Reaction mass of trimethyl-3-[(1-oxooctadecyl)amino]propylammonium methyl sulphate, Propane-1,2-diol and Trimethyl-3-[(1-oxohexadecyl)amino]propylammonium methyl sulphate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26th April to 10th May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: OECD/OCDE 442E In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

2018

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2017

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of trimethyl-3-[(1-oxooctadecyl)amino]propylammonium methyl sulphate, Propane-1,2-diol and Trimethyl-3-[(1-oxohexadecyl)amino]propylammonium methyl sulphate

EC Number:

947-471-7

Molecular formula:

C22H47N2O.CH3O4S - C3H8O2 - C24H51N2O.CH3O4S

IUPAC Name:

Reaction mass of trimethyl-3-[(1-oxooctadecyl)amino]propylammonium methyl sulphate, Propane-1,2-diol and Trimethyl-3-[(1-oxohexadecyl)amino]propylammonium methyl sulphate

Test material form:

solid

Remarks:

yellowish-brown solid

In vitro test system

Details on the study design:

-Nominal concentrations: 3.6 µg/ml, 3.0 µg/ml, 2.5 µg/ml, 2.1 µg/ml, 1.7 µg/ml, 1.4 µg/ml, 1.2 µg/ml, 1.0 µg/ml
-Positive control: 1-Chloro-2,4-Dinitrobenzene
-Solvents/Vehicles: Dimethyl sulfoxide (DMSO), Sodium chloride solution (saline 0.9 %)
-Cell or test system: the THP-1 cell line is an immortalized human monocytic leukemia cell line
-THP-1 Master culture: subcultured MC2 master culture was used in the preliminary and main tests
-Mainteinance (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 and 0.05 mM 2-mercaptoethanol.
-Flow cytometer: Apogee Flow Cytometer
-Evaluation softwares: Apogee Histogram Software for flow cytometry measurements and MS Excel for further calculations were used

PRELIMINARY TEST:
-Preparation of the cells: For testing, THP-1 cells were seeded at a density of either 0.1 × 10^6 cells/ml or 0.2 × 10^6 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 maintenance medium at 2 × 10^6 cells/ml. Then, cells were distributed into 24 well flat-bottom plate with 500 µl cell supsension / well (1 × 10^6 cells/well).
-Preparation master and working solutions: Master solutions (MS) were prepared with saline 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 saline. These master solutions were then further diluted 50 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.
-Solvent/vehicle control: The solvent/vehicle control used for the test item was culture medium.
-Test item exposure: The culture medium or working solutions described above were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well. The treated plates were then incubated for 24 ± 0.5 hours at 37° C under 5 % CO2. The plates were sealed with microplate covers prior to the incubation to avoid evaporation of test item.
-Propium iodide (PI) Staining: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.
-Cytotoxicity measurement by flow cytometry and estimation of CV75 value: 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:
-Test item dilutions: Saline 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 saline (eight concentrations). The master solutions were then further diluted 50-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.
-Solvent/vehicle controls: Culture medium was used as negative control for the test item and to assess the impact of DMSO. DMSO was tested as a solvent control 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.
-Positive control: 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.
-Application of test item and control substances: Test item and control substances prepared as working solutions (500 μl) were mixed with 500 μl of suspended cells (1 × 106 cells) at 1:1 ratio in a single replicate, and cells were incubated for 24±0.5 hours
-Fluorescein Isothiocyanate (FITC) staining: After 24 ± 0.5 hours of exposure, cells were transferred from the 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 relative fluorescence intensity (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 and 10,000 living cells could not be acquired in 60 seconds, 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 according to the following equation:
RFI = (MFI of chemical-treated cells – MFI of chemical- treated isotype control cells/MFI of solvent/vehicle-treated control cells – MFI of solvent/vehicle-treated isotype control cells) x100
The calculated cell viabilities from the isotype control (ctrl) cells (which are stained with mouse IgG1 isotype antibodies) were also noted. The PI uptake was analysed on channel FL-3. Cell viability was determined by the Apogee Histogram Software by gating out PI positive cells, and the calculated percentage of PI negative cells was displayed on the software.
-Optional Effective Concentration determination: For the test items predicted as positive, optionally two Effective Concentration (EC) values may be determined: the EC150 for CD86 and EC200 for CD54, the concentrations at which the test items induced a RFI of 150 or 200. These EC values potentially could contribute to the assessment of sensitising potency when used in integrated approaches such as IATA. They can be calculated by the following equations:
EC150 (for CD86) = Bconcentration + [(150 - BRFI) / (ARFI - BRFI) × (Aconcentration - Bconcentration)]
EC200 (for CD54) = Bconcentration + [(200 - BRFI) / (ARFI - BRFI) × (Aconcentration - Bconcentration)]
where: Aconcentration is the lowest concentration in μg/mL with RFI > 150 (CD86) or 200 (CD54)
Bconcentration is the highest concentration in μg/mL with RFI < 150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI > 150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI < 150 (CD86) or 200 (CD54)
For the purpose of more precisely deriving the EC150 and EC200 values, three independent runs for CD86/CD54 expression measurement may be required. The final EC150 and EC200 values are then determined as the median value of the ECs calculated from the three independent runs. When only two of three independent runs meet the criteria for positivity, the higher EC150 or EC200 of the two calculated values is adopted.

Results and discussion

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.
The DMSO controls had negative outcomes compared to the medium control for both markers in all runs, meaning that the RFI values of CD86 and CD54 marker expression was never over the positive criteria. The cell viabilities of medium and DMSO controls were higher than 90 % in all runs (taken cell viabilities of the IgG1 isotypic control). All three runs met the respective acceptance criteria, thus they were considered valid.
For medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control was over 105 % in all three runs.

In vitro / in chemico

Results

Results 1

Run / experiment:

other: other: 3 independent runs / average value reported

Parameter:

other: h-CLAT for CD86/ EC150 value (µg/ml)

Value:

2.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Run / experiment:

other: other: 3 independent runs / average value reported

Parameter:

other: h-CLAT for CD54/ EC200 value (µg/ml)

Value:

2.5

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:

The increase in CD86 marker expression (RFI) was greater than 150 % at one or two of the tested doses (with >50 % of cell viability) compared to the respective negative controls in all three runs. Also, effective concentration for CD86 expression (EC150) was determined, since clear dose response could be observed.

The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several higher concentrations (with >50 % of cell viability) in all three independent valid runs. Also, effective concentration for CD54 expression (EC200) was determined, since clear dose response could be observed.

Any other information on results incl. tables

Positive and negative control data

sample	concentration	RFI		viability ( % ) ‑ IgG1
		CD86	CD54	IgG
Medium	-	100	100	91.8
DMSO	0.20 %	98	91	91
DNCB	4.0 μg/ml	586	329	77.2
Medium	-	100	100	93
DMSO	0.2 %	70	56	91.4
DNCB	4.2 μg/ml	219	269	51.7
medium	-	100	100	94.7
DMSO	0.2 %	80	95	94.9
DNCB	4.4 μg/ml	532	537	62

Outcome of the individual runs of the main tests

Obtained CV75 value (µg/ml)	Result of the individual runs for CD86 (positive/negative)			h-CLAT prediction for CD86 expression	Result of the individual runs for CD54 (positive/negative)			h-CLAT prediction for CD54 expression
3	p	p	p	postive	p	p	p	positive

Effective concentrations calculated in the independent runs

		RFI for CD86			RFI for CD54
Linear interpolation	EC150/ EC200 (µg/ml)	2.7	2.2	2.8	2.8	2.1	2.5
Median EC150/EC200 (µg/ml)		2.7			2.5

Summary of the h-CLAT results for the test item

Obtained CV75 value (µg/ml)	h-CLAT result for CD86 (positive/negative) and obtained EC150 value (µg/ml)	h-CLAT result for CD54 (positive/negative) and obtained EC200 value (µg/ml)	h-CLAT result obtained (sensitizer/ non-sensitizer)
3	positive	positive	sensitizer
	(2.7 µg/ml)	(2.5 µg/ml)

Applicant's summary and conclusion

Interpretation of results:

other: the study is evaluated in a weight of evidence approach

Conclusions:

Since both CD86 and CD54 marker expressions gave prevailingly positive results, the overall h-CLAT prediction was concluded positive.
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.
The study is evaluated in a weight of evidence approach.

Executive summary:

In the course of this study the skin sensitization potential of the test item was studied.

The extent of cytotoxicity induced on THP-1 cells by the test item was studied in three dose finding tests. The maximal final concentrations used on the plates for the test item previously dissolved in saline were 1002 µg/ml (first run) and 8.4 µg/ml (second and third run). In the second dose finding test the concentration range was lowered due to the high level of cytotoxicity observed in the first run. An average CV75 value of 3.0 µg/ml was calculated and it was 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 3.8 µg/ml – 1.0 µg/ml. 

The increase in CD86 marker expression (RFI) was greater than 150 % at one or two of the tested doses (with >50 % of cell viability) compared to the respective negative controls in all three runs. Also, effective concentration for CD86 expression (EC150) was determined, since clear dose response could be observed.

The increase of CD54 marker expression (RFI) was greater than 200 % compared to the negative controls at several higher concentrations (with >50 % of cell viability) in all three independent valid runs. Also, effective concentration for CD54 expression (EC200) was determined, since clear dose response could be observed.

Since both CD86 and CD54 marker expressions gave prevailingly positive results, the overall h-CLAT prediction was concluded positive.

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.