Aluminum magnesium sodium oxide (Al10.33Mg0.67Na1.67O17)

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), according to OECD 442C,

- Keratinocyte Activation Assay (LuSens), according to OECD 442D, and

- Dendritic Cell Line Activation Assay (h-CLAT), according to OECD 442E.

Each test was conducted under GLP.

The results were as follows:

- DPRA: positive

- LuSens: negative

- h-CLAT: negative

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb 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:

04 February 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, 10978-52 H_K
- Purity: 82%
- Homogenity: homogeneous by visual inspection

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protect against humidity (store dry), ambient
- Stability under test conditions: guranteed by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: good homogenity was achieved with DMSO


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: stirring and ultra-sonic preparation
- Preliminary purification step: 4x stock concentration of highest concentration and further serial dilutions in DMSO
- Final dilution of a dissolved solid: 1:1.2 serial dilution of maximum concentration

FORM AS APPLIED IN THE TEST: suspension, unsolved but homogenous

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 RS Synthesis, Louisville KY, USA) 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 was 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 emulsion in de-ioinzed water)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide; visually turbid or precipitating samples were centrifuged and/or filtrated prior to injection into the HPLC

VEHICLE
- Vehicle: de-ionized water
- Reason for choice of the vehicle: The test substance was not soluble in one of the vehicles used for the assay (acetonitrile solutions in water, isopropanol, acetone, propanol, methanol, ethanol, N,N-dimethylformamide and tetrahydrofuran were tried). In de-ionized water a homogeneous suspension was achieved.

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, cysteine) or 1:50 (K-peptide, lysine)

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). Due to solubility issues, the maximum soluble test-substance concentration was tested additionally. The test substance was dissolved in de-ionized water at a concentration of 0.0108% (0.108 mg/mL) and was incubated as such without any further dilution in a 1:10 and 1:50 ratio with the cysteine and lysine peptide.
- 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 table 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 table 1)
- Possible interference of the test substance with the peptides was detected by performing a co-elution control for each tested concentration. The samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

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.

Run / experiment:

other: test substance with a 100 mM stock concentration

Parameter:

other: mean peptide depletion (cysteine and lysine peptides)

Value:

4.46

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: due to limited solubility: inconclusive

Run / experiment:

other: test substance with a maximum soluble stock concentration

Parameter:

other: mean peptide depletion (cysteine and lysine peptides)

Value:

0.82

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: due to limited solubility: inconclusive

Other effects / acceptance of results:

OTHER EFFECTS:
Due to the limited solubility of the test substance at 100 mM with both peptides the result could be underpredictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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. 9a+b

The samples of the 100 mM stock concentration with the peptides were suspensions at the time of preparation. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the C-containing peptide. Samples with the K-containing peptide were suspensions.

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	-0.69	0.25	0.43	0.00	0.60
Test substance 100mM stock concentration	3.01	3.67	7.38	4.69	2.36
Test substance maximum soluble stock concentration	0.31	1.07	1.34	0.91	0.53
PC: EGDMA in H2O	63.36	81.15	78.01	74.17	9.50

Table 7: Peptide depletion for K-peptide

Reaction with lysine-peptide	peptide depletion [%]
	sample 1	sample 2	sample 3	mean	SD
NC: H2O	-1.50	-1.62	3.12	0.00	2.70
Test substance 100mMstockconcentration	-0.81	3.13	10.37	4.23	5.67
Test substance maximum soluble stock concentration	-0.61	1.51	1.33	0.74	1.18
PC: EGDMA in H2O	6.84	5.93	11.25	8.01	2.84

Table 8: Mean peptide depletions

	Cysteine-Peptide  mean depletion [%] SD [%]		Lysine-Peptide  mean depletion [%] SD [%]		mean of both depletions [%]
Test substance 100mM stock concentration	4.69	2.36	4.23	5.67	4.46
Test substance maximum soluble stock concentration	0.91	0.53	0.74	1.18	0.82
PC: EGDMA in H2O	74.17	9.50	8.01	2.84	41.09

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

	C-peptide concentration	K-peptide concentration
	[mM]	[mM]
Min	0.441	0.459
Max	0.510	0.528
Mean	0.479	0.504
SD	0.014	0.013
n	22	21

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	5.76
Max	0.323	93.44	0.481	16.01
Mean	0.167	65.87	0.456	9.35
SD	0.083	13.54	0.018	2.39
n	18		18

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

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 limited solubility of the test substance, the samples with both peptides were suspensions at the 100 mM stock concentration and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

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 UV-detection at 220 nm.

The test substance was formulated at 100 mM in de-ionized water. Further, the maximum soluble concentration of the test substance in de-ionized water (0.0108%) was tested. Three samples of each test-substance concentration 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, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed for each tested concentration and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The test substance was not soluble in one of the vehicles used for the assay at a concentration of 100 mM: The test substance was a suspension in de-ionized water at 100 mM and dissolved at a concentration of 0.0108% (maximum soluble concentration). The samples of the 100 mM stock concentration with the peptides were suspensions at the time of preparation. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the C-containing peptide. Samples with the K-containing peptide were suspensions. The samples of the 0.0108% concentration were solutions with both peptides at the time of preparation and after the 24-hour incubation time. No co-elution of test substance and peptides was present.

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 limited solubility of the test substance, the samples with both peptides were suspensions at the 100 mM stock concentration and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

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:

May - Aug 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 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, 10978-52 H_K
- Purity: 82%
- Homogenity: homogeneous by visual inspection

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protect against humidity (store dry), ambient
- Stability under test conditions: guranteed by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: good homogenity was achieved with DMSO


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: stirring and ultra-sonic preparation
- Preliminary purification step: 4x stock concentration of highest concentration and further serial dilutions in DMSO
- Final dilution of a dissolved solid: 1:1.2 serial dilution of maximum concentration

FORM AS APPLIED IN THE TEST: suspension, unsolved but homogenous

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 showed good homogeneity in the preparation.

SELECTION OF CONCENTRATIONS
- In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 10 concentrations of the test-substance preparation (0.6 μg/mL up to 6098 μg/mL corresponding to final test-substance ingredient concentrations of 0.5 μg/mL up to 5000 μg/mL taking the purity of 82.0% into account) and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA.
- No relative viability below 75% was noticed, the absolute cell count number was reduced at concentrations of 610 μg/mL up to 6098 μg/mL. This is a clear indication of a cytotoxic effect: the viability value appears to be high but the value is based on a limited number of cells that are still available. Relative viability was calculated based on the total cell count. The CV75 value (=estimated concentration that affords 75% cell viability) of the test substance, based on relative viability by total cell counts, was determined by linear regression from the concentration response curve to be 706 μg/mL.
The total cell counts should not be reduced in any tested concentration. Therefore, the CV75 value was chosen to be the highest concentration for the main experiments. The additional concentrations were obtained by a 1:1.2 serial dilution series of the maximum concentration.

TEST CONCENTRATIONS for the 1st and 3rd experiment (µg/mL; not corrected for purity/contents)
706
588
490
409
340
284
236
197

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 ± 0.5 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 ± 0.5 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 anti-human CD86, FITC anti-human CD54 or FITC mouse IgG1 antibodies; for details see table 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.
Relative cell viability:
- From the independent replicates of a test substance concentration a mean is calculated after PI staining.
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 guavaSoft software InCyte (Millipore) was used. Data evaluation is 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 is calculated. The results are 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 50%.
- 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 and experiment 3

Parameter:

other: EC200% for CD54 [µg/mL]

Remarks:

concentration resulting in a RFI of 200%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: experiment 1 and experiment 3

Parameter:

other: EC150% for CD86

Remarks:

concentration resulting in a RFI of 150%

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 table 3

The test substance was unsolved but homogeneous in culture medium (2 x stock preparations and final concentrations) at the time of application and after 24 hours.

Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 and an EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable.

Considering PI intercalation into the DNA, no relative viability below 75% was noticed. However, the absolute cell count number was reduced at higher concentrations. This is a clear indication of a cytotoxic effect: the viability value appears to be high but the value is based on a limited number of cells that are still available. Hence, evaluation of viability is performed based on the total cell count. The CV75 value (= estimated concentration that affords 75% cell viability), based on relative viability by total cell counts, was determined by linear regression from the concentrationresponse curve to be 706 μg/mL (test substance as provided by the sponsor).

Table 2: Summary of h-CLAT Main Experiments: mean values of RFI CD86, RFI CD54 and relative viability.

1^stexperiment					3^rdexperiment
Concentration (test substance) [µg/mL]	RFI CD86	RFI CD54 mean [%] SD [%]	Viability (based on PI)	(based on total cell count)	Concentration (test substance) [µg/mL]	RFI CD86	RFI CD54 mean [%] SD [%]	Viability (based on PI)	(based on total cell count)
	mean [%]	mean [%]	rel. viability [%]	rel. viability [%]		mean [%]	mean [%]	rel. viability [%]	rel. viability [%]
197	103	45	101	93	197	107	120	104	103
236	77	77	101	96	236	96	125	104	102
284	92	119	99	89	284	121	159	100	93
340	76	85	101	91	340	120	197	98	90
409	85	57	101	90	409	125	78	100	92
490	71	68	101	85	490	122	100	102	94
588	101	63	100	76	588	91	81	101	90
706	57	119	101	68	706	135	147	101	87
VC	100	100	100	100	VC	100	100	100	100
LA 1000 µg/mL	63	108	100	99	LA 1000 µg/mL	65	162	100	98
DNCB 4 µg/mL	347	471	67	54	DNCB 4 µg/mL	262	677	69	59

Table 3: Historic control data of h-CLAT. Data not including present study.

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]		rel. viability mean [%]
Min	27	24		88		95
Max	140	198		97		108
Mean	75	115		93		99
SD	19	37		2		1
n (experiments)			89

 

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]		rel. viability mean [%]
Min	153	201		52		56
Max	879	1705		84		86
Mean	295	526		66		69
SD	112	245		7		6
n (experiments)			89

 

Vehicle Control (culture medium)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]
Min	52	43		90
Max	148	157		98
Mean	100	100		94
SD	25	23		2
n (experiments)			89

 

Vehicle Control (DMSO)	CD86 RFI [%]	CD54 RFI [%]		viability mean [%]		rel. viability mean [%]
Min	31	21		89		99
Max	149	193		98		110
Mean	101	108		95		101
SD	28	37		2		1
n (experiments)			89

Table 4: Reactivity check, performed with each new-thawed cells prior to use for a study, using NiSO4, LA, DNCB and the vehicle contr

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel. viability mean [%]
Min	60	69	99
Max	90	169	101
Mean	72	113	100
SD	8	24	0
n (experiments)		22

 

Nickel(II)sulfate hexahydrate (NiSo4100 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]
Min	182	1105	61
Max	424	3925	91
Mean	301	2266	82
SD	70	709	7
n (experiments)		22

 

Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	rel. viability mean [%]
Min	212	223	54
Max	428	1049	93
Mean	308	537	86
SD	59	210	9
n (experiments)		22

 

Interpretation of results:

GHS criteria not met

Conclusions:

In summary, after 24 hours of exposure to test substance CD86 and CD54 expression was not induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance does not induce 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 several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. Although no relative viability below 75% was noticed, the absolute cell count number was reduced at higher concentrations. This is a clear indication of a cytotoxic effect: the viability value appears to be high but the value is based on a limited number of cells that are still available. Hence, evaluation of relative viability has to be performed based on the total cell count. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance, based on relative viability by total cell counts, was determined by linear regression from the concentration response curve to be 706 μg/mL.

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 2 valid experiments were performed. A further experiment was invalid due to not covered acceptance criteria and is not included in this report.

At concentrations used in the main experiment the test substance was unsolved but homogeneous in culture medium (2 x stock preparations and final concentrations) at the time of application and after 24 hours. Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 and an EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable. The use of sufficient high concentrations is demonstrated by the relative viability based on total cell counts, which is reduced below 90%. Relative viability based on PI staining can not be taken into account as the value only appears to be high but is based on a limited number of cells that are still available.

In summary, after 24 hours of exposure to test substance CD86 and CD54 expression was not induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance does not induce dendritic cell activation.

Reference 3

Endpoint:

skin sensitisation: in vitro

Remarks:

ARE-Nrf2 Luciferase Test Method (LuSens)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jul - Aug 2017

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:

February 2015

Deviations:

no

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, 10978-52 H_K
- Purity: 82%
- Homogenity: homogeneous by visual inspection

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: protect against humidity (store dry), ambient
- Stability under test conditions: guranteed by the sponsor
- Solubility and stability of the test substance in the solvent/vehicle: good homogenity was achieved with DMSO


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: stirring and ultra-sonic preparation
- Preliminary purification step: 4x stock concentration of highest concentration and further serial dilutions in DMSO
- Final dilution of a dissolved solid: 1:1.2 serial dilution of maximum concentration

FORM AS APPLIED IN THE TEST: suspension, unsolved but homogenous

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), as for the 4x stock solution
- Reason for the vehicle: 4% DMSO in culture medium (DMEM + 1% FBS) was used because good homogeneity of the preparation was achieved.

SELECTION OF CONCENTRATIONS
- Cells were exposed to several concentrations of the test-substance preparation (0.6 µg/mL up to 2439 µg/mL corresponding to final test substance concentrations of 0.5 µg/mL (corresponding to 0.8 µM) up to 2000 µg/mL (corresponding to 3306 µM) taking the purity/contents of 82% 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 426 µg/mL (corresponding to 704 µM).
- 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 of the 1st experiment (µg/mL and corresponding concentration in unit µM; not corrected for purity/contents)
[µg/mL]; [μM]
614; 1015
512; 846
426; 705
355; 587
296; 489
247; 408
206; 340
171; 283

The 1st experiment was invalid as the relative viability of the positive control did not reach the acceptance criteria. However, in the test-substance concentrations, no relative viability below 70% was observed in the 1st experiment. Therefore, higher concentrations were tested in the 2nd experiment and 16 concentrations were applied, using two culture plates.

TEST CONCENTRATIONS of the 2nd experiment (µg/mL and corresponding concentration in unit µM; not corrected for purity/contents)
[µg/mL]; [μM]
Plate 1
1273; 2104
1061; 1754
884 ;1461
737; 1218
614; 1015
512; 846
426; 705
355; 587
Plate 2
296; 489
247; 408
206; 340
171; 283
143; 236
119; 197
99; 164
83; 137

No cytotoxicity below 70% was observed in all concentrations of the 2nd experiment and no luciferase activity above 1.5 fold induction of statistical significance with respect to the vehicle control was observed in two consecutive concentrations. Hence, higher concentrations were tested in further experiments and 16 concentrations were applied, using two culture plates.
The concentrations selected for the 3rd until 6th experiment are not in the factor 1.2 in relation to the concentrations selected for the 1st and 2nd experiment. Hence, concentrations of the 3rd until 6th experiment do not match the concentrations of the 1st and 2nd experiment. This deviation does not impair the validity of the study, as the results of the 4th and 6th experiment, with matching concentrations, are sufficient for evaluation of the LuSens.

TEST CONCENTRATIONS of the 3rd until 6th experiment (µg/mL and corresponding concentration in unit µM; not corrected for purity/contents)
[µg/mL]; [μM]
Plate 1
2439; 4031
2033; 3360
1694; 2800
1411; 2333
1176; 1944
980 ;1620
817; 1350
681; 1125
Plate 2
567; 938
473; 781
394; 651
328; 543
274; 452
228; 377
190; 314
158; 262

EXPERIMENTAL PROCEDURE
- No. of replicates: 6 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 ± 1 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-Glo-preparation (= 100 µL One-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 3 independent replicates a mean is calculated.
Luciferase fold induction:
- From 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 2, 4 and 6 within plate 1 and 2

Parameter:

other: EC1.5 [µM]

Remarks:

1.5-fold luciferase induction

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 if different from the ones specified in the test guideline: given in table(s) 4

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

3 valid and evaluable experiments (2nd, 4th and 6th experiment) were performed. The other experiments were invalid due to not met acceptance criteria or a technical error and are not reported.

Attached are the results of the three valid experiments and the historic control data of the LuSense-Assay.

Table 1: Summary of main experiments. 3 valid and evaluable experiments (2nd, 4th and 6th experiment) were performed.

Concentration (test substance)		2^ndexperiment, plate 1 fold induction rel. viability [%]			t-test			Concentration (test substance)		2^ndexperiment, plate 2 fold induction rel. viability [%]			t-test
µg/mL	µM	mean	mean	p-value		markers		µg/mL	µM	mean	mean	p-value		markers
355	587	0.74	92	0.002		**		83	137	1.05	96	0.108		n.s.
426	705	0.78	95	0.000		**		99	164	0.89	89	0.148		n.s.
512	846	0.72	98	0.000		**		119	197	0.83	89	0.019		*
614	1015	0.76	92	0.000		**		143	236	0.85	87	0.035		*
737	1218	0.73	88	0.000		**		171	283	0.79	91	0.000		**
884	1461	0.70	91	0.003		**		206	340	0.80	91	0.025		*
1061	1754	0.76	87	0.000		**		247	408	0.72	86	0.000		**
1273	2104	0.69	90	0.000		**		296	489	0.76	86	0.000		**
VC		1.00	100	-		-		VC		1.00	100	-		-
EGDMA								EGDMA
(18 µg/mL bzw. 90.8 µM)		5.00	86	0.000		**		(18 µg/mL bzw. 90.8 µM)		4.91	88	0.000		**
LA								LA
(450 µg/mL bzw. 5000 µM)		1.01	107	0.409		n.s.		(450 µg/mL bzw. 5000 µM)		0.94	107	0.145		n.s.
Concentration (test substance)		4^thexperiment plate 1 fold induction rel. viability [%]			t-test			Concentration (test substance)		4^thexperiment plate 2 fold induction rel. viability [%]			t-test
µg/mL	µM	mean	mean	p-value		markers		µg/mL	µM	mean	mean	p-value		markers
681	1125	0.84	96	0.049		*		158	262	0.90	96	0.000		**
817	1350	0.92	80	0.000		**		190	314	0.95	102	0.270		n.s.
980	1620	0.93	88	0.026		*		228	377	1.01	97	0.368		n.s.
1176	1944	0.93	81	0.088		n.s.		274	452	0.94	96	0.146		n.s.
1411	2333	0.99	80	0.469		n.s.		328	543	0.96	91	0.170		n.s.
1694	2800	0.92	79	0.000		**		394	651	0.95	95	0.266		n.s.
2033	3360	1.12	43	0.007		**		473	781	0.95	92	0.144		n.s.
2439	4031	1.13	59	0.052		n.s.		567	938	0.96	90	0.311		n.s.
VC		1.00	100	-		-		VC		1.00	100	-		-
EGDMA								EGDMA
(18 µg/mL bzw. 90.8 µM)		4.25	88	0.000		**		(18 µg/mL bzw. 90.8 µM)		3.01	94	0.000		**
LA (450 µg/mL bzw. 5000 µM)		1.03	100	0.212		n.s.		LA (450 µg/mL bzw. 5000 µM)		0.97	104	0.214		n.s.
Concentration (test substance)		6^thexperiment plate 1 fold induction rel. viability [%]			t-test			Concentration (test substance)		6th experiment plate 2 fold induction rel. viability [%]			t-test
µg/mL	µM	mean	mean	p-value		markers		µg/mL	µM	mean	mean	p-value		markers
681	1125	1.21	97	0.134		n.s.		158	262	1.30	99	0.043		*
817	1350	1.24	87	0.127		n.s.		190	314	1.37	97	0.004		**
980	1620	1.26	81	0.020		*		228	377	1.32	91	0.021		*
1176	1944	1.19	79	0.023		*		274	452	1.06	100	0.117		n.s.
1411	2333	1.43	62	0.033		*		328	543	1.08	97	0.195		n.s.
1694	2800	1.42	60	0.003		**		394	651	1.18	93	0.247		n.s.
2033	3360	1.37	37	0.078		n.s.		473	781	1.10	86	0.223		n.s.
2439	4031	1.49	22	0.030		*		567	938	1.08	85	0.145		n.s.
VC		1.00	100	-		-		VC		1.00	100	-		-
EGDMA								EGDMA
(18 µg/mL bzw. 90.8 µM)		5.21	81	0.000		**		(18 µg/mL bzw. 90.8 µM)		6.86	96	0.000		**
LA								LA
(450 µg/mL bzw. 5000 µM)		0.85	108	0.000		**		(450 µg/mL bzw. 5000 µM)		0.89	103	0.057		n.s.

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

Negative Control (LA 450 µg/mL)	fold induction	rel. viability [%]
Min	0.74	76
Max	1.25	127
Mean	0.96	104
SD	0.10	10
n	172

 

Positive Control (EGDMA 18 µg/mL)	fold induction	rel. viability [%]
Min	3.53	70
Max	10.38	135
Mean	6.56	96
SD	1.55	15
n	172

 

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	172

 

Basal expression	fold induction	rel. viability [%]
Min	0.62	119
Max	1.57	186
Mean	1.00	160
SD	0.15	13
n	172

Interpretation of results:

GHS criteria not met

Conclusions:

After 48 hours of exposure to 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 test substance does not have a keratinocyte activating potential.

Executive summary:

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 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 to be 426 μg/mL (704 μM) (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 3 valid experiments were performed. Three further experiments were invalid due to not met acceptance criteria or a technical error and are not included in this report.

At concentrations used in the main experiment the test substance was unsolved but homogeneous in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations) at the time of application and after 48 hours.

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

In summary, after 48 hours of exposure to 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 test substance does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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 formulated at 100 mM in de-ionized water. Further, the maximum soluble concentration of the test substance in de-ionized water (0.0108%) was tested. Three samples of each test-substance concentration 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, in order to detect possible interference of the test substance with the peptides, a co-elution control was performed for each tested concentration and the samples were analyzed by measuring UV absorbance at 258 nm in order to calculate the area ratio 220 nm / 258 nm.

The test substance was not soluble in one of the vehicles used for the assay at a concentration of 100 mM: The test substance was a suspension in de-ionized water at 100 mM and dissolved at a concentration of 0.0108% (maximum soluble concentration). The samples of the 100 mM stock concentration with the peptides were suspensions at the time of preparation. Visual observation after the 24-hour incubation time revealed precipitates in the samples of the test substance with the C-containing peptide. Samples with the K-containing peptide were suspensions. The samples of the 0.0108% concentration were solutions with both peptides at the time of preparation and after the 24-hour incubation time. No co-elution of test substance and peptides was present.

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 limited solubility of the test substance, the samples with both peptides were suspensions at the 100 mM stock concentration and that the result could therefore be under-predictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

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 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 to be 426 μg/mL (704 μM) (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 3 valid experiments were performed. Three further experiments were invalid due to not met acceptance criteria or a technical error and are not included in this report.

At concentrations used in the main experiment the test substance was unsolved but homogeneous in 4% DMSO in culture medium 3 (4 x stock preparations) and in 1% DMSO in culture medium 3 (final concentrations) at the time of application and after 48 hours.

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

In summary, after 48 hours of exposure to 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 test substance does not have a keratinocyte activating potential.

h-CLAT:

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 several concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. Although no relative viability below 75% was noticed, the absolute cell count number was reduced at higher concentrations. This is a clear indication of a cytotoxic effect: the viability value appears to be high but the value is based on a limited number of cells that are still available. Hence, evaluation of relative viability has to be performed based on the total cell count. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance, based on relative viability by total cell counts, was determined to be 706 µg/mL 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 2 valid experiments were performed. A further experiment was invalid due to not covered acceptance criteria and is not included in this report.

At concentrations used in the main experiment the test substance was unsolved but homogeneous in culture medium (2 x stock preparations and final concentrations) at the time of application and after 24 hours. Calculation of an EC150% (the concentration resulting in a RFI of 150%) for CD86 and an EC200% (the concentration resulting in a RFI of 200%) for CD54 was not applicable. The use of sufficient high concentrations is demonstrated by the relative viability based on total cell counts, which is reduced below 90%. Relative viability based on PI staining can not be taken into account as the value only appears to be high but is based on a limited number of cells that are still available.

In summary, after 24 hours of exposure to test substance CD86 and CD54 expression was not induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance does not induce dendritic cell activation.

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 amended for the tenth time in Regulation (EU) No 2017/776. As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.