Sodium 1-hydroxyethanesulphonate

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: positive

- h-CLAT: positive

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:

21 Jun 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))

Version / remarks:

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
- Source and lot/batch No.of test material: BASF SE; batch No.: Lab-sample from Dec 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- 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: Test 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 137.4 g/mol and a purity/contents of 53.9%) preparation in de-ionized water. At the time of the conduct of the DPRA preliminary information on the content of the test substance was available and used for calculation of 100 mM concentration (53.9 g/ 100 g). However, the final content of the test substance is reported to be 50.7 g /100 g. Hence, considering the final purity, a concentration of ca. 94 mM was used as stock concentration of the DPRA.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted 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 (K­ containing 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. 3). 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

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:

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. 9a+b

The test substance was dissolved in de-ionized water at a concentration of ca. 100 mM.

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 6: Peptide depletion for C-peptide

Reaction with cysteine- peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean             SD
NC: H2O	-1.32	0.94	0.38	0.00             1.18
Test substance	-8.79	-9.60	-3.84	-7.41            3.12
PC: EGDMA in H2O	84.38	89.50	92.78	88.89            4.24

Table 7: Peptide depletion for K-peptide

Reaction with lysine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean             SD
NC: H2O	1.17	-0.65	-0.52	0.00             1.01
Test substance	0.21	1.10	-1.47	-0.05            1.31
PC: EGDMA in H2O	6.60	8.61	4.92	6.71             1.84

Table 8: Mean peptide depletions

	Cysteine-Peptide   mean depletion [%]          SD[%]		Lysine-Peptide   mean depletion [%]          SD[%]		mean of both depletions[%]
Test substance	-7.41	3.12	-0.05	1.31	0.00
PC: EGDMA in H2O	88.89	4.24	6.71	1.84	47.80

Table 9a: Historic control data of vehicle control (de-ionized water) (not including present study)

	C-peptide concentration	K-peptide concentration
	[mM]	[mM]
Min	0.432	0.459
Max	0.510	0.528
Mean	0.475	0.504
SD	0.017	0.016
n	17	15

Table 9b: 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	0.403	5.93
Max	0.266	93.44	0.481	16.01
Mean	0.153	67.87	0.455	9.39
SD	0.063	13.34	0.020	2.58
n	13		12

Interpretation of results:

GHS criteria not met

Conclusions:

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.
However, it should be noted, that due to the lower concentration of the stock concentration used (ca. 94 mM instead of 100 mM), the result could be under-predictive.

Executive summary:

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 UVdetection at 220 nm.

The test substance was dissolved at ca. 100 mM concentration in de-ionized water (At the time of the conduct of the DPRA preliminary information on the content of the test substance was available and used for calculation of 100 mM concentration (53.9 g/ 100 g). However, the final content of the test substance is reported to be 50.7 g /100 g. Hence, considering the final purity, a concentration of ca. 94 mM was used as stock concentration of the DPRA). 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:

The test substance was dissolved in de-ionized water at a concentration of ca. 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. 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 -7.41%. The mean K-peptide depletion, caused by the test substance was determined to be -0.05%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 0.00%.

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.

However, it should be noted, that due to the lower concentration of the stock concentration used, the result could be under-predictive.

Reference 2

Endpoint:

skin sensitisation: in vitro

Remarks:

human Cell Line Activation Test (h-CLAT)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Dec 20017 - Jan 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT))

Version / remarks:

2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF SE; batch No.: Lab-sample from Dec 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

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 (culture medium) to achieve the required 2x concentration of the highest test concentration (stock solution). Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations in the culture medium. The test-substance preparations were prepared by stirring.
- Visual inspection of each dilution step was performed.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in culture medium

Details on the study design:

TEST SYSTEM
- Cell line: THP-1 cells (human monocytic leukemia cell line); obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB- 202)

CONTROLS
- Vehicle control: culture medium
- Positive control: 1-chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 µg/mL in 0.2% DMSO in culture medium
- Negative control: Lactic acid (LA, CAS no.: 50-21-5), 1000 µg/mL in culture medium
- Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1

VEHICLE
- Vehicle: culture medium
- Reason for the vehicle: The test substance was soluble in culture medium.

SELECTION OF CONCENTRATIONS
- Cells were exposed to 11 concentrations of the test-substance preparation (12 µg/mL up to 9862 µg/mL corresponding to final test substance ingredient concentrations of 6 µg/mL up to 5000 µg/mL taking the purity of 50.7% into account)) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. 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 7899 µg/mL. The highest tested concentration in the 1st main experiment was 1.2- fold of the CV75 value. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

TEST CONCENTRATIONS (µg/mL; not corrected for purity/contents)
9479
7899
6583
5486
4571
3809
3174
2645

EXPERIMENTAL PROCEDURE
- No. of replicates: 3 independent experiments with duplicates of each test-substance concentration in each experiment
- Seeding of cells: 24-well plates (500 µL of 2.0 x 10^6 cells/mL cell suspensions). Prior to use of the cells for a study, a reactivity check was performed with each new-thawed cells, as proposed in the OECD test guideline, using Nickel(II)sulfate hexahydrate, lactic acid and 1- chloro-2,4-dinitrobenzene in order to demonstrate qualification of the cells for the assay.
- Application of test substance: Treatment was performed by adding 500 µL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL.
- Exposure duration: 24 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 24 hours in order to detect test-substance precipitates.
- Cell staining and flow cytometric analysis: After visual inspection the cells were transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL buffer (PBS (without Ca2+/Mg2+) + 0.1% BSA). Cells were incubated with 600 µL of 0.01% Globulins Chon fraction II,III at 4°C for 15 min to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots (approximately 0.3 x 10^6 cells/180 µL/group) in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 µL working antibody solution (FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies; for details see tab. 1) was added to each pellet. Cell staining was performed at 4°C for 30 min in the dark. After staining the cells were washed twice with 200 µL buffer and finally re-suspended in 200 µL buffer. Before analysis in flow cytometer the cells were stained with 5 µL of PI (50 µg/mL diluted in buffer (PBS (without Ca2+/Mg2+) + 0.1% BSA)) to yield a final concentration of 1.22 µg/mL PI.

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 independent replicates of a test substance concentration a mean is calculated.
Relative fluorescence intensity:
- Analysis of the membrane markers was performed in 10,000 living cells, determined by PI staining. Concentrations inducing viability less than 50% were not considered for further assessment of dendritic cell activation. For data analysis, the CXP software (Beckman Coulter) was used. Data evaluation was performed with mean fluorescence intensity (MFI) of chemical treated cells among the viable cells, with systematic isotype control use to quantify and remove non-specific antibody binding. After subtracting the MFI of the isotype control, the RFI of each surface marker on the treated cells as compared with the vehicle control cells was calculated. The results were expressed as relative fluorescence intensity (RFI) of % CD86 pos. or % CD54 pos. expression compared to the respective vehicle control.
EC150% and EC200% calculation:
- The concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment conducted. The calculation was performed by linear regression from the two concentrations directly above and below the EC150% / EC200% concentration.

EVALUATION CRITERIA
- A test substance is predicted to activate monocytic THP-1 cells when CD86 expression is increased ≥ 150% and/or CD54 expression increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% and reproduced in the same cell surface marker in at least two independent experiments.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 5000 µg/mL for the vehicle culture medium or 1000 µg/mL for 0.2% DMSO in culture medium) or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested).
- To be relevant for evaluation, the cell viability must be more than 50% in at least four 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 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86 < 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- The reactivity check of new thawed cells should produce the following result:
Positive response in CD86 and CD54 for NiSO4 and DNCB;
Negative response in CD86 and CD54 for LA.
- Positive, negative and vehicle control data should lie within the range of the historic data.

Run / experiment:

other: experiment 1

Parameter:

other: EC200% for CD54 [µg/mL]

Remarks:

concentration resulting in a RFI of 200%

Value:

4 693

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: experiment 2

Parameter:

other: EC200% for CD54 [µg/mL]

Remarks:

concentration resulting in a RFI of 200%

Value:

4 639

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Parameter:

other: EC200% for CD54 [µg/mL]

Remarks:

concentration resulting in a RFI of 200%

Value:

5 184

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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 culture medium (2 x stock preparations and final concentrations).

No precipitates were noticed in any concentration after 24 hours.

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable.

Table 1: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability. RFI above 150% (CD86) or 200% (CD54) with rel. viability ≥50% are indicated in bold.

1^stexperiment				2^ndexperiment
Concentration (test substance) [µg/mL]	RFI CD86	RFI CD54	Viability rel. viability [%]	Concentration (test substance) [µg/mL]	RFI CD86	RFI CD54	Viability rel. viability [%]
	mean [%]	mean [%]			mean [%]	mean [%]
2645	85	177	101	2645	109	166	100
3174	73	116	101	3174	102	147	100
3809	76	148	99	3809	111	187	99
4571	86	188	98	4571	101	196	99
5486	89	277	99	5486	101	251	98
6583	93	197	99	6583	110	448	97
7899	107	345	96	7899	128	294	95
9479	123	485	93	9479	133	347	92
VC	100	100	100	VC	100	100	100
LA 1000 µg/mL	74	111	101	LA 1000 µg/mL	74	144	100
DNCB 4 µg/mL	269	591	84	DNCB 4 µg/mL	256	1267	76
3^rdexperiment
Concentration	RFI CD86	RFI CD54	Viability
(test substance) [µg/mL]	mean [%]	mean [%]	rel. viability [%]
2645	103	145	100
3174	91	134	100
3809	102	164	100
4571	98	171	98
5486	107	214	99
6583	111	202	98
7899	134	218	96
9479	137	291	93
VC	100	100	100
LA 1000 µg/mL	74	119	100
DNCB 4 µg/mL	294	896	81

Table 2: Historic control data of LuSens. Data (not including present study)

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]		rel. viability mean [%]
Min	31	43		95		99
Max	134	184		99		101
Mean	72	112		98		100
SD	11	18		1		0
n (experiments)			180

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]		rel. viability mean[%]
Min	151	211		69		70
Max	528	1893		92		98
Mean	286	605		84		85
SD	64	271		4		4
n (experiments)			180

Vehicle Control (culture medium)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]
Min	56	61		95
Max	144	139		99
Mean	100	100		98
SD	20	13		1
n (experiments)			180

Vehicle Control (DMSO)	CD86 RFI [%]	CD54 RFI [%]		viability mean[%]		rel.viability mean[%]
Min	39	48		90		92
Max	149	189		99		101
Mean	107	108		98		100
SD	21	18		1		1
n (experiments)			180

Table 3: Reactivity check, performed with each new-thawed cells prior to use for a study, using NiSO₄,LA, DNCB and the vehicle control

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel.viability mean[%]
Min	60	69	99
Max	90	169	101
Mean	72	112	100
SD	8	25	0
n (experiments)		21

Nickel(II)sulfate hexahydrate (NiSo4100 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]
Min	182	1105	61
Max	424	3569	91
Mean	305	2187	82
SD	69	620	7
n (experiments)		21

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel.viability mean[%]
Min	212	223	54
Max	428	1049	93
Mean	312	537	86
SD	57	215	10
n (experiments)		21

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

After 24 hours of exposure to the test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 11 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test after 24-hour exposure THP-1 cells were stained with FITC labeled anti-human- CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid experiments were performed.

The following results were observed:

The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 4693 μg/mL (experiment 1), 4639 μg/mL (experiment 2) and 5184 μg/mL (experiement 3), respectively.

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable.

In summary, after 24 hours of exposure to the test substance CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Reference 3

Endpoint:

skin sensitisation: in vitro

Remarks:

ARE-Nrf2 Luciferase Test Method

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Sep - Nov 2017 (6 experiments in total)

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)

Version / remarks:

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
- Source and lot/batch No.of test material: BASF SE; batch No.: Lab-sample from Dec 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor.

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): diluted 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), 90.8 µM (= 18 µg/ml) in 1% DMSO in culture medium (DMEM + 1% FBS)
- Negative control: DL-Lactic acid (LA, CAS no.: 50-21-5), 5000 µM (= 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 (1.0 µM up to 3945 µM corresponding to final test substance concentrations of 0.5 µM up to 2000 µM taking the purity/contents of 50.7% 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 113 µM (1st pre-test) or 58 µM (2nd pre-test). Based on a third pre-test , the top concentration for the 1st main experiment was chosen to be 901 µM.

TEST CONCENTRATIONS (µM; not corrected for purity/contents)
901
750
625
521
434
362
302
251
209
175
145
121
101
84
70
58
49
41
34
28
23
20
16
14

EXPERIMENTAL PROCEDURE
- No. of replicates: 4 independent 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 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:
- 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.
- A test substance is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (= 2000 µM if molecular weight is applicable or 2000 µg/mL if molecular weight is not applicable) or up to the cytotoxicity limit (at least one concentration displaying viability below 70%).
- 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 5

Parameter:

other: EC1.5 [µM]

Remarks:

1.5-fold luciferase induction

Value:

157

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: experiment 6

Parameter:

other: EC1.5 [µM]

Remarks:

1.5-fold luciferase induction

Value:

84

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive 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. 2

The test substance was soluble in 4% DMSO in culture medium 3 (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.

Table 1: Summary of main experiments. 2 valid and evaluable experiments (5th and 6st experiment) were performed

Concentration (test substance) [µM]		5^thexperiment, plate 1				Concentration (test substance) [µM]		5^thexperiment, plate 2
	fold induction mean	rel. viability [%] mean	p-value (t-test)		markers (t-test)		fold induction mean	rel. viability [%] mean	p-value (t-test)		markers (t-test)
251	1.66	90	0.000		**	58	1.16	87	0.037		*
302	2.12	72	0.019		*	70	1.37	81	0.049		*
362	1.83	75	0.000		**	84	1.39	85	0.027		*
434	2.01	84	0.024		*	101	1.41	89	0.036		*
521	1.91	81	0.001		**	121	1.43	80	0.000		**
625	1.77	88	0.016		*	145	1.46	84	0.002		**
750	1.97	82	0.002		**	175	1.56	79	0.002		**
901	1.91	86	0.003		**	209	1.58	80	0.002		**
VC	1.00	100	-		-	VC	1.00	100	-		-
EGDMA 90.8 µM	5.79	93	0.001		**	EGDMA 90.8 µM	4.77	93	0.000		**
LA 5000 µM	0.97	91	0.251		n.s.	LA 5000 µM	0.92	92	0.070		n.s.

Concentration (test substance) [µM]	5^thexperiment, plate 3				Concentration (test substance) [µM]	6th experiment, plate 1
	fold induction mean	rel. viabibity [%] mean	p-value (t-test)	markers (t-test)		fold induction mean	rel. viability [%] mean	p-value (t-test)	markers (t-test)
14 16 20 23 28 34 41 49	1.06 1.19 1.14 0.99 1.09 1.36 1.37 1.34	101 84 92 89 96 90 88 80	0.156 0.025 0.163 0.382 0.002 0.000 0.030 0.021	n.s. * n.s. n.s. ** ** * *	251 302 362 434 521 625 750 901	1.89 1.95 1.94 2.29 2.16 2.45 2.34 2.46	83 72 81 79 79 81 81 92	0.012 0.000 0.030 0.018 0.002 0.000 0.008 0.002	* ** * * ** ** ** **
VC EGDMA 90.8 µM LA 5000 µM	1.00 6.29 1.09	100 95 96	- 0.000 0.113	- ** n.s.	VC EGDMA 90.8 µM LA 5000 µM	1.00 4.91 0.96	100 96 107	- 0.000 0.023	- ** *
Concentration (test substance) [µM]	6th experiment, plate 2				Concentration (test substance) [µM]	6th experiment, plate 3
	fold induction mean	rel. viability [%] mean	p-value (t-test)	markers (t-test)		fold induction mean	rel. viability [%] mean	p-value (t-test)	markers (t-test)
58 70 84 101 121 145 175 209	1.09 1.31 1.50 1.63 1.40 1.66 1.61 1.84	77 78 81 73 78 79 75 82	0.317 0.000 0.000 0.087 0.013 0.005 0.001 0.003	n.s. ** ** n.s. * ** ** **	14 16 20 23 28 34 41 49	1.11 1.00 1.08 1.17 1.33 1.43 1.24 1.28	92 79 92 87 89 98 88 87	0.017 0.498 0.254 0.059 0.023 0.019 0.000 0.044	* n.s. n.s. n.s. * * ** *
VC EGDMA 90.8 µM LA 5000 µM	1.00 4.93 0.93	100 94 107	- 0.000 0.171	- ** n.s.	VC EGDMA 90.8 µM LA 5000 µM	1.00 5.36 0.90	100 84 100	- 0.000 0.015	- ** *

Table 2: Historic control data of LuSens. Data (not including present study)

Negative Control (LA 450 µg/mL)	fold induction	rel. viability [%]
Min	0.70	76
Max	1.25	140
Mean	0.95	106
SD	0.10	9
n	415

Positive Control (EGDMA 18 µg/mL)	fold induction	rel. viability [%]
Min	3.01	70
Max	10.38	135
Mean	6.15	96
SD	1.50	15
n	415

Vehicle Control (1% DMSO)	fold induction	rel. viability [%]
Min	1.00	100
Max	1.00	100
Mean	1.00	100
SD	0.00	0
n	415

Basal expression	fold induction	rel. viability [%]
Min	0.59	98
Max	1.57	186
Mean	0.94	152
SD	0.16	15
n	415

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

After 48 hours of exposure to the test substance luciferase activity in LuSens cells was 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 has a keratinocyte activating potential.

Executive summary:

The keratinocyte activating potential of 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, pre-tests (non-GLP) were performed. Cells were exposed to several 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. 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 6 experiments were performed. However, the first 4 experiments were invalid. Only the 5th and 6st experiment were used for evaluation.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was 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 has a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

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 -7.41% and a mean K-peptide depletion of -0.05%.

The mean peptide depletion was calculated to be 0.00% as negative values were considered to be "zero". 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.

The EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was calculated in two independent valid experiments to be 157 μM and 84 μM, respectively.

In summary, after 48 hours of exposure to the test substance luciferase activity in LuSens cells was 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 has a keratinocyte activating potential.

h-CLAT:

In order to assess the potential to induce the cell membrane markers CD86 and CD54 expression the human monocytic leukemia cell line THP-1 was incubated with the testsubstance for ca. 24 hours at 37°C and membrane marker expression (CD86 / CD54) was measured by flow cytometry according to the OECD guideline 442E.

After 24-hour exposure THP-1 cells were stained with FITC labeled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid experiments were performed.

The EC200% (the concentration resulting in a RFI of 200%) for CD54 was calculated to be 4693 μg/mL (experiment 1), 4639 μg/mL (experiment 2) and 5184 μg/mL (experiment 3), respectively.

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 was not applicable. However, the induction of the expression of CD54 in at least two independent experiments is sufficient to evaluate the test substance as positive for the induction of dendritic cell activation.

Based on the results of all three tests it was concluded that the test substance is not peptide reactive and activates keratinocytes and dendritic cells. Therefore the test substance is predicted 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 considered to be classified for skin sensitization (category 1) under Regulation (EC) No. 1272/2008.