Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Remarks:

in vitro h-CLAT study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 14 May 2018 to 01 June 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (The human Cell Line Activation Test (h-CLAT)

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In vitro skin sensitisation integrated testing strategy approach

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

EC Number:

947-766-0

Molecular formula:

Since the test substance is a complex UVCB, no defined molecular formula is available.

IUPAC Name:

Quaternary ammonium compounds, N,N,N'-tris(hydroxyethyl)-N,N'-dimethyl-N'-C16-18 (even numbered) and C18 unsatd., alkyltrimethylenedi-, bis(Me sulfates) (salts)

Test material form:

solid: bulk

In vitro test system

Details on the study design:

(A) Test and reference substances

The solvent control was culture medium (RPMI-1640) as defined in the Guideline for test chemicals solubilised or stably dispersed in medium or saline. Formulations were prepared under subdued lighting with the aid of vortex mixing, ultra-sonication and warming at 37oC, where required to give the maximum required treatment concentration and subsequent dilutions were made using culture medium. 2,4-dinitrochlorobenzene (CAS No. 97-00-7) supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

a) Test Substance Formulation
Dose Finding Assay
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control. The CV75 value was used to determine the concentration of test items for the CD86/CD54 expression measurement. The test substance was soluble in culture medium at 271.56 mg/mL and in saline at 254.4 mg/mL. Accordingly, culture medium was chosen as the vehicle for this assay. The test substance was dissolved at 100 mg/mL in culture medium and then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 50-fold in culture medium giving final test concentrations ranging from 7.8 to 1000 μg/mL (working solutions). All concentrations tested in the initial dose finding assay were toxic, therefore a further dose finding assay was performed, using a concentration of 1 mg/mL in culture medium, and final test concentrations from 0.078 to 10 μg/mL (following dilution in culture medium). The test substance formulation was prepared shortly before testing.

b) CD86/CD54 Expression Measurement
The test substance was dissolved at 0.42 mg/mL in culture medium. Eight stock solutions were prepared by 1.2-fold serial dilutions and then further diluted 50-fold into the culture medium to give eight final test concentrations in a range from 1.17 to 4.20 μg/mL (equivalent to 0.335 x CV75 to 1.2 x CV75). The test substance formulation was prepared shortly before testing.

(B) Test System
a) Specifications
Human monocytic leukemia cell line, THP-1 (ATCC® TIB-202™, an immortalised human monocytic leukemia cell line, used as a surrogate for dendritic cells), was supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.

b) Identification
The test system was suitably labelled to clearly identify the study number, test substance, test substance concentration, positive and vehicle controls.

c) Cell Culture Maintenance
THP-1 cells were cultured, at 37ºC under 5% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 106 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 106 cells/mL. Cell density did not exceed 1 x 106 cells/mL.

d) Reactivity Check
This was performed using DNCB (CAS No. 97-00-7, ≥99% purity), nickel sulphate (CAS No. 10101-97-0, 99% purity) and lactic acid (CAS No. 50-21-5, 85% purity) two weeks after thawing. DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.

e) Plate Preparation
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 106 cells/mL for 48 hours or at a density of 0.1 x 106 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well flat-bottom plate (500 µL/well) (expression assay) or a 96-well flat-bottomed plate (80 µL/1.6 x 105 cells per well) (DRF assay(s)).

(C) Study Design
a) Dose Finding Assay
The test substance working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5% CO2. After the 24-hour incubation period, all cells from a 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.

Cell viability was calculated using the following equation:
Cell Viability = (number of living cells / total number of acquired cells) x100

The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = (75 - c) x Log (b) - (75-a) x Log (d) / a - c

Where:
a was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively.

b) CD86/CD54 Expression Measurement
One experiment (consisting of two independent runs) was needed to drive a prediction. Each independent run was performed on a different day. On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 106 cells per well). The test substance working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours at 37°C, 5% CO2. After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4ºC for 15 minutes. After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes). After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4ºC for 30 minutes. The stained cells were washed three times with an excess of FACS buffer, re- suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL). The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

(D) Data Evaluation
a) Analysis of Results
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test substance-treated cells were calculated according to the following equation:

RFI = [(MFI of test substance-treated cells – MFI of test substance-treated isotype control cells) / (MFI of solvent-treated cells – MFI of solvent-treated isotype control cells)] x100

The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was calculated using the following equation:
Cell Viability = [number of living cells / total number of acquired cells] x100

b) Prediction Model
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered NEGATIVE:
1) The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
2) The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%).

c) Calculation of Effective Concentration (EC) Values
For test substances predicted as POSITIVE, two EC values, the EC150 for CD86 and the EC200 for CD54, are calculated as follows:

EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Adose – Bdose)].

Where:
A concentration is the lowest concentration in µg/mL with RFI >150 (CD86) or 200 (CD54)
B concentration 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).

d) Assay Acceptance Criteria
1) The cell viabilities of medium and solvent control should be higher than 90%
2) In the solvent control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
3) For both medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
4) In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%
5) For the test substance, the cell viability should be more than 50% in at least four tested doses in each run.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: RFI (CD86) %

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC150 could not be calculated

Results 2

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: RFI (CD54) %

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: EC200: 2.3 μg/mL

Other effects / acceptance of results:

The cell viability of the solvent control (medium) was higher than 90% in each independent run. In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%). For the solvent control, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions. For the positive control, RFI values were ≥200% for CD54 in each independent run, and ≥150% for CD86 in Experiment 1. For the positive control, the RFI values for CD86 in Experiment 2 did not however exceed 150%, and therefore no assessment could be made using this marker. As all data for CD54 were valid and a clear positive prediction for the test substance has been obtained via the CD54 marker, it was not considered necessary or relevant to perform additional experimentation for CD86. For the test substance, the cell viability was more than 50% in all tested concentrations in each independent run.

Any other information on results incl. tables

Results

a) Dose Finding Assay

The cell viability results are given in Table 7.1 and 7.2 (Kindly refer the attached material section for the tables). The test substance was completely toxic at all concentrations tested in the initial dose range finding assay (7.8 to 1000 μg/mL), and therefore a second range finding assay was performed with test concentrations ranging from 0.078 to 10 μg/mL. A reduction in viability was observed at the highest three concentrations, resulting in a mean CV75 value for the test substance of 3.5 μg/mL.

 

b) CD86/CD54 Expression Results

Expression measurements were performed in a range from 1.17 to 4.20 μg/mL (0.335 x CV75 to 1.2 x CV75). Geometric mean fluorescence intensity (MFI) and viability results are given inTable 7.3 (Kindly refer the attached background material section for the table). The cell viability was >50% at all concentrations tested in both experiments. The relative fluorescence intensity (RFI) values for the test substance were calculated as follows:

 

Concentration (µg/mL)	RFI (CD86) Exp 1	Exp 2	RFI (CD54) Exp 1	Exp 2
1.17	88	106	120	111
1.41	83	115	127	121
1.69	90	122	154	125
2.03	107	121	195	154
2.43	111	138	228	184
2.92	129	141	431	229
3.50	137	160	541	307
4.20	133	176	933	427
Solvent control (medium)	100	100	100	100
Positive control	337	120	965	304

 

In Experiment 1, the RFI values for CD86 were <150% and the RFI values for CD54 were >200%. The RFI values for CD54 were again >200% in Experiment 2, and therefore the test substance gave a positive prediction in the assay. No RFI values for CD86 in Experiment 1 were greater than 150, therefore the EC150 could not be calculated. The EC200 for CD54 calculated by linear regression of endpoint assay data was 2.3 μg/mL.

Conclusion

The test substance, Quaternary ammonium compounds, N,N,N’-tris(hydroxyethyl)-N,N’- dimethyl-N’-tallow alkyltrimethylenedi-, bis(Me sulfates) (salts) EC # 297-495-0, CAS # 93572-63-5, was considered to be positive in the human Cell Line Activation Test.

Applicant's summary and conclusion

Interpretation of results:

other: positive

Conclusions:

Under the study conditions, the test substance was considered to be positive in the human Cell Line Activation Test.

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance, Quaternary ammonium compounds, N,N,N’-tris(hydroxyethyl)-N,N’-dimethyl-N’-tallow alkyltrimethylenedi-, bis(Me sulfates, according to the OECD Guideline 442E, in compliance with GLP. Human monocytic leukemia cell line, THP-1 (an immortalised human monocytic leukemia cell line, used as a surrogate for dendritic cells), was used in the assay. The test substance was soluble in culture medium (RPMI-1640) at 271.56 mg/mL.The THP cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 10E+6 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 10E+6 cells/mL. Cell density did not exceed 1 x 10E+6cells/mL. Dose finding assays were conducted to determine a concentration showing 75% THP-1 cell survival (CV75) compared to the solvent control. The CV75 value for the test substance was 3.5 μg/mL. Final test concentrations in a range from 1.17 to 4.20 μg/mL (1.17, 1.41, 1.69, 2.03, 2.43, 2.92, 3.50, 4.20 μg/mL) after dilution in medium were used for the expression measurements. Aliquots of 500 µL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 10E+6 cells per well. After a 24-hour treatment, the cells were transferred into sample tubes, collected by centrifugation, washed with FACS buffer and then blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) at 4°C for 15 min. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies at 4°C for 30 min. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry. The cell viability was >50% at all concentrations tested in both experiments. In Experiment 1, the RFI values for CD86 were <150% and the RFI values for CD54 were >200%. The RFI values for CD54 were again >200% in Experiment 2, and therefore the test substance gave a positive prediction in the assay. No RFI values for CD86 in Experiment 1 were greater than 150%, therefore the EC150 could not be calculated. The EC200 for CD54 calculated by linear regression of endpoint assay data was 2.3 μg/mL. All acceptance criteria of the h-CLAT assay parameters were met in each experiment, with the exception that for the positive control (DNCB), the RFI values for CD86 in Experiment 2 did not exceed 150%, and therefore no assessment could be made using this marker. All data for CD54 were valid and a clear positive prediction for the test substance has been obtained via the CD54 marker. Under the study conditions, the test substance was considered to be positive in the human Cell Line Activation Test (Hartmann, 2018).