Dipotassium methanedisulphonate

Endpoint summary

Description of key information

Skin sensitisation (in chemico): Weight of evidence. Test method according to the OECD 442C Guideline with GLP. The test item showed a mean depletion of lysine and cysteine peptides of 0.5%, reflecting no or minimal reactivity and thus a negative prediction of DPRA.

Skin sensitisation (in vitro): Weight of evidence. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test item may be considered as not skin sensitizer using the KeratinoSensTM test method.

Skin sensitisation: Weight of evidence. integrated testing strategy (ITS). Based on the“2 out of 3-sens ITS” approach as described in annex 1 of OECD guidance ENV/JM/MONO(2016)29, two negative results obtained in the tests addressing two different key events (KEs) means that dipotassium methanedisulphonate does not need to be classified as skin sensitizer.

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:

14 November 2017 - 18 November 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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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.

Positive control results:

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

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

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

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.

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.

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.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 December 2017 - 04 May 2018

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Details on study design:

REAGENTS AND MEDIA
-Fetal Bovine Serum (FBS): Gibco (Lot # 1789459, 1884722)
-DMSO: Qualigens (Lot # 2217110817, 2287381017)
-D-MEM with Glutamax: Gibco (Lot # 1855240, 1897167, 1930096)
-Ethylene Diamine Tetra Acetic Acid (EDTA): Sigma (Lot # SLBM9213V)
-DPBS (1X): Sigma (Lot # RNBF3314, RNBF8606)
-Trypsin-EDTA: Gibco (Lot #1774124, 1811292, 1894157)
-Geneticin: Gibco(Lot #1751936, 1898753)
-Culture medium: D-MEM with Glutamax supplemented with 9.1% fetal bovine serum and 500 μg/mL geneticin.
-Exposure medium: 10 µL test solution + 230 µL of DMEM with 1% FBS without geneticin + 10 µL of DMSO.
-Staining solution: 27 µL of MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in culture medium.
-Passage number: cells were used at passage 21 and 25 for both experiments.

CONTROLS
-Positive control: Trans cinnamaldehyde (CAS 14371-10-9; batch no STBF4119V; purity 99.4%)
-Negative (solvent) control: Distilled water

CELLS SEEDING.
-Cells suspension: Cells were maintained in culture medium. After cells attained 80-90% confluency, they were washed twice with DPBS containing 0.05% of EDTA. To each flask 2-3 mL of 0.05% Trypsin-EDTA was added and flasks were incubated at 37 ± 1ºC (usually 5–10 minutes). After cells were detached completely, they were resuspended in DMEM with 9.1% FBS without geneticin. Cell count was taken and cell density was adjusted to 8 x 10e4 cells/mL. 125 µL of this culture (containing approx. 10,000 cells) was then dispensed in each well of 96 well plates, leaving one cell empty (to assess background values).
-nº of culture plates: four; three 96-well white assay plates (for luciferase assay) and one 96-well flat bottom transparent plate (for cytotoxicity, MTT assay).
-Cell density: 10e4 cells per well.
-Incubation: the seeded plates were incubated for 24 hours in 5±1 % CO2 at 37±1ºC.

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Positive control: Trans cinnamaldehyde was dissolved in DMSO to achieve concentration of 200 mM. This solution was further diluted to final concentration of 6.4 mM by adding 32 µL of 200 mM solution to 968 µL of DMSO.
Negative control: Distilled water was used as negative (solvent) control.
Test Item Preparation: Serial dilutions were made using Distilled water to obtain 12 master concentrations of the test item to be tested (from 0.098 to 200 mM). The master concentrations were then further diluted 25 fold into culture medium containing serum, and finally used for treatment with a further 4 fold dilution factor so that the final concentrations of the tested chemical ranged from 0.98 to 2000 µM.

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
After 24 ± 2 hours of incubation, medium from all the 4 plates (for single test item) was aspirated and discarded. It was replaced with 150 µL of DMEM containing 1% FBS without geneticin. The 25 fold dilution of the 100X master plate was performed into a fresh plate (10 µL test solution + 230 µL of DMEM with 1% FBS without geneticin + 10 µL of DMSO). Resulting 4X plate was further distributed to assay plates: 50 µL of these stock solutions were added 3 white assay plates and one cytotoxicity plate already containing 150 µL of culture medium. Plates were then covered with petri-seal and incubated for 46 hours in 5 ± 1 % CO2 at 37 ± 1ºC.

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 µM to 2000 µM.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 µM.

REPLICATES: The study was composed of 2 independent repetitions. For each repetition the test item and the controls were replicated on three independent plates for the measurement of induction and one plate for the measurement of cytotoxicity. Two valid repetitions (experiment 1 and 2) were considered for final evaluation.

LUCIFERASE ACTIVITY.
-Procedure: After 48 ± 2 hours of incubation, the medium from 96 well white assay plates was aspirated and discarded. Cells were washed once with 150 µL of DPBS. After washing, 20 µL of passive lysis buffer was added in each well. Cells were then incubated for 30 ± 10 minutes at 37 °C. After incubation, the plates with cell lysate were placed in luminometer. 50 µL of 1X Luciferase substrate was added and luciferase activity was recorded for 2 seconds.

CITOTOXICITY ASSESSMENT.
-Procedure: Master mix was prepared by combining 27 µL of MTT (5 mg/mL in DPBS) and 200 µL of DMEM containing 1% FBS without geneticin. After 48 ± 2 hours of incubation, the medium from 96 well transparent plate was replaced with 227 µL above prepared master mix. The plate was covered with foil and incubated for 4 hours. After incubation, the medium was removed and 200 μL of a 10% SDS solution was added to each well. The plate was sealed and covered with a foil and placed in the incubator for overnight incubation. Following incubation the absorption at 570 nm was measured.

DEVIATIONS FROM OECD GUIDELINE: No.

Positive control results:

The gene induction for positive control was found to be >1.5 at concentrations of 16 µM, 32 µM and 64 µM in both the repetitions. The EC1.5 value for positive control was found to be 1.09 µM and 13.63 µM in experiment 1 and 2, respectively. The average gene induction for positive control at 64 µM was found to be 7.10 and 2.72 for experiment 1 and 2, respectively. Clear dose response with increasing gene induction at increasing dose was observed for trans cinnamaldehyde in experiment 1 and experiment 2.

Key result

Run / experiment:

other: Repetition 1

Parameter:

other: Imax

Value:

1.13

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Repetition 2

Parameter:

other: Imax

Value:

1.2

Vehicle controls validity:

valid

Negative controls validity:

valid

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 442D were performed, obtaining values that fall within the respective reference range for 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 14.89% and for repetition 2 was 10.35% which are less than 20%.

- Acceptance criteria met for positive control: The luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The EC1.5 value for positive control was found to be 1.09 µM and 13.63 µM in experiment 1 and 2, respectively, and the average gene induction for positive control at 64 µM was found to be 7.10 and 2.72 for experiment 1 and 2, respectively. The latter criterion is not fulfilled, however, clear dose response with increasing gene induction at increasing dose was observed for trans cinnamaldehyde in experiment 1 and experiment 2. Thus, the acceptance criteria are met.

Table 1: Mean Summary of Imax values of test item 

I_max

Test Item	Rep1	Rep2	Average
Dipotassium Methanedisulphonate	1.13	1.20	1.17

 

Table 2: Mean Summary of Imax, EC1.5, IC30 and IC50 values of Positive control

Trans-Cinnamaldehyde	Imax	EC1.5	IC50	IC30
Rep 1	7.10	1.09	-	-
Rep 2	2.72	13.63	-	-

Table 3: Summary of Mean Induction of the test item (Two Experiments)

Concentration (µM)	Dipotassium Methanedisulphonate
	Mean	SD
0.98	1.06	0.20
1.95	0.99	0.16
3.91	1.03	0.08
7.81	1.02	0.04
15.63	0.99	0.06
31.25	1.01	0.12
62.5	0.96	0.08
125	0.98	0.01
250	1.02	0.07
500	1.06	0.09
1000	1.02	0.08
2000	0.97	0.11

Interpretation of results:

other: KeratinoSensTM 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 442D.

Conclusions:

Under the experimental conditions the test substance may be considered as not skin sensitizer using the KeratinoSensTM test method.

Executive summary:

The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control trans cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control (distilled water) were placed in the seeded plates and incubated for 48 hours ± 2 hour at 37ºC, 5% CO2. The study was composed of 2 independent repetitions considered valid for final evaluation. All validity criteria were fulfilled. For the test item, calculated Imax values were lower than 1.5 in the 2 repetitions. Thus, a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation (in chemico): Weight of evidence. 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.

Skin sensitisation (in vitro): Weight of evidence. The KeratinoSensTM test method was performed for the test substance as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 1 plate (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 µM to 2000 µM, positive control trans cinnamaldehyde at 5 concentrations from 4 to 64 µM and negative control (distilled water) were placed in the seeded plates and incubated for 48 hours ± 2 hour at 37ºC, 5% CO2. The study was composed of 2 independent repetitions considered valid for final evaluation. All validity criteria were fulfilled. For the test item, calculated Imax values were lower than 1.5 in the 2 repetitions. Thus, a negative result can be predicted for skin sensitization using the KeratinoSensTM test method.

Skin sensitisation: Weight of evidence. integrated testing strategy (ITS). Based on the OECD Guidance Document on the reporting of defined approaches to be used within integrated approaches to testing and assessment for skin sensitization ((ENV/JM/MONO(2016)29), the “2 out of 3-sens ITS” approach as described in its annex 1 has been selected to allow the classification of the substance.

The combination of test methods used in this strategy covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E or the modified Myeloid U937 Skin Sensitisation Test (mMUSST)].

As there is no differential weighting of the individual test methods used and no predefined sequential order of testing, the order and information source from which data is obtained is not defined. Due to the higher complexity and resources needed (e.g. flow cytometer needed) to conduct the tests used for KE 3, the DPRA (KE1) and Nrf2-ARE-based tests (KE2) will usually be conducted first.

Based on the current knowledge, information obtained from peptide reactivity, whether obtained from in chemico or in silico methods, seems to show the highest predictive power and may provide more weight to the overall assessment of skin sensitisation (Natsch et al., 2013, Urbisch et al., 2015).

According to this approach two concordant results addressing two different key events (KEs) indicate the sensitising potential, i.e. two positive results indicate a sensitiser, two negative results indicate a non-sensitiser.

Thus, dipotassium methanedisulphonate does not need to be classified as skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is not classified for skin sensitization according to CLP Regulation no. 1272/2008.