nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 February 2017-17 February 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

non-animal testing is the default requirement. In vivo methods can only be used if the in chemico or in vitro test methods are not adequate for the substance or cannot be used for classification and risk assessment.

Test material

Test material information

Constituent 1

Reference substance name:

nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

EC Number:

946-436-3

Molecular formula:

UVCB

IUPAC Name:

nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide

impurity 1

Reference substance name:

(p-chlorophenoxy)isobutyroyl chloride

EC Number:

255-286-1

EC Name:

(p-chlorophenoxy)isobutyroyl chloride

Cas Number:

41267-93-0

Molecular formula:

C10H10Cl2O2

IUPAC Name:

2-(4-chlorophenoxy)-2-methylpropanoyl chloride

impurity 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

solid

Details on test material:

Identification: Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide
Appearance: Light yellow lumps
Test item storage: At room temperature; Store in closed container

Specific details on test material used for the study:

UVCB

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Controls:
negative control DMSO (vehicle)
positive control: Ethylene dimethacrylate glycol
On each plate three blank wells were tested (no cells and no treatment).

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). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages. 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.

Test item preparation
No correction was made for the composition/purity of the test item. The test item was dissolved in DMSO to a final concentration of 40 mg/mL. The compound formed a clear colourless solution at 40 mg/mL. The test item was vortexed and sonicated (10 minutes with a maximum temperature of 25.0°C). From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 400, 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391 and 0.195 μg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitate was observed at the start or end of the treatment period (study plan deviation 1).
In experiment 2 was the test item dissolved in DMSO at 0.625 mg/mL. The test item was vortexed and sonicated (11 minutes with a maximum temperature of 28.0°C). From the stock 11 spike solutions in DMSO were prepared (1.3-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%).
These solutions were diluted 4-fold in the assay resulting in final test concentrations of 6.25, 4.81, 3.70, 2.84, 2.19, 1.68, 1.29, 1.00, 0.766, 0.589, 0.453 and 0.439 μg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. No precipitate was observed at the start or end of the treatment period (study plan deviation 1).

Media
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/mL).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Environmental conditions
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 72 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.0°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 and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

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 substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0oC in the presence of 5% CO2. In total 2 experiments were performed.

Luciferase activity measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega 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 luminometer to assess the quantity of luciferase (integration time one second).

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) 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 to each well. After shaking, the absorption was measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis
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.
Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: Fold induction =( Lsample-Lblank) / (Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well Lblank is the luminescence reading in the blank well containing no cells and no treatment Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb-Ca) x [(1.5-Ia)/ (Ib-Ia)] + Ca
where:
Ca is the lowest concentration in μM with > 1.5 fold induction Cb is the highest concentration in μM with < 1.5 fold induction Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction
(mean of three replicate wells) Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells).

Viability is calculated by Equation 3:
Equation 3:Viability = [(Vsample - Vblank) / (Vsolvent-vblank)] x 100
where:
Vsample is the MTT-absorbance reading in the test chemical well Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05).
ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data (study plan deviation 2). The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

Data interpretation
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 μM or 200 μg/mL should be considered as inconclusive.

Acceptance criteria
-The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 μM).
-The EC1.5 should be between 5 and 125 μM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
-Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Results and discussion

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.72 and the EC1.5 16.3 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 4.05 and the EC1.5 20.4 μM.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Experiment 1 (0.195-400 μg/mL; 2-fold dilution series)

Parameter:

other: Imax

Value:

1.78

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: A luminesce activity induction compared to the vehicle control was only observed at 1.56 μg/mL with an Imax of 1.78. Since the test item was toxic at this concentration (49.5% viability), no EC1.5 was calculated.

Remarks:

The dilution series for experiment 2 were adapted due to the toxicity observed and to carefully investigate the effect observed at 1.56 μg/mL.

Results 2

Key result

Run / experiment:

other: Experiment 2 (0.349-6.25 μg/mL; 1.3-fold dilution series)

Parameter:

other: Imax

Value:

1.21

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No luminesce activity induction compared to the vehicle control was observed at any of the test concentrations. The Imax was 1.21 and therefore no EC1.5 could be calculated.

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
Both experiments 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 (16.3 μM and 20.4 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.72-fold and 4.05-fold in experiment 1 and 2, respectively).
-Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.2% and 9.8% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item is classified as inconclusive in the KeratinoSensTM assay since only negative results (<1.5-fold induction, no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) were observed at test concentrations with a viability of >70% and these test concentrations were <1000 μM.

Executive summary:

The substance was tested for skin sensitisation properties in the KeratinoSens™ assay according to OECD TG 442D.

The test item was dissolved in dimethyl sulfoxide at 40 mg/ml. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.195 – 400 μg/ml (2-fold dilution series). Due to toxicity, the test concentrations used for the second experiment were 0.349 – 6.25 μg/ml.

Two independent experiments were performed.

Both experiments 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 (16.3 μM and 20.4 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.72-fold and 4.05-fold in experiment 1 and 2, respectively).

-Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.2% and 9.8% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed toxicity with an IC30 of 0.848 μg/mL and an IC50 of 1.54 μg/mL in experiment 1, and an IC30 of 5.26 μg/mL and IC50 value of 5.55 μg/ml in experiment 2.In experiment 1, a luminesce activity induction compared to the vehicle control was only observed at 1.56 μg/mL with an Imax of 1.78. Since the test item was toxic at this concentration (49.5% viability), the induction was not relevant and no EC1.5 was calculated.

The dilution series for experiment 2 was adapted to a range of 0.349 – 6.25 μg/mL (1.3-fold dilution series) due to the toxicity of the compound and the induction observed at 1.56 μg/mL. No induction of the luciferase activity >1.5-fold compared to the vehicle control was measured at any of the test concentrations in experiment 2. Therefore no EC1.5 value was calculated. The maximum luciferase activity induction (Imax) was 1.21-fold in experiment 2.

The test item is classified as inconclusive in the KeratinoSensTM assay since only negative results (<1.5-fold induction, no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) were observed at test concentrations with a viability of >70% and these test concentrations were <1000 μM.

Finally, it is concluded that the KeratinoSensTM assay is valid and that Nitrate amine salt of N-[3-dimethylaminopropyl]- C14-C20 amides, saturated, reaction products with ethylene oxide is classified as inconclusive under the experimental conditions described in this report.