1-Propanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N-methyl-N-[2-[(1-oxo-9-octadecen-1-yl)amino]ethyl]-3-sulfo-, methyl sulfate, sodium salt (1:1:1)

Endpoint summary

Description of key information

In an in vitro skin sensitisation assay (ARE-Nrf2 Luciferase Test Method, KeratinoSens) according to OECD Guideline TG 442 D, the test item did induce luciferase activity in at least two independent experimental runs. In an in vitro assay according to OECD TG 442E (h-CLAT) addressing the third key event of skin sensitisation, activation of dendritic cells was observed. Based on the results from the in vitro studies and considering the AOP “2 out of 3” the test item in predicted as skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

21 January 2019 - 31 January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD/OCDE 442E IN VITRO SKIN SENSITISATION ASSAYS ADDRESSING THE KEY EVENT ON ACTIVATION OF DENDRITIC CELLS ON THE ADVERSE OUTCOME PATHWAY FOR SKIN SENSITISATION – Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°158: human Cell Line Activation Test (h CLAT)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

Cell line used: THP-1 cells, Human Acute Monocytic Leukemia, LOT 63558119
Maintenance (culture) medium for THP-1 cells: RPMI-1640 modified medium (with 25 mM HEPES buffer) supplemented with 10 (v/v) % fetal bovine serum (FBS), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.05 mM L-glutamine solution and 0.05 mM 2-mercaptoethanol.
Material and conditions:
- For cell culture standard laboratory equipment was used.
- Flow cytometer: Apogee Flow Cytometer (APOGEE A60 UNIVERSAL ANALIZER)
- Buffer: Phosphate Buffered Saline (DPBS)
- Blocking Solution 0.01% Globulins Cohn fraction II,III
- Antibodies: FITC antibody solutions (50 μL of pre-mixed solution per sample):
- FITC labelled CD86 antibody solution - 6 μL of antibody to 44 μL of FACS buffer
- FITC labelled CD54 antibody solution - 3 μL of antibody to 47 μL of FACS buffer
- FITC labelled-mouse IgG1 antibody solution - 3 μL of antibody to 47 μL of FACS buffer
- Reagent for cytotoxicity test: Propidium iodide (PI)solution: 12.5 μg/mL of propidium iodide solution in DPBS

Controls used:
- Vehicle control: DMSO, sodium chloride solution
- Positive control: 1- chloro-2,4-dinitrobenzene (DNCB, CAS no.: 97-00-7)

Preliminary tests:
For testing, THP-1 cells were seeded at a density of either 0.1 × 10E6 cells/mL or 0.2 × 10E6 cells/mL, and precultured in culture flasks for 72 hours or 48 hours respectively. On the day of testing, cells were harvested from the culture flasks and resuspended with fresh maintance medium at 2 × 10E6 cells/mL. Then, cells were distributed into 24 well flat-bottom plate with 500 µL / wells (1 × 10E6 cells/well).
Master solutions (MS) were prepared with DMSO as follows: Eight master solutions (eight concentrations) were prepared of the test item stock solution, by two-fold and 1.2-fold serial dilutions using DMSO. These master solutions were then further diluted 250 fold into culture medium to obtain the working solutions (WS). The working solutions were finally used for exposure by adding an equal volume of working solution (500 µL) to the volume of THP-1 cell suspension (500 µL) in the 24-well plate to achieve a further two-fold dilution as the final concentration of the test item. The solvent/vehicle control used for the test item was 0.2 % DMSO in culture medium. After 24±0.5 hours of exposure, cells were transferred into sample tubes and 600 μL of FACS buffer was added to each sample. Cells were then collected by centrifugation (250 g, 5 min, 4 ºC). The supernatants were discarded and the remaining cells were washed again with 600 μL of FACS buffer. Finally, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added for each sample. The PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Cell viability was analyzed by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.

Main Test (CD86 and CD54 expression):
DMSO was used to dissolve the test item for the stock solution (SS) in the main tests, as well. The test item was first diluted to the concentration corresponding to the CV75 × 1.2 value determined in the dose finding assay. For the master solutions (MS), 1.2-fold serial dilutions were made from the stock solution using DMSO (eight concentrations). The master solutions were then further diluted 250-fold into the culture medium to obtain the working solutions (WS). These working solutions were finally used for exposure with a further final two-fold dilution factor in the plate. DMSO is tested as a solvent control for the test item and also for the positive control at a single final concentration in the plate of 0.2 %, so it underwent the same dilution as described for the working solutions. Culture medium was used as another negative control to assess the impact of DMSO. DNCB was used as the positive control for CD86/CD54 expression measurement at a final nominal concentration of 4.0 μg/mL in the plate. To obtain a 4.0 μg/mL concentration a 2 mg/mL stock solution of DNCB in DMSO were prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution. The working solution then was diluted 2-fold when added to the cells. Test item and control substances prepared as working solutions (500 μL) were mixed with 500 μL of suspended cells (1 × 10E6 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24 ± 0.5 hours. After 24 ± 0.5 hours of exposure, cells were transferred from 24-well plate into sample tubes, then 1 mL of FACS buffer was added to each sample and cells were collected by centrifugation (250 g, 5 min, 4 ºC). The washing step was repeated once more with 1 mL of FACS buffer. After washing, cells were blocked with 600 μL of 1 × blocking solution and incubated at 4°C for 15 min. After blocking, cells were split in three aliquots of 200 μL into sample tubes. After centrifugation (250 g, 5 min, 4 ºC), cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 (isotype) antibodies and incubated at 4° C for 30 min. The antibodies described in the h-CLAT DB-ALM protocol 158° were used. After washing twice with 150 μL of FACS buffer, cells were resuspended in 400 μL of FACS buffer and 20 μL of 1 × PI solution was added to each sample. The expression levels of CD86 and CD54, and cell viability were analysed using flow cytometry

Flow cytometry measurement and RFI determination:
The expression of CD86 and CD54 was analysed with flow cytometry with the acquisition channel FL-1. A total of minimum 10,000 living cells (PI negative) were acquired. When the cell viability was low, up to 30,000 cells including dead cells were acquired or data of one minute after the initiation of the analysis. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for positive control (ctrl) cells and chemical-treated cells were calculated.

Acceptance criteria:
- The cell viabilities of medium and solvent/vehicle controls should be higher than 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 more than 50%.
- In the solvent controls, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 > 150 and CD54 > 200).
- For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%.
- For the test item resulting in negative outcome, the cell viability at the 1.2 x CV75 should be less than 90% (when HSC is used instead of CV75, the data for the test chemical is accepted regardless the cell viability at the highest dose).
- For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.

Prediction model:
If the RFI of CD86 is equal to or greater than 150% at any tested dose (>50 % of cell viability) in at least 2 independent runs, AND/OR if the RFI of CD54 is equal to or greater than 200 % at any tested dose (>50 % of cell viability) in at least 2 independent runs, the test item prediction is considered as positive. Otherwise it is considered as a negative. In case the first two independent runs are not concordant a third run needs to be performed and the final prediction will be based on the mode of conclusions from the three individual runs.

Positive control results:

The positive control gave expected results for both markers, meaning that the RFI values of both CD86 and CD54 expression was over the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each run.

Key result

Run / experiment:

other: 1st

Parameter:

other: RFI value for CD54 [%]

Value:

281

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 47 µg/mL

Key result

Run / experiment:

other: 2nd

Parameter:

other: RFI value for CD54 [%]

Value:

414

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 48 µg/mL

Key result

Run / experiment:

other: 3rd

Parameter:

other: RFI value for CD54 [%]

Value:

362

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 40 µg/mL

Key result

Run / experiment:

other: 1st

Parameter:

other: RFI value for CD86 [%]

Value:

52

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 47 µg/mL

Key result

Run / experiment:

other: 2nd

Parameter:

other: RFI value for CD86 [%]

Value:

35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 48 µg/mL

Key result

Run / experiment:

other: 3rd

Parameter:

other: RFI value for CD86 [%]

Value:

79

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration: 40 µg/mL

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

Preliminary tests (Dose finding assays):
Two independent runs for the dose finding assay were performed to determine the test item concentration that results in 75 % cell viability (CV75) compared to the solvent/vehicle control determined in the trial forming. In the first run the highest final test item concentration on the plate was the highest soluble concentration corresponding to 250 µg/mL. Since high level of cytotoxicity was observed, the concentration range was shifted and the highest final concentration of 63 µg/mL was used on the plate. In the first run a 2-fold serial dilution was used while in the second run a 1.2-fold dilution was used when preparing the master solutions.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory demonstrated technical proficiency by correctly obtaining the expected h-CLAT prediction for the 10 proficiency substances recommended in OECD 442E and by obtaining CV75, EC150 and EC200 values that fell within the respective reference ranges. Moreover, a historical database of reactivity check results positive controls and solvent/vehicle controls was generated and has been maintained to confirm the reproducibility of the test method over time.

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

Table 1: Positive and negative control results

	sample	concentration	RFI		viability (IgG1)
			CD86	CD54	IgG
Exposure date:   25 January 2019	DMSO	0.2%	115	117	93.0
	DNCB	4.4 μg/mL	454	429	70.5
Exposure date:   29 January 2019	DMSO	0.2 %	79	71	93.2
	DNCB	4.2 μg/mL	909	591	74.7
Exposure date:   31 January 2019	DMSO	0.2 %	85	82	91.6
	DNCB	4.0 μg/mL	489	435	70.6

The positive control gave expected results for both markers,meaning that the RFI values of both CD86 and CD54 expression was over

the positive criteria (CD86 ≥ 150 % and CD54 ≥ 200 %) and the respective cell viabilities were more than 50 % in each run.

Table 2: Results 1st run

Test item		Cells batch Test item weight in (g)		THP-1 (ATCC) MC2 20180205-20190103 0.0283			Exposure date Analysis date		24 January. 2019 25 January,2019		1st run	VALID
Sample	Conc. (µg/mL)	MFI (geo Mean)			Corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)	MFI >105% to IgG
		CD86	CD54	isotyp	CD86	CD54	vs medium control	vs DMSO control	vs medium control	vs DMSO control	IgG	CD86	CD54
Control	-	3226	2604	2141	1085	463		100		100	92.9	151%	122%
DMSO	0.2%	3350	2647	2104	1246	543		115		117	93.0	159%	126%
DNCB	4.4	8102	4778	2447	5655	2331		454		429	70.5
Test item	57	4128	6285	3316	812	2969		65		547	14.0
	47	3800	4675	3151	649	1524		52		281	56.8
	39	3664	4372	2954	710	1418		57		261	76.2
	33	3540	4169	2886	654	1283		52		236	83.3
	27	3302	3330	2601	701	729		56		134	92.4
	23	3293	3501	2725	568	776		46		143	92.3
	19	3287	3641	2642	645	999		52		184	92.2
	16	3247	3111	2391	856	720		69		133	93.4

Table 3: Results 2nd run

Test item		Cells batch Test item weight in (g)		THP-1 (ATCC) MC2 20180205-20190103 0.0285			Exposure date Analysis date		28 January. 2019 29 January,2019		2nd run	VALID
Sample	Conc. (µg/mL)	MFI (geo Mean)			Corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)	MFI >105% to IgG
		CD86	CD54	isotyp	CD86	CD54	vs medium control	vs DMSO control	vs medium control	vs DMSO control	IgG	CD86	CD54
Control	-	3110	2514	2058	1052	456		100		100	92.9	151%	122%
DMSO	0.2%	2926	2419	2097	829	322		79		71	93.2	140%	115%
DNCB	4.2	9837	4207	2305	7532	1902		909		591	74.7
Test item	57	3488	4576	3035	453	1541		55		479	49.0
	48	3416	4459	3126	290	1333		35		414	66.2
	40	3329	4024	3188	141	836		17		260	79.4
	33	3185	3483	2738	447	745		54		231	87.7
	27	3061	3444	2684	377	760		45		236	89.2
	23	3059	3288	2516	543	772		66		240	91.2
	19	2998	3066	2407	591	659		71		205	92.4
	16	3006	2814	2458	548	356		66		111	93.8

Table 4: Results 3rd run

Test item		Cells batch Test item weight in (g)		THP-1 (ATCC) MC2 20180205-20190103 0.0285			Exposure date Analysis date		30 January. 2019 31 January,2019		3rd run	VALID
Sample	Conc. (µg/mL)	MFI (geo Mean)			Corrected MFI		RFI (CD86)		RFI (CD54)		Viability (%)	MFI >105% to IgG
		CD86	CD54	isotyp	CD86	CD54	vs medium control	vs DMSO control	vs medium control	vs DMSO control	IgG	CD86	CD54
Control	-	3640	2986	2146	1494	840		100		100	91.4	170%	139%
DMSO	0.2%	3324	2747	2055	1269	692		85		82	91.6	162%	134%
DNCB	4.0	8579	5384	2372	6207	3012		489		435	70.6
Test item	57	4095	9712	2916	1179	6796		93		982	23.2
	48	4068	6552	3114	954	3438		75		497	45.5
	40	4020	5519	3013	1007	2506		79		362	66.0
	33	3850	4763	2874	976	1889		77		273	79.6
	27	3679	4496	2766	913	1730		72		250	83.7
	23	3425	4003	2597	828	1406		65		203	90.6
	19	3359	3796	2601	758	1195		60		173	90.0
	16	3356	4163	2495	861	1668		68		241	90.3

Table 5: EC150 and EC200 VALUES FOR THE TEST ITEM CLASSIFIED AS SENSITISER

		RFI for CD54
Sample		Conc. (µL/mL)	Log2 (conc.)	RFI	Conc. (µL/mL)	Log2 (conc.)	RFI	Conc. (µL/mL)	Log2 (conc.)	RFI
				1			2			3
Test item		57	5.8	547	57	5.8	479	57	5.8	982
		47	5.6	281	48	5.6	414	48	5.6	497
		39	5.3	261	40	5.3	260	40	5.3	362
		33	5.0	236	33	5.0	231	33	5.0	273
		27	4.8	134	27	4.8	236	27	4.8	250
		23	4.5	143	23	4.5	240	23	4.5	203
		19	4.2	184	19	4.3	205	19	4.3	173
		16	4.0	133	16	4.0	111	16	4.0	241
Linear regression	EC150/ EC200			30.8			18.9			-
Log-linear extrapolation	Log 2 (EC150/ EC200)			-			-			2.6
	EC150/ EC200			-			-			6.2
Median EC150/EC200		18.9

Table 6: Summary of the h-CLAT results for the test item

Name of the Test item	Obtained CV75 value (µg/mL)	h-CLAT result for CD86 (positive/negative)	h-CLAT result for CD54 (positive/negative) and obtained EC200 value (µg/mL)	h-CLAT result obtained (sensitizer/ non-sensitizer)
Test item	47.2	negative	positive (18.9µg/mL)	sensitizer

Interpretation of results:

other: activation of dendritic cells

Remarks:

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

Based on these results and the h-CLAT prediction model, the test item demonstrated an in vitro sensitizing potential under the experimental conditions of human Cell Line Activation Test.

Executive summary:

A study according to OECD TG 442E was performed to determine the skin sensitization potential of the test item.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. The default stock concentration used for test items dissolved in DMSO was set to the highest soluble concentration (125 mg/mL) that could be reached during the formulation trial. In the second dose finding test a lower concentration range was used due to the high level of cytotoxicity observed in the first run. An average CV75 value of 47.2 µg/mL was calculated and used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent runs between 57 µg/mL – 16 µg/mL. The increase in CD86 marker expression (RFI) was never equal to or greater than 150 % at any tested doses (with >50 % of cell viability) compared to the respective negative controls in all of the independent runs. Therefore, all three valid runs were concluded negative for CD86 marker expression. The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at most of the tested concentrations (with >50 % of cell viability) in all three independent valid runs. Based on the concordant results of the three individual runs for CD54 expression, prediction was concluded as positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation and log-linear extrapolation. The EC200 value for CD54 was ~19 µg/mL. All three runs were considered valid in the main study. Since CD54 marker expressions gave positive results, the overall h-CLAT prediction was concluded positive.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 March 2019 - 03 April 2019

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:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM (INVITTOX) Protocol n°155: KeratinoSens™

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system):
Formulation of the Test Item
The test item was formulated and examined in the test as follows: first, solubility of the test item was evaluated in DMSO at the concentration corresponding to 200 mM. Since the test chemical has no defined molecular weight (MW), a default concentration of 40 mg/mL or 4% (w/v) was used (same way as a 200 mM stock solution would be used). The test item could be properly dissolved in DMSO after approximately 5 minutes of vortexing, and formed a clear, yellowish and homogenous solution at 40 mg/mL corresponding to 200 mM. In ultrapure water the test item and solvent formed a foamy suspension. Since DMSO is the preferred vehicle, and the formula fulfilled all requirements, it was chosen as the appropriate solvent of the test item.

Cell or Test System:
The KeratinoSens™ cell line is a transgenic cell line with a stable Luciferase construct insertion.
Name: KeratinoSens™ cell line
Description: immortalised adherent cell line derived from human keratinocytes (HaCaT) transfected with selectable plasmids
Supplier: Givaudan Schweiz AG
Lot Number: 20160415

Preparation of Media:
Maintance (culture) medium: DMEM supplemented with 9.0 (v/v) % fetal bovine serum (FBS) and ~ 500 μg/mL G418.
Thawing medium: DMEM containing 9.1 (v/v) % FBS without G418
Exposure medium: DMEM containing 1 (v/v) % FBS without G418

Principle of the KeratinoSens™ method:
The KeratinoSens™ method is an in vitro assay that quantifies the extent of luciferase gene induction following 48 hours incubation time of the KeratinoSens™ reporter cells with the test chemicals. Luciferase gene induction is measured in the cell lysates by luminescence detection using a light producing luciferase substrate (Luciferase Reagent). Cytotoxicity and the relative luminescence intensity of luciferase substrate in the lysates are measured and luciferase induction compared to solvent/vehicle control is calculated.
KeratinoSens™ cells were derived from HaCaT human keratinocytes and transfected with selectable plasmids containing luciferase gene under the transcriptional control of the AKR1C2 ARE gene sequence, upstream of the SV40 promoter. AKR1C2 is known to be one of the genes up-regulated upon contacting skin sensitisers in dendritic cells. Therefore, this method is able to mimic the activation of the Keap1-Nrf2-ARE regulatory pathway, and is relevant for the assessment of the skin sensitisation potential of test chemicals. A prediction model is used, to support the discrimination between sensitisers and non-sensitisers.

Test Chemical Master Solutions:
Based on the test chemical stock solution made of DMSO two fold serial dilutions were made using the solvent to obtain twelve 100 × master concentrations of the test chemical creating a 100 × master plate. The 100 × master concentrations were further diluted 25 fold into exposure medium to obtain the 4 × master plate, by adding 10 μL of the 100 × master concentrations to 240 μL exposure medium.

Positive control:
The positive control used was Trans-Cinnamaldehyde for which a series of five 100 × master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 200 mM stock solution) and diluted as described for the 4 × master solutions. The final concentration of the positive control on the treated plates ranged from 4 to 64 μM.

Negative control:
The negative (solvent) control used was DMSO, for which six wells per plate were prepared. It underwent the same dilution as described for the master and working solution concentrations in 7.3.1, so that the final negative (solvent) control concentration was 1% DMSO in exposure medium on the treated plates.
This DMSO concentration is known not to affect cell viability and corresponds to the same concentration of DMSO found in the tested chemical and in the positive control.

Preparation of cells:
For testing cells were 80 - 90% confluent and care was taken to ensure that cells were never grown to full confluence. One day prior to testing cells were harvested in thawing medium, and distributed into 96-well plates (10 000 cells/well) homogenously. For each individual test in the study, three replicates were used for the luciferase activity measurements, and one parallel replicate for the cell viability assay. One well per plate was left empty to assess background values. Cells were grown for 24 ± 0.5 hours in 96-wells microplates at 37 ± 1 °C in the presence of 5 % CO2.

Exposure:
After the 24 hour incubation time, thawing medium was replaced with fresh exposure medium. The 4 × master solutions of the test chemical and control substances were added to each well in a way that an additional 4 fold dilution was achieved on the plate for the final concentrations to be established (50 μL of 4× master solution to 150 μL of exposure medium). The treated plates were then incubated for about 48 ± 1 hours at 37 ± 1 °C in the presence of 5 % CO2. Care was taken to avoid cross-contamination between wells by covering the plates with a foil prior to the incubation with the test chemical.

Luciferase activity measurements:
After the 48 hour exposure time with the test chemical and control substances, cells were washed with DPBS (270 μL), and 1× lysis buffer (20 μL) for luminescence readings was added to each well for 20 minutes at room temperature (on all three plates). Plates with the cell lysate were then placed in the luminometer for reading. First the luciferase substrate (50 μL) was added to each well and after one second, the luciferase activity was integrated for 2 seconds.

Cytotoxicity
For the cell viability assay, medium was replaced after the 48 hour exposure time with MTT working solution (200 μL) and cells were incubated for 4 hours at 37 ± 1 °C in the presence of 5 % CO2. The MTT working solution was then removed and cells were solubilised by the addition of isopropanol (50 μL). After shaking for 30 minutes the absorption was measured at 570 nm with a spectrophotometer.

Acceptance Criteria:
For each test chemical and positive control substance, in order to derive a prediction, at least two independent tests, each containing three replicates for the luminescence measurements and one for viability measurement, were needed. In case of discordant results between the two independent tests, a third test containing four replicates should have been performed. Each independent test was to be performed on a different day with fresh stock solution of test chemicals and independently harvested cells. Cells may have come from the same passage however. KeratinoSens™ prediction should be considered in the framework of an IATA and in accordance with the limitations stated in the OECD test guideline.
The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations. The EC1.5 value of the positive control should be within two standard deviations of the historical mean of the testing facility or between 7 μM and 30 μM (based on the validation dataset). In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of Trans-Cinnamaldehyde should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
Historical control mean for EC1.5 value of the positive control: 14.6 ± 9.1 μM
Acceptance range for EC1.5 value of the positive control: 5.6 μM – 23.7 μM
Finally, the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO should be below 20 % in each test which consists of 6 wells tested in triplicate.

Prediction model:
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 tests, otherwise the KeratinoSens™ prediction is considered negative:
- the Imax is equal or higher than 1.5 fold and statistically significantly different as compared to the negative/solvent control (as determined by a two-tailed, unpaired Student’s T-test);
- the cellular viability is higher than 70 % at the lowest concentration with induction of luciferase activity ≥ 1.5 fold;
- the EC1.5 value is less than 1000 μM (or < 200 μg/mL for test chemicals with no defined molecular weight);
- there is an apparent overall dose-response for luciferase induction (or a biphasic response).

Positive control results:

The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 μM and 9 μM in the two individual tests which fell into the corresponding acceptance range established based on the historical control data: 5.6 μM – 23.7 μM. In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 μM was 28.35 and 83.88 fold. Although these values are out of the 2 – 8 fold induction range, the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control, therefore the tests were considered valid.

Key result

Run / experiment:

other: 1st run

Parameter:

other: average fold Luciferase induction

Value:

2.71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 63 µM

Key result

Run / experiment:

other: 2nd run

Parameter:

other: maximal fold Luciferase induction

Value:

2.31

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 31 µM

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
Prior to routine use of the test method described in the OECD Test Guideline 442D, our laboratory demonstrated technical proficiency, using the 10 Proficiency Substances listed in APPENDIX IA - ANNEX 1 of TG 442D.

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

Table 1: Average fold induction, significance and viability (%) values for the positive control in the first test

		Trans-Cinnamaldehyde
Concentration (µM)		4	8	16	32	64
average induction		1.32	1.66	1.64	2.61	28.35
significance		0.064	0.004	0.002	0.000	0.022
viability		100%	90%	92%	94%	75%

 

Table 2: Average fold induction, significance and viability (%) values for the positive control in the second test

		Trans-Cinnamaldehyde
Concentration (µM)		4	8	16	32	64
average induction		1.11	1.38	2.27	3.38	83.88
significance		0.567	0.103	0.022	0.002	0.014
viability		92%	95%	91%	45%	5%

Table 3: Summary of the KeratinoSens™ results for the test item

Significant induction above 1.5-fold (yes/no)	Average EC 1.5 (μM)	Average IC30 / IC50 (µM)	Average Imax (fold)	Showing clear dose response (yes/no)	KeratinoSens™ result obtained (sensitizer / non-sensitizer)
yes	19	78 / 92	309	yes	sensitizer

 

Table 4: Measured luminescence values for the test item and controls ( First test 25 February 2019 – 28 February 2019)

		Test item
Concentration (µM)		1.0	2.0	4	8	16	31	63	125	250	500	1000	2000
Plate ID	20190225-1055-2	1.11	1.49	1.33	1.66	1.45	1.76	3.32	3.92	-0.01	-0.02	-0.02	-0.02
	20190225-1055-3	0.86	1.04	1.41	1.40	1.44	1.65	2.86	3.82	-0.01	-0.01	-0.01	-0.01
	20190225-1055-4	2.15	0.82	1.05	1.09	1.10	1.95	1.93	3.65	0.00	0.00	0.00	0.00
average induction		1.37	1.12	1.26	1.39	1.33	1.79	2.71	3.80	-0.01	-0.01	-0.01	-0.01
significance		0.444	0.523	0.096	0.028	0.042	0.012	0.004	0.000	0.000	0.000	0.000	0.000
viability		92%	78%	76%	87%	81%	84%	93%	1%	2%	1%	0%	0%

 

Table 5: Measured luminescence values for the test item and controls ( Second test 28 February 2019 – 03 March 2019)

		Test item
Concentration (µM)		1.0	2	4	8	16	31	63	125	250	500	1000	2000
Plate ID	20190228-1020-2	1.12	1.29	1.39	1.72	1.70	2.85	2.48	0.60	-0.02	-0.03	-0.02	-0.03
	20190228-1020-3	1.09	1.15	1.26	1.41	1.14	2.06	2.46	0.24	-0.05	-0.05	-0.05	-0.05
	20190228-1020-4	1.13	1.20	1.42	1.16	1.66	2.02	2.18	-0.04	-0.05	-0.05	-0.05	-0.05
average induction		1.11	1.21	1.36	1.43	1.50	2.31	2.38	0.27	-0.04	-0.04	-0.04	-0.04
significance		0.505	0.229	0.133	0.019	0.128	0.001	0.002	0.013	0.001	0.001	0.001	0.001
viability		99%	147%	97%	132%	152%	130%	89%	3%	1%	-1%	-1%	1%

Interpretation of results:

other: activation of keratinocytes

Remarks:

The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Conclusions:

Based on these results and the KeratinoSens™ prediction model, the test item was concluded positive therefore having a sensitizing potential under the experimental conditions of KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method).

Executive summary:

In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD TG 442D.

For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both tests met the acceptance criteria. The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 μM and 9 μM in the two individual tests respectively. In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 μM was 28.35 and 83.88 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations in the first test however, there was cytotoxicity (viability < 70 %) induced at 32 and 64 μM in the second test.

For the test item twelve doses were used in two independent tests between 2000 μM to 1 μM. There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at concentrations corresponding to 63 μM and greater in both tests. In the first test the IC30 and IC50 values were 79 μM and 92 μM while in the second test 77 μM and 91 μM respectively. The fold induction exceeded the threshold of 1.5-fold compared to the respective negative controls in both independent tests. EC1.5 values were 22 μM and 16 μM in the first and second tests respectively. Both tests were concluded positive for luciferase gene induction.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

For the evaluation of the skin sensitisation potential of the test substance a Weight of Evidence approach was used. A Keratinocyte Activation Assay (KeratinoSens) was performed to evaluate the keratinocyte activation potential and a h-CLAT Assay to determine the activation of dendritic cells.

KeratinoSens

In the course of this study the skin sensitization potential of the test item was studied using the KeratinoSens™ method (ARE-Nrf2 Luciferase Test Method) according to OECD TG 442D.

For the test chemical and positive control substance, in order to derive a prediction two independent tests were sufficient to be conducted, since the results of those tests were concordant and both tests met the acceptance criteria.The luciferase activity induction obtained with the positive control, Trans-Cinnamaldehyde was statistically significant above the threshold of 1.5 at four concentrations in the first test and at three concentrations in the other test. The EC1.5 value of the positive control was 6 μM and 9 μM in the two individual tests respectively. In addition, the average induction in the parallel plates for Trans-Cinnamaldehyde at 64 μM was 28.35 and 83.88 fold and the dose response could be clearly seen with increasing luciferase activity induction at increasing concentrations for the positive control. There was no cytotoxicity (cell viability lower than 70 %) induced by the positive control at any of the concentrations in the first test however, there was cytotoxicity (viability < 70 %) induced at 32 and 64 μM in the second test.

For the test item twelve doses were used in two independent tests between 2000 μM to 1 μM. There was cytotoxicity induced (viability < 70 %) by the test item in KeratinoSens™ cells compared to the solvent/vehicle control at concentrations corresponding to 63 μM and greater in both tests. In the first test the IC30 and IC50 values were 79 μM and 92 μM while in the second test 77 μM and 91 μM respectively. The fold induction exceeded the threshold of 1.5-fold compared to the respective negative controls in both independent tests. EC1.5 values were 22 μM and 16 μM in the first and second tests respectively. Both tests were concluded positive for luciferase gene induction.

h-CLAT

A study according to OECD TG 442E was performed to determine the skin sensitization potential of the test item.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in two dose finding tests. The default stock concentration used for test items dissolved in DMSO was set to the highest soluble concentration (125 mg/mL) that could be reached during the formulation trial. In the second dose finding test a lower concentration range was used due to the high level of cytotoxicity observed in the first run. An average CV75 value of 47.2 µg/mL was calculated and used for setting the dose-range for measuring CD86 and CD54 expression in the main test. Eight doses were used in three independent runs between 57 µg/mL – 16 µg/mL. The increase in CD86 marker expression (RFI) was never equal to or greater than 150 % at any tested doses (with >50 % of cell viability) compared to the respective negative controls in all of the independent runs. Therefore, all three valid runs were concluded negative for CD86 marker expression.The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at most of the tested concentrations (with >50 % of cell viability) in all three independent valid runs. Based on the concordant results of the three individual runs for CD54 expression, prediction was concluded as positive. Thus, the effective concentration for CD54 expression (EC200) was determined by linear interpolation and log-linear extrapolation. The EC200 value for CD54 was ~19 µg/mL.All three runs were considered valid in the main study.Since CD54 marker expressions gave positive results, the overall h-CLAT prediction was concluded positive.

Conclusion

Based on the results from the in vitro studies the test item in predicted as skin sensitizer.

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is considered to be classified for skin sensitisation, Category 1, H317 (May cause an allergic skin reaction) under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.