Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

23 February - 19 April 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:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)

EC Number:

916-222-4

Molecular formula:

C51H99N2O6.CH3O4S

IUPAC Name:

Reaction mass of N,N'-ethylenebis[N-methyl-2-[(1-oxo-9-octadecenyl)oxy]-N-[2-[(1-oxo-9-octadecenyl)oxy]propyl]propylammonium] dimethyl disulphate and [2-[bis(2-hydroxypropyl)amino]ethyl]bis(2-hydroxypropyl)(methyl)ammonium methyl sulphate, dioleate (ester)

additive 1

Reference substance name:

Propane-1,2-diol

EC Number:

200-338-0

EC Name:

Propane-1,2-diol

Cas Number:

57-55-6

Molecular formula:

C3H8O2

IUPAC Name:

propylene glycol

Specific details on test material used for the study:

Test Item : AGENT 5088-127A
Chemical Name : Reaction mass of [2-[bis(2-hydroxypropyl) amino] ethyl] bis(2- hydroxypropyl)(methyl)ammonium methylsulphate, dioleate (ester) [&]
N N’-ethylenebis[N-methyl-2-[(1-oxo-9-octadecnyl)oxy]-N-[2-[{1-oxo-9-octadecenyl}oxy]propyl]propylammonium] dimethyldisulphide
EC No. : 916-222-4
Purity as per COA : 100%
Physical Appearance : Solid
Batch No. : 5124-143-001
Manufactured Date : 20.12.2017
Expiry date : 20.12.2020

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The ARE-Nrf2 Luciferase test method using KeratinoSens™ is an in vitro method which quantifies luciferase gene induction following 48 h treatment with test chemicals. Contact sensitizers which activate Nrf2-ARE regulatory pathways in cells, up-regulate the luciferase gene induction in G16210_KeratinoSens Page 13/37 Copy No. 2/2 KeratinoSens™ which are stably transfected with Luciferase gene under transcriptional control of promoter fused with ARE element. Luciferase gene induction is measured in the cell lysates by luminescence detection using a well-established light producing luciferase substrate. Fold induction of Luciferase activity is then calculated and used in a prediction model which allows assigning the test chemical to discriminate between sensitizers and non-sensitizers.

Commercially available DMEM was supplemented with the following
• Fetal Bovine Serum (FBS) to a final concentration of 9.1% in
medium.
• Geneticin final concentration 500 μg/mL.
• Penicillin 100 IU/mL
• Streptomycin 100 μg/mL

Treamtment Medium
Commercially available DMEM was supplemented with FBS to a final concentration of 1% in the medium. The medium was stored at 2 - 8°C until use.

Positive Control Solution
A volume of 5.3 μL of Cinnamaldehyde was mixed with 194.7 μL of DMSO to a stock concentration of 200 mM. A final concentration of 6.4 mM was prepared by further diluting 32 μL of the 200 mM stock solution in 968 μl of DMSO.

Luciferase Substrate
Each bottle containing Lyophilized luciferase powder was mixed with 10 mL of luciferase assay solution and mixed well. Preparation was done before use in the assay.

Passive Lysis Buffer (1x)
One part of passive lysis buffer (5x) was mixed to four parts of milli-Q water.

Solubility Test
Solubility check was done for the highest concentration 40 mg/mL was done by mixing 48 mg of the test item with 1.2 mL of DMSO and voxtexed.

Test Solution Preparation
Test item solution was prepared on 4 days i, e on 19th, 21st, 23rd and 24th March 2018. On all the four days test item solution at a final concentration of 40 mg/mL was prepared in DMSO. The test item solution at 40 mg/mL was prepared

Results and discussion

Positive control results:

Experiment 1: The positive control Cinnamaldehyde caused a dose related induction of the luciferase activity.The EC1.5 was 15.41 μM.
Experiment 2: The positive control Cinnamaldehyde caused a dose related induction of the luciferase activity.The EC1.5 was 10.78 μM.
Experiment 3: The positive control Cinnamaldehyde caused a dose related induction of the luciferase activity.The EC1.5 was 16.07 μM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.83

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

2.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 3

Parameter:

other: Imax

Value:

1.46

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5

Value:

6.25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 5

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5

Value:

25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 6

Key result

Run / experiment:

other: 3

Parameter:

other: EC1.5

Value:

12.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

Any other information on results incl. tables

Maximal Gene Induction (Imax)

The maximal gene induction (Imax) shows the dynamic range of gene induction by the test item and positive control. As the prediction model rates any chemical positive with an Imax, which is statistically significant above solvent control and above the threshold of 1.5, the absolute value of the Imax is not very important for the prediction.

The maximum gene induction Imax was calculated as below

Test Item       Imax                                                 Imax

Rep 1       Rep 2       Rep 3              (avg)

S047-01       1.83*       2.25*       1.46                  1.85

* Imax at the cytotoxic concentration

EC 1.5 Concentration values

For each experiment, EC1.5 value is calculated automatically in the summary sheet, this already indicates that the gene induction is statistically significant at the corresponding concentration according to a T-test. EC 1.5 concentration values for the test item was tabulated below.

Test Item              Imax                                          Concentration (μg/mL)                     Cytotoxicity

Rep 1        Rep 2       Rep 3     Rep 1       Rep 2       Rep 3            Rep 1       Rep 2       Rep 3

S047 - 01              1.83       2.25             1.46      6.25       25              12.5              Cytotox   Cytotox        Noncytotox

There was statistically siginificant induction in two of the three repetitions but at the cytotoxic concentrations. No significant induction at the noncytotoxic concentration was observed.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on these results it is concluded that the test item AGENT 5088-127A is a non-sensitizer in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSensTM assay).

Executive summary:

The potential of the test item AGENT 5088-127A to cause skin sensitisation was evaluated in ARE-Nrf2 Luciferase Test (KeratinoSensTM Assay).

KeratinoSensTM assay is an in vitro cell based assay in which the keratinoSensTM cells were exposed to the test item for 48 hours and subsequently measured for luciferase activity using a luminometer. The assay was run in 96 well plates, and test item was tested at 12 concentrations ranging form 0.195 μg/mL to 400 μg/mL along with the vehicle control, control blank and the reference compound cinnamic aldehyde ranging from 4 μM to 64 μM. Each plate was tested in parallel in triplicate for analysis of luciferase induction and one additional replicate plate was used for cytotoxicity assessment. The full test in triplicate analysis was independently conducted three times. The cells were exposed to the test concentrations for 48 ± 2 hours at 37 °C in a carbondioxide incubator. After the exposure time luciferase substrate was added and the luminescence activity at each concentration was integrated for 1500 ms (1.5 Seconds). The viability assay plates containing the cells were treated with treatment medium containing MTT solution. After overnight incubation, the absorbance was read at 600 nm using a photometer. The luminescence reading and the absorbance reading obtained were analyzed in an excel sheet provided by Givaduan. The test item AGENT 5088-127A did not show any statistically significant induction above the threshold of 1.5 or 50 % at any of the non-cytotoxic concentration over the solvent control. Under the same circumstances the positive control cinnamaldehyde showed statistically significant induction over the solvent control confirming the sensitivity of the assay.

Based on these results it is concluded that the test item AGENT 5088-127A is a non-sensitizer in the in vitro skin sensitization assay by ARE-Nrf2 Luciferase test method (KeratinoSens assay).