Tetraamminezinc(2+) carbonate

Endpoint summary

Description of key information

Skin sensitisation: Based on the results of an in chemico/in vitro test strategy the test item is not peptide reactive (DPRA), activates keratinocytes(LuSens) and activates dendritic cells (h-CLAT). Therefore, the substance is predicted to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 December 2016 - 27 April 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: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Details on the study design:

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)
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.

Material and technical equipment:
HPLC: Liquid chromatograph Thermo Scientific, Dionex Ultimate 3000consisting of the following modules:
Pump: HPG-3400RS
Autosampler: WPS-3000TSL
Column oven: TCC-3000
UV-Detector: DAD-3000
Column: ZORBAX SB-C18 2.1 x 100 mm, 3.5 μm with guard column
SecurityGuard Ultra Cartridges, UHPLC C18 for 4.6 mm ID(Phenomenex)
HPLC mobile phase A: H2O/ACN/TFA 950/50/1 V/V/V
HPLC mobile phase B: ACN/H2O/TFA 950/50/0.85 V/V/V

Reagents for preparing the buffers for pH 7.5 phosphate buffer (used for solving C-containing peptide):
Sodium phosphate, monobasic monohydrate, CAS no. 10049-21-5
Sodium phosphate, dibasic heptahydrate, CAS no. 7782-85-6 for pH 10.2 ammonium acetate buffer (used for solving K containing peptide):
Ammonium acetate, CAS no. 631-61-8
Ammonium hydroxide, 28% – 30%, CAS no. 1336-21-6

Controls:
Negative control (NC): vehicle control = de-ionized water
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5), prepared as 50 mM emulsion in de-ionized water
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Test substance preparation:
The test substance was prepared as a 100 mM preparation in de-ionized water (considering a molecular weight of 193.54 g/mol and a purity/contents of 16.9%). After short stirring the test substance was a suspension in de-ionized water and the preparation was stirred until and during sample preparation.
In addition, the undiluted test substance was tested (no preparation required).
Vehicle: de-ionized water
Reason for the vehicle: The test substance contains 82.7% water and was homogeneously further diluted in de-ionized water.

Experimental procedure:
Per concentration three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at
220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

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 was used for preparing the calibration samples and the test-substance and control samples.

Preparation of calibration samples:
The calibration samples were prepared from the peptide stock solutions in 20% deionized water in the respective buffer (= dilution buffer) using serial dilutions.
(0.534, 0.267, 0.134, 0.067, 0.033, 0.017, 0.000 mM peptide)

Preparation of the test-substance samples:
C-peptide
750 μL C-peptide stock-solution
200 μL solvent (vehicle)
50 μL test-substance (preparation)
(or PC-preparation or solvent (VC))

K-peptide
750 μL K-peptide stock-solution
250 μL test-substance (preparation)
(or PC-preparation or solvent (VC))

The samples were incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Data evaluation:
For evaluation of peptide depletions peak areas at 220 nm are used. When samples were additionally analyzed by measuring UV absorbance at 258 nm, the area ratio 220 nm/ 258 nm may be calculated and serve as a measure of peak purity. The ratio of a pure peptide peak should be consistent over all samples (100% ± 10% of the mean of the vehicle controls). However, due to small peak areas calculation of the area ratio may not be possible for all samples.

Calculation of the peptide concentrations:
For each peptide and test run 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 concentration (C-containing and K-containing peptide depletion; example calculation for C-containing peptide). 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.

Acceptance criteria of the DPRA:
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). In addition the positive control should cause depletion of both peptides comparable to historic data.

Evaluation of results:
Evaluation criteria of DPRA; cysteine 1:10 / lysine 1:50 prediction model.
Mean peptide depletion [%] Reactivity Evaluation
> 42.47 high reactivity positive
> 22.62 ≤ 42.47 moderate reactivity positive
> 8.11 ≤ 22.62 low reactivity positive
> 4.65 ≤ 8.11 no to low reactivity borderline1
≤ 4.65 minimal or no reactivity negative

In the case mean peptide depletion [%] cannot be determined due to invalid K-peptide depletion (e.g. insolubility of the K-peptide samples or interference in the samples of the K-peptide) but valid C-peptide depletion is available, evaluation is performed as follows:

Evaluation criteria of DPRA; cysteine 1:10 prediction model.
C peptide depletion [%] Reactivity Evaluation
> 98.24 high reactivity positive
> 23.09 ≤ 98.24 moderate reactivity positive
> 17.28 ≤ 23.09 low reactivity positive
> 10.50 ≤ 17.28 no to low reactivity borderline2
≤ 10.50 minimal or no reactivity negative

1 The „borderline“-evaluation was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation given in OECD TG442C. OECD TG442C defines mean depletions ≤ 6.38 as “negative” (minimal or no reactivity) and mean depletions > 6.38 ≤ 22.62 as “positive” (low reactivity).
2 The „borderline“-evaluation was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation given in OECD TG442C. OECD TG442C defines mean depletions ≤ 13.89 as “negative” (minimal or no reactivity) and mean depletions > 13.89 ≤ 23.09 as “positive” (low reactivity).

Limitations of the evaluation by insolubility and gravimetric procedure:
For test substances that are not completely soluble by visual observation in the sample preparations containing the peptides immediately after preparation or after 24 hours, or when a gravimetric procedure is applied (with the exception of application of the undiluted test substance (liquids) or the maximal soluble test-substance concentration (solids)), the result may be under-predictive due to limited availablity of the test substance. In this case mean peptide reactivity < 8.11% (cysteine 1:10 / lysine 1:50 prediction model) or < 17.28% (cysteine 1:10 prediction model) is interpreted as “inconclusive”. However, a mean peptide depletion > 8.11% or > 17.28% is considered as “positive”.

Key result

Run / experiment:

other: 1st run

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

1.38

Negative controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

35.26%

Run / experiment:

other: 1st run

Parameter:

other: peptide depletion (%)

Remarks:

cysteine peptide

Value:

2.76

Negative controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

61.86%

Run / experiment:

other: 2nd run

Parameter:

other: peptide depletion (%)

Remarks:

cysteine peptide

Value:

0

Negative controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

61.81%

Run / experiment:

other: 1st run

Parameter:

other: peptide depletion (%)

Remarks:

lysine peptide

Value:

0

Negative controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

8.67%

Run / experiment:

other: 2nd run

Parameter:

other: peptide depletion (%)

Remarks:

lysine peptide

Value:

0

Negative controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

11.13%

Key result

Run / experiment:

other: 2nd run

Parameter:

other: mean peptide depletion of both peptides (%)

Value:

0

Negative controls validity:

valid

Remarks:

0%

Positive controls validity:

valid

Remarks:

36.47%

Other effects / acceptance of results:

OTHER EFFECTS:
Llimited solubility of the test substance with the cysteine-peptide.

ACCEPTANCE OF RESULTS:
The acceptance criteria were met. The positive control caused depletion of both peptides comparable to historic data:

Historic control data of negative control / vehicle control (not including present study).
De-ionized water
Historic period: Feb 2014 - Mar 2016
C-peptide concentration [mM] K-peptide concentration [mM]
Min 0.441 0.488
Max 0.510 0.528
Mean 0.479 0.506
SD 0.015 0.009
n 19 19

Historic control data of positive control (not including present study):
EGDMA, 50 mM in de-ionized water
Historic period: Feb 2014 - Mar 2016
C-peptide concentration [mM] C-peptide depletion [%] K-peptide concentration [mM] K-peptide depletion [%]
Min 0.032 44.32 0.427 5.76
Max 0.323 93.44 0.481 16.01
Mean 0.164 66.47 0.459 9.27
SD 0.072 14.31 0.014 2.45
n 16 16

Table 1: DPRA, test run 1. Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide mean depletion     [%]               SD [%]	Lysine-Peptide mean depletion [%]         SD [%]	mean of both depletions [%]
Collacral TAZ 100 mM	2.76               0.98	-1.13         1.51	1.38
PC: EGDMA in H20	61.86              2.84	8.67         1.72	35.26

 

Table 2: DPRA, test run 2. Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide mean depletion [%]        SD [%]	Lysine-Peptide mean depletion [%]        SD [%]	mean of both depletions [%]
Collacral TAZ undiluted	-2.79        0.59	-0.13       0.44	0.00
PC: EGDMA in H20	61.81       7.68	11.13       1.83	36.47

Interpretation of results:

other: not protein binding

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 January 2017 - 27 April 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

Qualifier:

according to guideline

Guideline:

other: ESAC Opinion on the BASF-coordinated Performance Standards-based validation of the LuSens test method for skin sensitisation testing, ESAC Opinion No. 2016-04

Version / remarks:

24 June 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Ramirez T, Mehling A, Kolle SN, Wruck CJ, Teubner W, Eltze T, Aumann A, Urbisch D, Ravenzwaay BV, Landsiedel R.., LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification.

Version / remarks:

28 Dec 2014 (Toxicol In Vitro.)

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: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Details on the study design:

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

Material:
Luminometer: TriStar² Multimode reader LB 942 (Berthold)
Spectralphotometer: TriStar² Multimode reader LB 942 (Berthold)
Culture medium 1: D-MEM (Cat. No. Biochrom FG 0445)+ 10 % FBS + 1 % Penicillin /
Streptomycin, Puromycin dihydrochloride 25 μL (Sigma)
Culture medium 2: D-MEM (Cat. No. Biochrom FG 0445) + 10 % FBS
Culture medium 3: D-MEM (Cat. No. Biochrom FG 0445) + 1 % FBS
Buffer: Phosphate Buffered Saline (PBS) w/o Ca2+ / Mg2++ 0.05 % EDTA
Phosphate Buffered Saline (PBS) w/o Ca2+ / Mg2+
Phosphate Buffered Saline (PBS) with Ca2+ / Mg2+
Lysisbuffer: 10 g SDS, 99.6 mL DMSO, 0.4 mL glacial acetic acid
Luciferace reagent : SteadyGloLuciferase Assay, (Promega)
MTT Solution: Thiazolyl Blue Tetrazolium Bromide (Sigma) 5 mg/mL with PBS without Mg2+/Ca2+ (stock solution) ; diluted 1 :10 with medium 3 for the assay

Controls for the LuSens:
Negative control (NC): DL-Lactic acid (LA, CAS no.: 50-21-5), 450 μg/mL in 1% DMSO in culture medium 3
Positive control (PC): Ethylene glycol dimethacrylate (EGDMA, CAS no. 97-90-5), 18 μg/mL in 1% DMSO in culture medium 3
Vehicle control (VC): 1% DMSO in culture medium 3
Blank control: Culture medium 3 without cells
Basal control: Culture medium 3 with cells

Test substance preparation:
The test substance was weighed and topped up with the chosen vehicle (4% DMSO in culture medium 3) to achieve the required 4x concentration of the highest concentration (stock solution). Further concentrations were prepared as 4x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate). Reason for the vehicle: 4% DMSO in culture medium 3 was used because good homogeneity of the preparation was achieved.

Experimental procedure:
Before substance incubation, cells were seeded in 96-well microtiter plates (120 μL of 0.83 x 10E5 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. Two independent, valid experiments were performed. In each experiment, three replicates of each test-substance concentration were tested. After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 μL medium 3. The test substance was prepared as described. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 μL) to the cells (final DMSO concentration in the test medium = 1%).

The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. 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.

Luciferase assay:
The cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Subsequently 200 μL of Steady-Glo-preparation (= 100 μL Steady-Glo- Mix and 100 μL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability: MTT assay
0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+/Mg2+)
and medium 3) 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.

Luciferase fold induction:
fold induction test substance = test substance treated cells - background without cells / mean vehicle control treated cells - background without cells
From the three independent replicates a mean is calculated.

Statistical analyses:
A pair-wise comparison of the concentration groups, positive control and negative control group with the vehicle control was performed using the Welch
t-test (one-sided) for the hypothesis of equal means.

Calculation of EC1.50:
If applicable, the concentration resulting in a positive response (1.50 fold-induction of statistical significance and viability >70%) is calculated from each experiment conducted. The calculation is performed by linear regression from the two concentrations directly above and below the EC1.50 concentration (b = axis intercept; m = slope).
EC1.50 = 1.50-b/m

Acceptance criteria
A tested concentration is not further evaluated when relative viability is less than 70%. The cell viability of VC 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. In addition, positive, negative and vehicle control data should lie within the range of the historic data.

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

positive:
statistically significant induction of luciferase activity ≥ 1.50 with respect to the vehicle control in at least two consecutive concentrations that do not
reduce viability below 70% of two independent experiments and at least one of these concentrations must lie above the borderline area (> 1.65)
negative:
no statistically significant induction of luciferase activity ≥ 1.50 with respect to the vehicle control in at least two consecutive concentrations that do not reduce viability below 70% of two independent experiments and at least one of these concentrations must lie below the borderline area (< 1.35). The use of sufficiently high concentrations must be demonstrated by at least one concentration displaying viability below 70% or the application of the maximum (applicable) concentration. In addition, the borderline criteria must not be met.
borderline (1):
induction of luciferase activity with respect to the vehicle control lies in the historic margin of deviation of the cut off value 1.50 (1.35 – 1.65) in at least two consecutive concentrations that do not reduce viability below 70% of two independent experiments
inconclusive:
after conduct of at least two and a maximum of three independent valid and evaluable experiments no final evaluation is possible (e.g. one experiment positive, negative and borderline, respectively)

(1) The „borderline“-evaluation was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation given in the publication of Ramirez et al., 2014.

Key result

Run / experiment:

other: 189 µg/mL test substance concentration (1st experiment)

Parameter:

other: fold induction

Value:

6.18

Vehicle controls validity:

valid

Remarks:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.89

Key result

Run / experiment:

other: 227 µg/mL test substance concentration (1st experiment)

Parameter:

other: fold induction

Value:

4

Vehicle controls validity:

valid

Remarks:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.89

Key result

Run / experiment:

other: 272 µg/mL test substance concentration (1st experiment)

Parameter:

other: fold induction

Value:

5.27

Vehicle controls validity:

valid

Remarks:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.89

Key result

Run / experiment:

other: 131 µg/mL test substance concentration (2nd experiment)

Parameter:

other: fold induction

Value:

4.91

Vehicle controls validity:

valid

Remarks:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.14

Key result

Run / experiment:

other: 157 µg/mL test substance concentration (2nd experiment)

Parameter:

other: fold induction

Value:

4.41

Vehicle controls validity:

valid

Remarks:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.14

Key result

Run / experiment:

other: 189 µg/mL test substance concentration (2nd experiment)

Parameter:

other: fold induction

Value:

6.36

Vehicle controls validity:

valid

Remarks:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.14

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The acceptance criteria were met in all experiments.
The positive and negative and vehicle control data is comparable to historic data.

Basal Expression fold induction rel. viability [%]
Min 0.56 96.2
Max 1.38 185.8
Mean 0.89 150.5
SD 0.16 15.4
n 325

Negative Control fold induction rel. viability [%]
Min 0.70 75.8
Max 1.38 137.5
Mean 0.96 106.7
SD 0.11 9.2
n 325

Positive Control fold induction rel. viability [%]
EGDMA
Min 3.49 70.1
Max 10.38 136.1
Mean 6.61 103.4
SD 1.39 12.4
n 325

Vehicle fold induction rel. viability [%]
DMSO
Min 1.00 100.0
Max 1.00 106.7
Mean 1.00 100.0
SD 0.0 0.0
n 325

Table 1: Results of preliminary cytotoxicity assessment. The final test substance concentrations were calculated considering a purity/contents of 16.9%.

Concentration (final test substance) [µg/mL]	Concentration (test substance) [µg/mL]	mean OD570-690 of 3 replicates	mean rel. viability [%]
VC	VC	0.328	100.0
0.5	3.0	0.342	104.2
1.0	5.9	0.342	104.4
5.0	30	0.347	105.9
10	59	0.351	107.1
50	296	0.364	111.0
100	592	-0.004	-1.2
500	2959	0.012	3.7
1000	5917	0.016	4.7
2000	11834	0.035	10.7

Table 2: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 1).

Concentration (test substance) µg/mL	1st experiment
	fold induction			rel. viability [%]		t-test
	mean	SD		mean	SD	p-value	markers
189	6.18	0.96		95.9	4.6	0.005	**
227	4.00	0.26		76.1	9.9	0.001	**
272	5.27	0.55		82.6	1.9	0.003	**
326	9.74	0.75		57.4	7.0	0.001
392	8.71	2.61		12.9	7.4	0.018	*
470	1.13	1.10		3.8	0.8	0.426	n.s.
564	0.09	0.09		-3.0	12.2	0.000	**
677	0.00	0.09		4.1	1.1	0.000	**
VC	1.00	0.14		100.0	6.6	-	-
EGDMA (18 µg/mL)	7.89	0.37		86.0	11.9	0.000	**
LA (450 µg/mL)	0.92	0.24		99.0	7.5	0.252	n.s.

Table 3: Mean values and standard deviations of luciferase induction and rel. viability as well as p-values of t-test (experiment 2).

Concentration (test substance)   (µg/mL)	2nd experiment
	fold induction			rel. viability [%]		t-test
	mean	SD		mean	SD	p-value	markers
131	4.91	0.62		79.5	3.8	0.004	**
157	4.41	0.26		72.3	13.3	0.000	**
189	6.36	0.72		77.6	15.0	0.003	**
227	9.01	0.80		59.9	5.1	0.002	**
272	6.65	0.85		18.8	1.5	0.004	**
326	2.29	2.52		7.6	1 s	0.235	n.s.
392	0.09	0.32		-2.2	13.5	0.017	*
470	-0.04	0.15		5.7	0.9	0.001	**
VC	1.00	0.19		100.0	5.4	-	-
EGDMA (18 µg/mL)	7.14	0.33		71.0	10.6	0.000	**
LA (450 µg/mL)	1.15	0.19		103.5	12.0	0.082	n.s.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19 January 2017 - 27 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for Testing of Chemicals 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: Kampagne 02/2014
- Expiration date of the lot/batch: February 18, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature

Details on the study design:

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

Additional material and technical equipment:
Flow cytometer: FC 500 MPL (Beckman Coulter) with CXP Software (Beckman Coulter) (pre-test only)
Guava easyCyteTM HT (Millipore) with guavaSoft software InCyte (Millipore) (main experiments)
Culture medium: RPMI 1640: with L-glutamine, 25mM HEPES (Gibco)+ 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma)
Blocking Solution: 0.01% Globulins Cohn fraction II,III (Sigma)
Antibodies:
FITC anti-human CD54 (DAKO/DAK-F714301)
FITC mouse IgG1 (DAKO/DAK-X092701)
FITC anti-human CD86 (BD Pharmingen 555657)
Reagent for cytotoxicity test: Propidium iodide (Sigma)

Controls for the h-CLAT:
Negative control (NC): Lactic acid (LA, CAS no.: 50-21-5), 1000 μg/mL in culture medium
Positive control (PC): 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7), 4.0 μg/mL in 0.2% DMSO in culture medium
Vehicle control (VC): 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.

Test substance preparation:
The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2x concentration of the highest concentration (stock solution).
Further concentrations were prepared as 2x concentrations by serial 1:1.2 dilution according to the planned concentrations in culture medium.

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

Preparation of the cells:
THP-1 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium supplemented with 10% fetal bovine serum (heat inactivated), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.05 mM 2-mercaptoethanol under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) until for 5 passages but not longer than passage 30 prior to testing. Prior to use of the cells for a study, a reactivity check is performed with each new-thawed cells, as proposed in the OECD draft 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.

For substance incubation, cells were seeded in 24-well plates (500 μL of 2.0 x 10E6 cells/mL cell suspensions). Two independent, valid experiments were performed. In each experiment, duplicates of each test-substance concentration were tested.

Test-substance application:
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 10E6 cells/mL. The plates were placed into the incubator under standard culture conditions for the exposure period of 24 hours.Each test-substance concentration was visually inspected directly after application and after the exposure period of 24 hours.

Cell staining and flow cytometric analysis:
After visual inspection the cells were collected by centrifugation and washed twice with 1 mL buffer. 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 106 cells/180 μL/group) in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 μL working antibody solution 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) to yield a final concentration of 1.22 μg/mL PI.

Data evaluation:
The CV75-value (relative survival rate) is calculated by linear regression. This value is the substance concentration at which cell viability is 75% compared to the vehicle control (b = axis intercept; m = slope).
Cell viability is determined by PI staining. From the independent replicates of a test substance concentration a mean is calculated.

Relative fluorescence intensity:
Analysis of the membrane markers is performed in 10000 living cells, determined by PI staining. Concentrations inducing viability less than 50% are not considered for further assessment of dendritic cell activation. For data analysis, the CXP software (Beckman Coulter) or guavaSoft software InCyte (Millipore) is 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. The RFI of CD86 or CD54 is calculated by the following equation:

RFI [%]= MFI of chemical treated cells MFI of chemical - treated isotype control cells/ MFI of vehicle control cells -MFI of vehicle isotype control cells X 100

Calculation of EC150% and EC200%:
If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) is calculated for each cell surface marker from each experiment conducted. The calculation is performed by linear regression from the two concentrations directly above and below the EC150% / EC200% concentration (b = axis intercept; m = slope).

Acceptance criteria of the h-CLAT:
A tested concentration is not to be further evaluated when relative viability is less than 50%. 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.
In addition, positive, negative and vehicle control data should lie within the range of the historic data.

Evaluation of results:
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. In each experiment, at least one concentration must lie above the borderline area (> 170% (CD86) or 220% (CD54)). 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 maximum applicable concentration or up to the cytotoxicity limit (viability less than 90% at the highest concentration tested). In addition, the borderline criteria must not be met. To be relevant for evaluation, the cell viability must be more than 50% in at least four tested concentrations of an experiment.

Evaluation:
positive
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 and at least one concentration must lie above the borderline area (> 170% (CD86) or 220% (CD54))
negative
CD86 expression is not increased ≥ 150% and/or CD54 expression is not increased ≥ 200% at any concentration in relation to vehicle control that do not reduce viability below 50% in at least two independent experiments and at least one concentration must lie below the borderline area (< 130% (CD86) or < 180% (CD54)). The use of sufficiently high concentrations must be demonstrated by at least one concentration displaying viability below 90% or the application of the maximum (applicable) concentration.
In addition, the borderline criteria must not be met.
borderline (1)
RFI lies in the historic margin of deviation of ± 20% around the cut off value (130% - 170% for CD86; 180% - 220% for CD54) at concentrations that do not reduce viability below 50% of two independent experiments
inconclusive
after conduct of at least two and a maximum of three independent valid and evaluable experiments no final evaluation is possible (e.g. one experiment positive, negative and borderline, respectively)
(1) The „borderline“-evaluation was determined statistically using historic BASF data and hence considers the variance of the test method. This evaluation is an amendment to the evaluation given in the OECD test guideline

Key result

Run / experiment:

other: 364 µg/mL test substance concentration (1st experiment)

Parameter:

other: RFI CD86 mean (%)

Value:

162

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

valid

Remarks:

85.6%

Positive controls validity:

valid

Remarks:

299%

Key result

Run / experiment:

other: 437 µg/mL test substance concentration (1st experiment)

Parameter:

other: RFI CD86 mean (%)

Value:

156

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

valid

Remarks:

85.6%

Positive controls validity:

valid

Remarks:

299%

Key result

Run / experiment:

other: 524 µg/mL test substance concentration (1st experiment)

Parameter:

other: RFI CD86 mean (%)

Value:

149.6

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

valid

Remarks:

85.6%

Positive controls validity:

valid

Remarks:

299%

Key result

Run / experiment:

other: 364 µg/mL test substance concentration (1st experiment)

Parameter:

other: RFI CD54 mean (%)

Value:

1 298

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

valid

Remarks:

95.5%

Positive controls validity:

valid

Remarks:

603.4%

Key result

Run / experiment:

other: 437 µg/mL test substance concentration (1st experiment)

Parameter:

other: RFI CD54 mean (%)

Value:

1 400.1

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

valid

Remarks:

95.2%

Positive controls validity:

valid

Remarks:

603.4%

Key result

Run / experiment:

other: 524 µg/mL test substance concentration (1st experiment)

Parameter:

other: RFI CD54 mean (%)

Value:

1 458.8

Vehicle controls validity:

valid

Remarks:

100%

Negative controls validity:

valid

Remarks:

95.2%

Positive controls validity:

valid

Remarks:

603.4%

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The acceptance criteria mentioned were met in all experiments. The positive and negative and vehicle control data is comparable to historic data (Historic data are presented in table 4),

Table 1: Results of preliminary cytotoxicity assessment. The final test substance concentrations were calculated considering the purity/contents of 16.9%.

Concentration (final test substance) [µg/mL]	Concentration (test substance) [µg/mL]	%PI negative cells replicate 1	%PI negative cells replicate 2	mean viability of duplicates	rel. viability mean [%]
VC	VC	98.4	98.2	98.3	100.0
0.5	3.0	98.1	98.7	98.4	100.1
1.0	5.9	98.3	98.0	98.1	99.8
5.0	30	98.3	98.2	98.3	99.9
10	59	98.1	97.9	98.0	99.7
50	296	96.9	95.5	96.2	97.9
100	592	87.3	81.4	84.3	85.8
500	2959	4.5	4.7	4.6	4.7
1000	5917	1.3	1.2	1.3	1.3
2000	11834	1.2	2.4	1.8	1.8
5000	29586	16.7	27.4	22.0	22.4

The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve to be 906 μg/mL (test substance as provided by the sponsor). The higher rel. viability value, observed at the highest test substance concentration, is attributed to an artifact caused by test substance particles.

Table 2: h-CLAT. RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 1st experiment. Due to a technical error, one value is available for RFI CD86 at concentrations of 629 µg/mL and 755 µg/mL, only.

1st experiment
Concentration	RFI CD86			RFI CD54		Viability
(test substance)	mean [%]	SD [%]		mean [%]	SD [%]	rel. viability			SD
[µg/mL]						[%]			of viability
364	162.6	21.7		1298.4	189.1	96.2			1.7
437	156.0	19.0		1400.1	187.0	95.6			1.5
524	149.6	11.8		1458.8	63.9	78.2			35.9
629	92.2	-		1926.7	40.3	87.7			2.7
755	118.0	-		1493.3	68.9	81.0			3.5
906	89.1	18.2		1243.3	117.5	69.3			2.9
1088	54.0	3.9		752.4	160.9	46.6			3.0
1305	36.0	0.7		429.4	109.1	33.1			9.7
VC	100.0	30.4		100.0	26.8	100.0			2.1
LA 1000 µg/mL	85.6	10.5		95.2	31.4	99.6			0.7
DNCB 4 µg/mL	299.0	65.4		603.4	235.2	62.2			3.9

Table 3: h-CLAT. RFI CD86, RFI CD54 and rel. viability. Mean values and standard deviations of 2nd experiment.

2nd experiment
Concentration	RFI CD86			RFI CD54		Viability
(test substance)	mean [%]	SD [%]		mean [%]	SD [%]	rel. viability			SD
[µg/mL]						[%]			of viability
364	274.5	82.7		2041.6	40.8	92.8			2.2
437	244.9	109.4		2463.3	146.6	90.6			1.4
524	259.9	92.0		2967.7	275.0	87.3			1.8
629	145.7	40.0		2234.1	81.8	78.1			2.0
755	121.8	19.4		1318.4	20.8	65.3			1.6
906	120.3	30.4		1354.0	66.7	53.4			3.0
1088	71.6	10.3		849.7	14.3	25.2			1.4
1305	37.8	9.4		586.6	10.2	10.7			1.9
VC	100.0	29.7		100.0	35.4	100.0			1.8
LA 1000 µg/mL	109.6	35.0		163.9	21.3	99.4			1.4
DNCB 4 µg/mL	302.5	67.3		542.1	114.1	70.1			4.5

 

Table 4: Historic control data of h-CLAT. Data shown of test period Feb 2016 until Apr 2017 (not including present study).

Negative Control (LA 1000 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]	rel. viability mean [%]
Min	35.1	24.2	87.7	94.7
Max	131.0	194.1	97.1	108.4
Mean	75.5	115.5	92.5	98.8
SD	18.0	35.7	1.9	1.5
n (experiments)		68
Positive Control (DNCB 4 µg/mL)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]	rel. viability mean [%]
Min	152.9	201.1	52.4	55.6
Max	879.4	1704.7	84.2	86.3
Mean	284.7	524.3	64.9	68.7
SD	114.0	259.4	6.4	6.3
n (experiments!		68
Vehicle Control (culture medium)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]
Min	52.3	44.2	89.9
Max	147.7	155.8	97.8
Mean	100.2	100.1	93.9
SD	25.2	21.7	1.9
n (experiments)		68
Vehicle Control (DMSO)	CD86 RFI [%]	CD54 RFI [%]	viability mean [%]	rel. viability mean [%]
Min	33.6	34.6	89.0	98.7
Max	149.4	192.6	97.5	109.7
Mean	100.8	109.5	94.3	100.7
SD	28.0	35.6	1.7	1.3
n (experiments)		68

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A combination of three in chemico/in vitro methods, addressing key events of the adverse outcome pathway (AOP) for skin sensitization (OECD, 2012) as defined by the OECD, were part of the in vitro Skin Sensitization Turnkey Testing Strategy to access the skin sensitization potential of the test item.

DPRA

The reactivity of the test item towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). 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. Two test runs were performed. The test substance was formulated at a 100 mM concentration in de-ionized water (1st test run) and applied undiluted (2nd test run). Per test-substance treatment, 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. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

Further, with both tested concentrations 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 samples of the 100 mM preparation as well as the undiluted test substance with the cysteine peptide were suspensions at the time of preparation. In the case of the undiluted test substance, precipitation was noticed, additionally. After 24 hours, suspensions and precipitation were noticed in the samples of both test substance concentrations. The samples with the lysine-peptide were solutions at the time of preparation and after 24 hours at both test substance concentrations. No co-elution of test substance and peptides was present.

The mean C-peptide depletion, caused by the test substance was determined to be 2.76% (100 mM preparation) and -2.79% (undiluted test substance).The mean K-peptide depletion, caused by the test substance was determined to be -1.13% (100 mM preparation) and -0.13% (undiluted test substance). Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 1.38% (100 mM preparation) and 0% (undiluted test substance).

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test item 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 the C-containing peptide were suspensions and that the result could therefore be underpredictive. Following OECD TG 442C a “negative” result should be considered “inconclusive” in this case.

LuSens

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 a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined to be 392 μg/mL. In the main test luciferase activity was measured after 48-hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed.

At concentrations used in the main experiment the test substance was soluble in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration.

After 48 hours of exposure to 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 test substance has 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 was performed. Cells were exposed to 10 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 to be 906 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.

At concentrations used in the main experiment the test substance was soluble in culture medium (final concentrations). No precipitates were noticed in any concentration. After 24 hours of exposure to test substance CD86 and 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 test substance induces dendritic cell activation.

Conclusion

Taken together, the test item is not peptide reactive, activates keratinocytes and activates dendritic cells. Applying the evaluation criteria, the test item is predicted to be a skin sensitizer (BASF, 2017).

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes. As a result the substance is considered to be classified for skin sensitisation Cat.1 (H317: "May cause an allergic skin reaction") under Regulation (EC) No 1272/2008,as amended for the tenth time in Regulation (EU) No 2017/776.