Sodium methyl sulphate

Endpoint summary

Description of key information

The skin sensitizing potential of the test substance was assessed using an in vitro OECD guideline testing strategy comprising the following assays:

- Direct Peptide Reactivity Assay (DPRA),

- Keratinocyte Activation Assay (LuSens), and

- Dendritic Cell Line Activation Assay (h-CLAT).

Each test was conducted under GLP according to the respective OECD guideline.

The results were as follows:

- DPRA: negative

- LuSens: negative

- h-CLAT: waived

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: part of in vitro skin sensitization testing strategy

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

04 Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: KIRSCHAZ2-00182
- Content: 99.1 g/100 g (Based on preliminary information 98% was used for calculation of 100 mM concentration which was slightly lower than the contents determined, finally. As the assay was “negative”, this did not influence the result of the study, obviously.)
- Molecular weight: 134.09 g/mol
- Log KOW: -3.71 (calculated)
- Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system): The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in the vehicle (after short stirring). Due to the use of deionized water as vehicle the verification of the stability of the test substance in the vehicle was not required.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as ca. 100 mM (considering a molecular weight of 134.09 g/mol and a purity/contents of 98%) preparation in de-ionized water on a weight per volume basis within 4 hours of preparation of the test substance samples.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in de-ionized water

Details on the study design:

TEST SYSTEM
- Synthetic peptides: Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol); Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
- Source: The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and/or RS Synthesis, Louisville KY, USA and/or JPT Peptide Technologies GmbH, Berlin, Germany) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
- Preparation of peptide stock solutions: Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K­containing peptide). The peptide stock solution were used for preparing the calibration samples, the test-substance and control samples.

CONTROLS
- vehicle control: de-ionized water: Set A) performance control (analyzed together with the calibration samples without incubation); Set B) Stability control (placed at the very start and ending of the sample list for HPLC analysis); Set C) for calculation of the peptide depletion (analyzed with the samples)
- Positive control: ethylene glycol dimethacrylate (EGDMA; CAS no. 97-90-5) (prepared as a 50 mM solution in de-ioinzed water)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide

VEHICLE
- Vehicle: de-ionized water
- Reason for choice of the vehicle: The test substance was soluble in de-ionized water (tested prior to the assay).

SAMPLE PREPARATION
- Peptide stock solutions were mixed with the test substance or positive control or vehicle control at a ration of 1:10 (C-peptide) or 1:50 (K-peptide)

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 (for each peptide)
- The test substance was prepared at a ca. 100 mM concentration. The C-containing peptide was incubated with the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containg peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).
- Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis
- Samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours
- The remaining non-depleted peptide concentration was determined by HPLC with gradient elution and UV-detection at 220 nm about 24 hours after sample preparation (for details on HPLC conditions see tab. 2). The analysis time itself did not exceed 30 hours.
- Calibration samples of known peptide concentration (dissolved in 20% de-ionized water in the respective buffer), prepared from the respective peptide stock solution used for test-substance incubation, were measured before analysis of the test-substance samples with the same analytical method (for details see tab. 1)

DATA EVALUATION (for detailed formulas see "Any other information on material and methods")
Calculation of the peptide concentrations:
- For each peptide a calibration curve is generated from the measured peak areas of the calibration samples of known peptide concentration. The peptide concentration of the samples is calculated with the respective calibration curve using linear regression (b = axis intercept; m = slope).
Calculation of the peptide depletion:
- The mean peptide depletion for each of the two peptides is calculated as the mean value of the three samples conducted for each peptide and test substance. When a negative value for C- or K-containing peptide depletion is obtained the value is considered zero for calculation of the mean peptide depletion. The mean peptide depletion of a test substance is calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

ACCEPTANCE CRITERIA
- The standard calibration curve should have an r² >0.99.
- The negative control (vehicle control) samples of sets A and C should be 0.50 mM +/- 0.05 mM.
- The CV of the nine vehicle controls B and C should be < 15%.
- Since the mean peptide depletion for each peptide is determined from the mean of three single samples, the variability between these samples should be acceptably low (SD < 14.9% for % cysteine depletion and < 11.6% for % lysine depletion).
- The positive control should cause depletion of both peptides comparable to historic data.

Parameter:

other: mean peptide depletion [%]

Value:

0.13

Vehicle controls validity:

valid

Negative controls validity:

other: negative control = vehicle control

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: see tab. 8a+b

Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

Table 5: Peptide depletion for C-peptide

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean; SD
NC: H2O	-0.50	0.43	0.08	0.00; 0.47
Test substance	0.09	0.61	0.05	0.25; 0.32
PC: EGDMA in H2O	58.91	62.10	64.57	61.86; 2.84

Table 6: Peptide depletion for K-peptide

Reaction with lysine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean             SD
NC: H2O	0.98	-2.00	1.02	0.00      1.74
Test substance	0.20	-1.42	0.53	-0.23      1.04
PC: EGDMA in H2O	8.93	6.83	10.24	8.67     1.72

Table 7: Mean peptide depletions

	Cysteine-Peptide   mean depletion [%]          SD [%]		Lysine-Peptide   mean depletion [%]          SD [%]		mean of both depletions [%]
Test substance	0.25	0.32	-0.23	1.04	0.13
PC: EGDMA in H2O	61.86	2.84	8.67	1.72	35.26

Table 8a: Historic control data of vehicle control (de-ionized water) (not including present study)

	C-peptide concentration	K-peptide concentration
	[mM]	[mM]
Min	0.441	0.488
Max	0.510	0.528
Mean	0.479	0.506
SD	0.015	0.009
n	19	19

Table 8b: Historic control data of positive control (EGDMA, 50 mM in de-ionized water) (not including present study)

	C-peptide concentration [mM]	C-peptide depletion [%]	K-peptide concentration [mM]	K-peptide depletion [%]
Min	0.032	44.32	0.427	5.76
Max	0.323	93.44	0.481	16.01
Mean	0.164	66.47	0.459	9.27
SD n	0.072 16	14.31	0.014	2.45 16

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

However, in the current case for the test substance the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

DPRA:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with synthetic peptides for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in de-ionized water. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the

respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The following results were obtained in the DPRA:

Visual observation after the 24-hour incubation time did not reveal precipitates in any samples of the test substance with the peptides. No co-elution of test substance and peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be 0.25%. The mean K-peptide depletion, caused by the test substance was determined to be -0.23%.

Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.13%. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Remarks:

ARE-Nrf2 Luciferase Test Method

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Nov 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

other: part of in vitro skin sensitization testing strategy

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Feb 2015

Deviations:

yes

Remarks:

Cell viability assay: 2 hours incubation with MTT reagent

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: KIRSCHAZ2-00182
- Content: 99.1 g/100 g (Based on preliminary information 98% was used for calculation of 100 mM concentration which was slightly lower than the contents determined, finally. As the assay was “negative”, this did not influence the result of the study, obviously.)
- Molecular weight: 134.09 g/mol
- Log KOW: -3.71 (calculated)
- Proposed reaction mechanism for protein binding by OECD toolbox (non-GLP system): The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Solubility and stability of the test substance in the solvent/vehicle: The test substance was soluble in the vehicle. Because the test-substance preparation was applied shortly after preparation, no analysis of the test substance in the vehicle was performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared within 4 hours of application. The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium (DMEM + 1% FBS) to achieve the required 4x concentration of the highest test concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate). The test-substance preparations were prepared by stirring.
- Visual inspection of each dilution step was performed.
- Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.

FORM AS APPLIED IN THE TEST (if different from that of starting material): dissolved in 4 % DMSO in culture medium (DMEM + 1% FBS)

Details on the study design:

TEST SYSTEM
- Cell line: LuSens (human transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen, Germany)

CONTROLS
- Vehicle control: 1% DMSO in culture medium (DMEM + 1% FBS)
- Positive control: ethylene glycol dimethacrylate (EGDMA, CAS no.: 97-90-5), 18 µg/ml in 1% DMSO in culture medium (DMEM + 1% FBS)
- Negative control: DL-Lactic acid (LA, CAS no.: 50-21-5), 450 µg/ml in 1% DMSO in culture medium (DMEM + 1% FBS)
- Blank control: culture medium (DMEM + 1% FBS) without cells
- Basal control: culture medium (DMEM + 1% FBS) with cells

VEHICLE
- Vehicle: 4% DMSO in culture medium (DMEM + 1% FBS)
- Reason for the vehicle: The test substance was soluble in 4% DMSO in culture medium (DMEM + 1% FBS)

SELECTION OF CONCENTRATIONS
- Cells were exposed to several concentrations of the test-substance preparation (0.5 μg/mL up to 2020 μg/mL corresponding to final test substance concentrations of 0.5 μg/mL up to 1980 μg/mL taking a purity/contents of 98% into account) and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve to be 734 μg/mL. The highest tested concentration in the 1st main experiment was 1.23 fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

TEST CONCENTRATIONS (not corrected for purity/contents)
1268 μg/mL
1057 μg/mL
881 μg/mL
734 μg/mL
611 μg/mL
510 μg/mL
425 μg/mL
354 µg/mL

EXPERIMENTAL PROCEDURE
- No. of replicates: 2 independent valid experiments with 3 replicates of each test-substance concentration in each experiment
- Seeding of cells: Cells were seeded in white (for luciferase assay) and clear (for cell viability assay) 96-well microtiter plates (120 µL of 0.83 x 10^5 cells/mL cell suspensions) in culture medium (DMEM + 10% FBS) and incubated for 24 hours.
- Application of test substance: After cell adaption for 24 hours, cell culture medium was exchanged by DMEM + 1% FBS. The test substance preparations (4x concentrations) were applied in a ratio of 1:4 to the cells (final DMSO concentration in the test medium = 1%). For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.
- Exposure duration: 48 hours under standard culture conditions (plates were sealed with semi-permeable plate sealers to prevent evaporation of the test substance)
- Each test-substance concentration was visually inspected directly after application and after the exposure period of 48 hours in order to detect test-substance precipitates.
- Luciferase assay: After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Subsequently 200 µL of One-/Steady-Glo-preparation (= 100 µL One-/Steady-Glo- Mix and 100 µL PBS (without Ca2+/Mg2+)) per well were added and cells shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.
- Determination of cell viability: Cell culture medium was aspirated from all wells. The cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Thereafter 200 μL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+) and medium (DMEM + 15 FBS)) was added to each well of the 96-well microtiter plate and incubated for further 2 hours after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 μL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

DATA EVALUATION (for details on formulas see "Any other information on materials and methods")
CV75 calculation:
- The CV75-value (relative survival rate) is calculated by linear regression. This value is the substance concentration at which relative cell viability is 75% compared to the vehicle control.
Cell viability:
- From the 3 independent replicates a mean is calculated.
Luciferase fold induction:
- From the 3 independent replicates a mean is calculated.
EC1.5 calculation (if applicable):
- The concentration resulting in a positive response (1.50 fold-induction of statistical significance and viability >70%) was calculated from each experiment conducted. The calculation was performed by linear regression from the two concentrations directly above and below the EC1.50 concentration.

STATISTICAL ANALYSES
- For the statistical evaluation of luciferase fold-induction the Welch t-test (one-sided) was used.

EVALUATION CRITERIA
- A test substance is concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeds a 1.50 fold-induction of statistical significance with respect to the vehicle control at concentrations that do not reduce viability below 70% in at least two consecutive concentrations of two independent experiments. In each experiment, at least one of these concentrations must lie above the borderline area (> 1.65).
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 µg/mL) or maximum applicable concentration or up to the cytotoxicity limit (at least one concentration displaying viability below 70%). In addition, the borderline criteria must not be met.
- To be relevant for evaluation, the cell viability must be more than 70% in at least three tested concentrations of an experiment.

ACCEPTANCE CRITERIA
- A tested concentration was not further evaluated when relative viability is less than 70%.
- The cell viability of vehicle control cells must yield at least 85%.
- The mean of the positive control EGDMA should achieve ≥2.50 fold-induction and the mean of the LA <1.50 and the mean of the viability must be ≥70%.
- The CV [%] of the luminescence in the vehicle control wells for each plate should be below 20%.
- The mean of the basal expression of the cells must be <1.50 fold-induction as compared to the solvent control.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Run / experiment:

other: experiment 1 + 2

Parameter:

other: EC1.5 [µM]

Remarks:

luciferase induction (with respect to vehicle control)

Value:

1.5

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:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values: see tab. 3

The test substance was soluble in 4% DMSO in culture medium (DMEM + 1% FBS) (4 x stock preparations) and in 1% DMSO in culture medium (DMEM + 1% FBS) (final concentrations).

No precipitates were noticed in any concentration after 48 hours.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

Table 2: Summary of main experiments. 2 valid and evaluable experiments were performed

Concentration (test substance) µg/mL	fold induction		1^stexperiment rel. viability [%]		t-test
	mean	SD	mean	SD	p-value		markers
354	1.23	0.15	93.3	6.7	0.049		*
425	0.86	0.08	62.1	6.5	0.029		*
510	0.91	0.15	73.6	11.3	0.206		n.s.
611	0.96	0.30	71.4	6.5	0.421		n.s.
734	1.11	0.08	70.6	5.7	0.078		n.s.
881	1.11	0.19	67.2	11.3	0.219		n.s.
1057	1.11	0.11	53.4	13.4	0.124		n.s.
1268	1.48	0.08	68.5	9.6	0.000		**
VC	1.00	0.21	100.0	8.2	-		-
EGDMA (18 µg/mL)	7.64	0.46	104.1	7.6	0.000		**
LA (450 µg/mL)	0.95	0.13	116.5	4.5	0.245		n.s.

Concentration (test substance) µg/mL	fold induction		2^ndexperiment rel. viability [%]		t-test
	mean	SD	mean	SD	p-value		markers
354	1.01	0.02	76.6	5.2	0.451		n.s.
425	1.12	0.09	71.3	14.0	0.064		n.s.
510	1.02	0.15	73.1	8.2	0.435		n.s.
611	1.05	0.14	71.5	4.5	0.298		n.s.
734	0.93	0.11	68.6	4.2	0.193		n.s.
881	0.99	0.16	69.2	3.3	0.482		n.s.
1057	0.90	0.04	60.7	7.5	0.024		*
1268	1.06	0.26	61.7	4.8	0.360		n.s.
VC	1.00	0.18	100.0	5.3	-		-
EGDMA (18 µg/mL)	9.88	0.78	114.6	9.5	0.000		**
LA (450 µg/mL)	1.00	0.07	122.0	7.6	0.472		n.s.

Table 3: Historic control data of LuSens. Data (not including present study)

Negative Control (LA 450 µg/mL)	fold induction	rel. viability [%]
Min	0.70	75.8
Max	1.38	137.5
Mean	0.96	106.8
SD	0.11	9.0
n	305

Positive Control (EGDMA 18 µg/mL)	fold induction	rel. viability [%]
Min	3.49	70.1
Max	10.38	136.1
Mean	6.53	104.0
SD	1.37	12.0
n	305

Vehicle Control (1% DMSO)	fold induction	rel. viability [%]
Min	1.00	100.0
Max	1.00	100.0
Mean	1.00	100.0
SD	0.00	0.0
n	305

Basal expression	fold induction	rel. viability [%]
Min	0.56	96.2
Max	1.38	185.8
Mean	0.88	149.7
SD	0.16	15.4
n	305

Executive summary:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

However, in the current case for the test substance the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

LuSens:

The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 734 μg/mL (corresponding to test substance as provided by the sponsor). In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed.

The following results were observed:

The test substance was soluble in 4% DMSO in culture medium (4 x stock preparations) and in 1% DMSO in culture medium (final concentrations). No precipitates were noticed in any concentration after 48 hours. Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments.

From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The objective was to assess the skin sensitizing potential of the test substance. A combination of the following three in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were

part of this in vitro Skin Sensitization Turnkey Testing Strategy:

• protein reactivity (DPRA),

• activation of keratinocytes (LuSens), and

• activation of dendritic cells (h-CLAT).

However, in the current case for Sodium methyl sulfate the results derived with DPRA and LuSens were sufficient for a final assessment. Therefore further testing in h-CLAT was waived.

DPRA:

In order to assess the reactivity towards peptides, synthetic cysteine (C)- or lysine (K)-containing peptides were incubated with the testsubstance for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UVdetection at 220 nm according to the OECD guideline 442C.

Therefor the test substance was dissolved at a ca. 100 mM concentration in de-ionized water and three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide).

The test substance caused a mean C-peptide depletion of 0.25% and a mean K-peptide depletion of -0.23% (negative depletions were considered to be "zero" for calculation of mean peptide depletion).

The mean peptide depletion was calculated to be 0.13%. Thus it was concluded, that the test substance shows minimal or no chemical reactivity towards peptides.

LuSens:

In order to assess the keratinocyte activating potential, a luciferase reporter cell line (LuSens cells) was incubated with the testsubstance for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer according to the OECD guidline 442D.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Based on the results of both tests it was concluded that the test substance is not peptide reactive and does not activate keratinocytes. Therefore the test substance is predicted not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.