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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Not definable, substance is an UVCB

Molecular formula:

Not definable, substance is an UVCB

IUPAC Name:

Not definable, substance is an UVCB

Test material form:

liquid: viscous

Details on test material:

Identification: Quaternium-70
Appearance: Amber gel
Batch: 0002000146
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until 02 April 2018 (retest date)

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

In vitro test system

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.

Results and discussion

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).

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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

Results 5

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

Results 6

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).

Applicant's summary and conclusion

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.