repetitions

Parameter:

other: %depletion in lysine peptide

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Run / experiment:

other: Mean of 3 repetitions

Parameter:

other: %depletion in cysteine peptide

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Run / experiment:

other: Mean of lysine and cysteine peptides

Parameter:

other: mean %depletion in peptides

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442C were performed, obtaining values that fall within the respective reference range for 8 out of the 10 for each peptide.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for calibration curve: yes, coefficient r2 was higher than 0.99 for lysine and cysteine.
- Acceptance criteria met for reference control A: yes, the mean concentration of peptide was 0.46 mM for lysine and 0.51 mM for cysteine which are equal to 0.50± 0.05 mM
- Acceptance criteria met for reference control B: yes, the CV (coefficient of variation) of the controls B was 0.51 % for Cysteine peptide and 1.44 % for Lysine peptide which are lower than 15%.
- Acceptance criteria met for reference control C: yes, the mean concentration of peptide was 0.53 mM for lysine and 0.49 mM for cysteine which are equal to 0.50± 0.05 mM. The CV (coefficient of variation) of the controls C was 1.46% for Cysteine peptide and 0.55% for Lysine peptide which are lower than 15%.
- Acceptance criteria met for positive control: Yes, SD of the 3 repetitions for each peptide was 0.56% for cysteine and 0.41% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively. The depletion mean rate was 78% for cysteine peptide and 61% for lysine peptide which are between 60.8% and 100% for the cysteine and between 40.2% and 69.4% for the lysine.
- Acceptance criteria met for variability between replicate measurements: Yes, SD of the 3 repetitions of the test item for each peptide was 1.45% for cysteine and 0.28% for lysine which are lower than 14.9% and 11.6% for cysteine and lysine respectively.

Any other information on results incl. tables

Table 3: Positive control and test item percent peptide depletion

Percent Peptide Depletion (Cysteine)
Positive Control Name/ Test Item Code	Rep-1 Area (AU)	Rep-2 Area (AU)	Rep-3 Area (AU)	Average	% Depletion	SD	RCV	Expected Depletion
Cinnamic aldehyde (17.11.17)	437677	417586	420800	425354	78	10792.06	2.54	60.8-100
Dipotassium Methanedisulphonate (17.11.17)	1946824	1923178	1890957	1920320	0	28042.97	1.46	-
Co-elution control- Dipotassium Methanedisulphonate (17.11.17)	ND	-	-	-	-	-	-	-
Percent Peptide Depletion (Lysine)
Cinnamic aldehyde (18.11.17)	681685	687649	695988	688441	61	7184.29	1.04	40.2-69
Dipotassium Methanedisulphonate (18.11.2017)	1734371	1733655	1742632	1736886	1	4989.04	0.29	-
Co-elution control- Dipotassium Methanedisulphonate (18.11.17)	ND	-	-	-	-	-	-	-

Keys: Rep = Replicate, AU = Arbitrary Units, SD = Standard Deviation, RCV = Relative Coefficient of Variability, ND = Not Detected, - = Not applicable

No co-elution occurred of the test item neither with lysine nor with cysteine peptides.

Applicant's summary and conclusion

Interpretation of results:

other: DPRA test result is considered in an overall weight of evidence assessment, i.e. as part of an integrated approach to testing and assessment (IATA) in accordance with OECD guideline 442C.

Conclusions:

The test item shows mean depletion of 1% for lysine and 0% for cysteine, i.e. an overall average of 0.5%, reflecting no or minimal reactivity and thus a negative prediction of DPRA skin sensitization test.

Executive summary:

A DPRA skin sensitization test was performed for dipotassium methanedisulphonate as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442C, under GLP conditions. The test item was prepared at 100 mM in Milli-Q water and Cinnamaldehyde (positive control) was prepared at 100 mM in acetonitrile. Reference controls A, B and C were prepared with acetonitrile and Milli-Q water (control C) in order to check the HPLC system suitability, the stability of peptide over time and the influence of the solvent on the peptide depletion respectively. Peptide solutions were prepared at 0.667 mM in phosphate buffer for cysteine and ammonium acetate buffer for lysine. Test item and positive control were incubated for 24 hours ± 2 hours at 25°C±2.5°C with the peptides solutions at 1:10 and 1:50 ratio for cysteine and lysine respectively, before HPLC analysis. Each sample was tested in triplicate for both peptides. All validity criteria were fulfilled. The test item shows mean depletion of 1% for lysine and 0% for cysteine, i.e. an overall average of 0.5%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.