Dimethyl[3-[(1-oxooctadecyl)amino]propyl][2-oxo-2-(tetradecyloxy)ethyl]ammonium chloride

Endpoint summary

Description of key information

The hazard assessment for skin sensitization is based on in-vitro tests addressing two separate key events in the Adverse Outcome Pathway (AOP):  

1. KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay), addressing the second key event relating to keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.

2. U-SENS™ method, addressing the third key event, i.e. the activation of dendritic cells (DC), typically assessed by the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937.

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:

key study

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:

Adopted February, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Test item: 207517/A
Purity/composition correction factor: No correction will be made for the purity or composition of the test item in this assay
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not indicated
Chemical name (IUPAC), synonym or trade name: Ceraphyl 70
Specific gravity/density 1 g/cm3 (25°C)
Solubility in vehicle:
- Water Not available
- Dimethyl sulfoxide Not indicated
Stability in vehicle:
- Water Not available
- Dimethyl sulfoxide Not indicated

Details on the study design:

1. The KeratinoSensTM test (an ARE-Nrf2 luciferase reporter assay) is proposed to address the second key event in this AOP takes place in the keratinocytes and includes inflammatory responses as well as gene expression associated with specific cell signaling (stress) pathways such as the antioxidant/ electrophile response element (ARE)-dependent pathways.
2. Test System: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Solubility test: the test item was dissolved in DMSO to a final concentration of 40 mg/mL, sonicated for10 min (Temp.: 16 – 20 °C) and warmed up to 37°C. The 100-fold dilution in DMEM glutamax of 40 and 20 mg/mL formed a homogeneous solution (slight precipitation). The 100-fold dilution of the 10 mg/mL DMSO stock in DMEM glutamax formed a clear solution. The final concentration of 400 µg/mL was selected as highest concentration for the main assay.
3. Positive Control: Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), stock solutions prepared in DMSO and diluted to a final concentration range from 7.8 to 250 µM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.
4. Vehicle Control: 1% DMSO in exposure medium and eighteen wells tested per plate.
5. Blank: On each plate three true blank wells were tested (no cells and no treatment).
6. Culture media:
- Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
- Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
- Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
7. Environmental conditions: humid atmosphere of 65 – 100%, containing 5.0 ± 0.5% CO2 in air in the dark at 35.4 – 37.0 °C. Temperature and humidity were continuously monitored throughout the experiment.
8. Plating of Cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+7 in experiment 1, P+13 in experiment 2 and P+7 in experiment 3.
9. Treatment of Cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. Initially, experiment 1, 2 and 3 did not pass all the acceptability criteria and therefore these parts of the study was repeated. In total 3 valid experiments were performed.
10. Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
11. Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.
12. Interpretation:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Positive control results:

The EC1.5 of the positive control was between 5 and 125 µM (92 µM, 83 µM and 106 µM in experiment 1, 2 and 3, respectively). A dose response was observed in all experiments and the induction at 250 µM was higher than 2-fold in experiment 1 and 2 (2.38-fold and 2.39-fold in experiment 1 and 2, respectively).

Run / experiment:

other: Experiment 1

Parameter:

other: toxicity expressed as IC30 (µg/mL)

Value:

5.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 1

Parameter:

other: luminescence activity induction expressed as EC1.5 (µg/mL)

Value:

6.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: toxicity expressed as IC30 (µg/mL)

Value:

14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: luminescence activity induction expressed as EC1.5 (µg/mL)

Value:

4.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 3

Parameter:

other: toxicity expressed as IC30 (µg/mL)

Value:

15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Run / experiment:

other: Experiment 3

Parameter:

other: luminescence activity induction expressed as EC1.5 (µg/mL)

Value:

4.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (92 µM, 83 µM and 106 µM in experiment 1, 2 and 3, respectively). A dose response was observed in all experiments and the induction at 250 µM was higher than 2-fold in experiment 1 and 2 (2.38-fold and 2.39-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (4.4%, 8.3% and 8.8% in experiment 1, 2 and 3, respectively).

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, Quaternium-70 is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report. The test item Quaternium 70 is a surfactant. Positive results in an in-vitro test with surfactants should be considered with caution.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 26 Feb 2018, and the experimental completion date was 30 Mar 2018.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)’

Version / remarks:

9 October 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Test item: 207517/A
Purity/composition correction factor: No correction will be made for the purity or composition of the test item in this assay
Test item handling: No specific handling conditions required
Stability at higher temperatures: Not indicated
Chemical name (IUPAC), synonym or trade name: Ceraphyl 70
Volatile: Not volatile
pH: Not available
Specific gravity/density: 1 g/cm3 (25°C)
Solubility in vehicle: DMSO: Not indicated
Stability in vehicle: DMSO: Not indicated

Details on the study design:

The U-SENS™ method is proposed to address the third key event in the Adverse Outcome Pathway (AOP), i.e. the activation of dendritic cells (DC), typically assessed by the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and non-sensitisers.

Test System U937 human monocytes.
Justification Inducible CD86-expressing cells
Source ATCC (American Type Culture Collection, Virginia, USA).
ATCC no.: CRL-1593.2TM.
Stock cultures of these cells are stored in liquid nitrogen (-196°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test. Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

Cell culture medium: Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).
Environmental conditions: All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 70 - 100 %) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.7 - 36.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Plating of Cells: Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.

Three valid experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 2 did not pass all the acceptability criteria and therefore this part of the study was repeated.

Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL. In the second and third experiment cells were treated with four and eight selected doses of test item, respectively. The three tests shared at least 2 concentrations. The concentrations selected in the second and third experiment were 100, 140, 180 and 200 µg/mL and 1.0, 10, 20, 50, 80, 100 and 200 µg/mL, respectively. In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.

Cell antibodies staining for IgG1 and CD86: Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot. The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

For each culture (IgG1 well and CD86 well), the percentage of viable cells (PI negative cells) was evaluated. The viability for each dose level is the mean of the IgG1 well and CD86 well.
The theoretical concentration at which the chemical induces 30% cytotoxicity (i.e., 70% viability) was calculated. Additionally, a stimulation index (S.I.) is calculated.

Positive control results:

The positive control (TNBS) showed a S.I. ≥ 465% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 111% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC150 in µg/mL

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations.

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC150 in µg/mL

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Quaternium-70 showed toxicity at all test concentrations, and CV70 was considered to be < 100 µg/mL

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: EC150 in µg/mL

Value:

2.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

Quaternium-70 showed toxicity, the calculated CV70 was 51 µg/mL

Other effects / acceptance of results:

All tests passed the acceptance criteria:
• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100% in all experiments).
• The mean viability of the triplicate DMSO vehicle control cells was above the threshold of 90% (100% in all experiments).
• The DMSO vehicle control mean value of its triplicate CD86 S.I. was smaller than 250% of the mean of the triplicate CD86 S.I. of untreated U937 cells in both experiments.
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.
• No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

Overview Stimulation index of CD86 and Cell Viability in
Experiment 1, 2 and 3 of Quaternium-70

 

Test items	Dose (µg/mL)	% Viability (Mean)*			CD86-IgG1 S.I.*			Colour Interference S.I.*
		Experiment			Experiment			Experiment
		1	2	3	1	2	3	1	2	3
Quaternium-70
	1	100	-	100	104	-	103	108	-	106
	10	100	-	99	100	-	359	105	-	148
	20	99	-	97	97	-	710	105	-	168
	50	100	-	71	112	-	1225	104	-	177
	80	-	-	48	-	-	1271	-	-	152
	100	100	51	38	63	1833	903	117	128	146
	140	-	45	53	-	2436	1231	-	128	223
	180	-	52	-	-	1604	-	-	119	-
	200	100	60	19	63	749	741	114	116	127

*    Red values are either below 70% viability or above 150 S.I..

-       Not Applicable

Overview Stimulation index of CD86 and Cell Viability in Experiment 1, 2 and 3

of the Positive (TNBS), Negative (LA), Untreated (RPMI) and Vehicle Control (DMSO)

Controls	% Viability (Mean)*				CD86-IgG1 S.I.*
	Experiment				Experiment
	1	2	3		1		2		3
LA1	100	100	100		110		79		97
LA2	100	100	100		99		67		77
LA3	100	100	100		97		71		111
TNBS1	99	100	100		302		425		465
TNBS2	99	100	100		334		525		619
TNBS3	99	100	100		340		475		489
DMSO	100	100	100		154		140		143
	IgG1 value (%)				CD86 basal expression (%)
	Experiment				Experiment
	1	2	3		1		2		3
RPMI1	0.6	0.6	0.9		13.0		3.9		5.2
RPMI2	0.6	0.9	0.7		11.4		2.4		5.1
RPMI3	0.7	0.9	1.0		13.2		3.3		4.7
RPMI Mean Viability				100%		100%		100%
RPMI Drift				8%		0%		-15%
LA Drift				-7%		-3%		28%

*          Red values are either below 70% viability, above 150 S.I

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In conclusion, Quaternium-70 is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Since the test item Quaternium 70 is a surfactant, positive results in in-vitro tests should be considered with caution.

Justification for classification or non-classification

Quaternium-70 is classified as positive for both increase in the expression levels of CD86 cell surface marker in the U937 cell line and activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. Quaternium 70 should be classified as a skin sensitizer category 1.