Registration Dossier

Administrative data

Description of key information

The test substance was determined to be positive in human Cell Line Activation (h-CLAT) test and negative in ARE-Nrf2 Luciferase test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to
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
Details on 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.
Key result
Parameter:
other: RFI (CD86) %
Run / experiment:
Experiment 1
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: EC150 could not be calculated
Key result
Parameter:
other: RFI (CD54) %
Run / experiment:
Experiment 1
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.

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.

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).

Endpoint:
skin sensitisation: in vitro
Remarks:
keratinosens study
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From 17 April 2018 to 18 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
These study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In vitro skin sensitisation integrated testing strategy approach
Details on study design:
(A) Test and reference substances
Dimethyl sulfoxide (DMSO) supplied by Sigma Aldrich Chemical Co. Ltd. was used as the negative control. Cinnamic aldehyde (CAS No. 14371-10-9), supplied by Sigma Aldrich Chemical Co. Ltd. was used as the positive control.

(a) Test Substance Formulation
Test formulations were prepared using purified water. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 200 mM. Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations (0.098, 0.195, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50,100 and 200 mM). Test formulations prepared using water were sterilised by filtration. The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 0.98 to 2000 µM (see Section 9 for details of deviations to Protocol). Formulations were prepared shortly before testing.

(b) Negative and Positive Control Substance Formulation
The negative control was diluted into culture medium containing serum so that the final concentration was 1%. An aqueous solvent was used, therefore DMSO was added to the treatment solutions at 1% (final concentration). The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 4 to 64 µM.

(B) Test system
a) Specifications
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland.

b) Identification
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.

c) Preparation of Cultures
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.

(C) Methods
(a) Treatment Plate Preparation
The cells were 80 - 90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated at 37±1°C, 5% (v/v) CO2, for 24±1 hours. For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

(b) Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test substance and control formulations were added. One well per plate was left empty (no cells and no treatment) to assess background values. Each plate was sealed and incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours. For each test substance and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of at least two independent repetitions each containing three replicates of each concentration. It should be noted that following the initial Experiment 2 treatments, the average coefficient of variation of the luminescence reading for the negative control (DMSO) was above 20%, therefore the data was rejected (data not reported) and the experiment was repeated. Discordant results were obtained between the two repetitions, therefore a third repetition containing three replicates was performed. Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from the same passage (Experiments 1 and 3) or different passages (Experiment 2).

(c) Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e.

(d) Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Protocol deviations
1) Treatment Plate Preparation
In Experiment 3 the cultures were incubated for 25 hours and 13 minutes prior to treatment. This deviation from Protocol did not affect the integrity or outcome of the study as all assay acceptance criteria were met in this experiment.

2) Test Substance Formulation
The Protocol specifies that the master concentrations are diluted 1 in 25 and then 1 in 4 in media. In Experiment 3, there was as an error in this final step for the highest two concentrations (500 and 1000 µM) for the cytotoxicity assessment. It was considered that the full aliquot of test substance formulation was not transferred to the well of the culture plate. It was considered that this deviation from Protocol did not affect the integrity or outcome of the study as cell viability was <70% at concentrations of 3.9 µM and above.

These study deviations neither affected the overall interpretation of study findings nor compromised the integrity of the study.
Key result
Parameter:
other: EC1.5 (μM)
Run / experiment:
Experiment 1
Value:
ca. 1.14
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
ca. 5.25
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Cell viability (%)
Run / experiment:
Experiment 1
Value:
ca. 87.17
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Dose response
Run / experiment:
Experiment 1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Yes: Dose response
Other effects / acceptance of results:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 32 µM in Experiments 1 and 3 and at a concentration of 32 µM in Experiment 2. The EC1.5 values for the positive control were 9.00, 20.53 and 12.90 µM in Experiments 1, 2 and 3, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.61, 3.12 and 4.91 in Experiments 1, 2 and 3, respectively. The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 6.15%, 17.95% and 11.51% in Experiments 1, 2 and 3, respectively. All assay acceptance criteria were met, with the exception that the average induction for the positive control at 64 μM in Experiment 1 was 10.61. The assay was considered valid as there was a clear dose response with the positive control.

Results

 

1) Calculation of Imax and EC1.5 Values

Luminescence readings and fold increases are given in Table 10.1 to Table 10.3 (Kindly refer the attached background material section). The overall maximal fold increases (Imax) were 5.25, 0.89 and 0.5 for Experiments 1, 2 and 3, respectively. The EC1.5 value was 1.14 for Experiment 1. There were no EC1.5 values for Experiments 2 or 3 as there were no statistically significant increases in induction.

 

2) Viability

MTT-absorbance readings are given in Table 10.4 (Kindly refer attached background material section).

The cell viability at the EC1.5 determining concentration was 87.17% in Experiment 1. The cell viability measurement was not applicable for Experiments 2 and 3 as there were no EC1.5 determining concentrations. The IC50values were 3.00, 8.27 and 3.38 µM in Experiments 1, 2 and 3, respectively. The IC30 values were 2.43, 5.08 and 1.78 µM in Experiments 1, 2 and 3, respectively.

Assay Acceptance

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 32 µM in Experiments 1 and 3 and at a concentration of 32 µM in Experiment 2. The EC1.5 values for the positive control were 9.00, 20.53 and 12.90 µM in Experiments 1, 2 and 3, respectively. The average induction in the two replicates for the positive control at 64 µM were 10.61, 3.12 and 4.91 in Experiments 1, 2 and 3, respectively. The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 6.15%, 17.95% and 11.51% in Experiments 1, 2 and 3, respectively. All assay acceptance criteria were met, with the exception that the average induction for the positive control at 64 μM in Experiment 1 was 10.61. The assay was considered valid as there was a clear dose response with the positive control.

Summary of results

Criteria

Experiment 1

Experiment 2

Experiment 3

Imax

5.25

0.89

0.5

Cell Viability

87.17%

Not calculated

Not calculated

EC1.5

1.14 µM

Not calculated

Not calculated

Dose Response

Yes

No

No


Conclusion

All four conditions required for a positive prediction were met in Experiment 1, however, none the four conditions required for a positive prediction were met in Experiments 2 and 3, therefore 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 negative in the ARE-Nrf2 Luciferase Test.


Conclusions:
Under the study conditions, the test substance was considered to be negative for skin sensitisation in the ARE-Nrf2 Luciferase 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 442D, in compliance with GLP. The test substance was dissolved in purified water to the final stock concentration (200 mM). Serial dilutions were then made using purified water to obtain 12 master concentrations of the test substance (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM. Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 h. After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 h at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e. After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 min at 25±2°C, loaded into the luminescence plate reader and read. Imax values in experiment 1, 2 and 3 were determined to be 5.25, 0.89 and 0.5, respectively. Cell viability was determined to be 87.17% in experiment 1, whereas it could not be calculated in experiment 2 and 3. EC1.5 was determined to be 1.14 µM in experiment 1, whereas it could not be calculated in experiment 2 and 3. All assay acceptance criteria were met, with the exception that the average induction for the positive control at 64 μM in Experiment 1 was 10.61. The assay was considered valid as there was a clear dose response with the positive control. All four conditions required for a positive prediction were met in Experiment 1, however, none of the four conditions required for a positive prediction were met in Experiments 2 and 3. Under the study conditions, the test substance was considered to be negative for skin sensitisation in the ARE-Nrf2 Luciferase Test (Dreher, 2018).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Study 1

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 442D, in compliance with GLP. The test substance was dissolved in purified water to the final stock concentration (200 mM). Serial dilutions were then made using purified water to obtain 12 master concentrations of the test substance (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations ranged from 0.98 to 2000 µM. Aliquots of 50 µL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 h. After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 h at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e. After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 min at 25±2°C, loaded into the luminescence plate reader and read. Imax values in experiment 1, 2 and 3 were determined to be 5.25, 0.89 and 0.5, respectively. Cell viability was determined to be 87.17% in experiment 1, whereas it could not be calculated in experiment 2 and 3. EC1.5 was determined to be 1.14 µM in experiment 1, whereas it could not be calculated in experiment 2 and 3. All assay acceptance criteria were met, with the exception that the average induction for the positive control at 64 μM in Experiment 1 was 10.61. The assay was considered valid as there was a clear dose response with the positive control. All four conditions required for a positive prediction were met in Experiment 1, however, none of the four conditions required for a positive prediction were met in Experiments 2 and 3. Under the study conditions, the test substance was considered to be negative for skin sensitisation in the ARE-Nrf2 Luciferase Test (Dreher, 2018).

Study 2

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).

Justification for classification or non-classification

Overall, based on the available evidence fromin silico(OECD QSAR Tool box v.4.1 profiling and prediction from DEREK NEXUS v6.0.1) together with in vitro studies (KeratinoSensTMassays and h-CLAT), the test substance was overall concluded to be non-sensitising to skin.​

Therefore, based on the available weight of evidence from in silico and in vitro studies, the test substance does not warrant classification for skin sensitisation according to EU CLP criteria (Regulation 1272/2008/EC).