Phosphoric acid, mono- and di-C12-18-(even numbered)-alkyl esters, sodium salts

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From May 02, 2017 to September 29, 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:

February, 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Test material

Test material information

Constituent 1

Reference substance name:

Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts

Molecular formula:

C12H25O4P1Na2 (representative mono ester - mono- C12 PSE, Na+); C24H50O4P1Na1 (representative di ester - di- C12 PSE, Na+);

IUPAC Name:

Phosphoric acid, mono- and di-C 12 -18-(even numbered)-alkyl esters, sodium salts

Test material form:

solid

In vitro test system

Details on the study design:

PRINCIPLE OF THE TEST:
The test method makes use of an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an Antioxidant response element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 (transcription factor involved in the antioxidant response pathway) dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Test chemicals are considered positive if they induce a statistically significant induction of the luciferase activity above a given threshold (i.e. > 1.5 fold or 50% increase), below a defined concentration which does not significantly affect cell viability (i.e. below 1000 µM and at a concentration at which the cellular viability is above 70%).

EXPERIMENTAL MATERIALS
- TEST SYSTEM SOURCE AND MAINTENANCE:
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 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.

SUBCULTURING
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 from the frozen stock.

CELL CULTURE MEDIUM:
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Maintenance medium: Dulbecco’s minimal 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 supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.

TEST SUBSTANCE, CONTROLS AND WORKING SOLUTIONS:
- A correction factor was used to correct for the purity of of the test substance
- Vehicle: The vehicle of the Test substance was ethanol (Extra pure, Merck, Darmstadt, Germany). The vehicle of the positive control was dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).
- Negative Control: The negative control is the vehicle of the test substance.
- Positive Control (RS582): Ethylene dimethacrylate glycol (Batch# SHBG0572V)
- Solvent control: The solvent control of the positive control was 1% DMSO in exposure medium. Eighteen wells were tested per plate. The solvent control of the test substance was 0.25% ethanol in exposure medium.
- On each plate three blank wells were tested (no cells and no treatment).

TEST SUBSTANCE SOLUBILITY TEST: A solubility test was performed. No solution or homogeneous suspension could be prepared in DMSO and milli-Q at 40 and 20 mg/mL. Therefore ethanol was tested as solvent. A concentration of 50 µg/mL was determined to be the limit of solubility. a stock of 20 mg/mL was prepared which was 400-fold diluted in the assay resulting in a top concentration in the assay of 50 µg/mL

DOSE LEVELS:
In the main experiments the stock solution of test substance followed series with ethanol and exposure medium to get following test concentrations in Experiment 1 and 2: 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.098, 0.049 and 0.024 µg/mL (final concentration ethanol of 0.25%).

NUMBER OF RUN: All concentrations of the test substance and positive controls were tested in triplicate.

EXPERIMENTAL DESIGN
TREATMENT OF CELLS: 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 cells were incubated overnight in the incubator. The passage number used was 20 in Experiment 1 and 22 in Experiment 2. 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 substance 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 h at 37±1.0ºC in the presence of 5% CO2.

Initially, experiment 1 did not pass all the acceptability criteria and therefore this part of the study was repeated.

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 two seconds).

CELL VIABILITY ASSAY: medium was replaced after the 48 h 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 h 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 at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

- ENVIRONMENTAL CONDITION: Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations were not considered to affect the study integrity.

Results and discussion

Positive control results:

The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity.
Experiment 1: The Imax was 2.40 and the EC1.5 85 µM.
Experiment 2: The Imax was 3.08 and the EC1.5 38 µM.
Both Experiment passed the acceptance criteria:

In vitro / in chemico

Results

Key result

Run / experiment:

other: Experiment 1 and 2

Parameter:

other: Imax value (overall maximal fold luciferase activity induction)

Remarks:

The maximum luciferase activity

Value:

0.87

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments (Imax <1.5)

Other effects / acceptance of results:

1. 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.
2. The EC1.5 of the positive control was between 5 and 125 µM (85 µM and 38 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.40-fold and 3.08-fold in experiment 1 and 2, respectively).
3. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.3% and 6.3% in experiment 1 and 2, respectively).
4. Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control Ethanol was below 20% (7.2% and 4.3% in experiment 1 and 2, respectively).
5. Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

No precipitation was observed at the start and end of the incubation period in the 96-well plates in either of Experiment 1 or 2

Experiment 1

·          The test substance showed toxicity. The calculated IC₃₀ was 0.30 µg/mL and the calculated IC₅₀ was 1.04 µg/mL.  

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The I_max was 0.87 and therefore no EC_1.5 could be calculated. 

 

Experiment 2

·          The test substance showed toxicity. The calculated IC₃₀ was 1.54 µg/mL and the calculated IC₅₀ was 2.87 µg/mL. 

·          No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The I_max was 1.37 and therefore no EC_1.5 could be calculated.  

The test substance showed toxicity (IC₃₀ values of 0.30 µg/mL and 1.54 µg/mL and IC₅₀ values of 1.04 µg/mL and 2.87 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 0.87-fold and 1.37-fold in experiment 1 and 2 respectively. The test substance is classified as inconclusive in the KeratinoSens^TM assay since negative results (<1.5-fold induction) were observed at test concentrations < 200 µg/mL.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under study conditions, the test substance was determined to be inconclusive for skin sensitisation potential

Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance using the ARE-Nrf2 Luciferase test method according to the OECD Guideline 442D, in compliance with GLP. Two independent experiments were performed. In both experiments, cells were incubated with the test substance at 12 different concentrations in a range of 0.024 – 50 µM. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control to determine test substance concentration needed for a statistically significant induction of luciferase activity above the threshold (i.e. EC1.5 value). In addition, the viability was assessed with an MTT assay. The EC1.5 value observed at test concentrations with a cell viability of >70% compared to the vehicle control was considered positive for skin sensitisation. The test substance showed toxicity (IC30 values of 0.30 µg/mL and 1.54 µg/mL and IC50 values of 1.04 µg/mL and 2.87 µg/mL in experiment 1 and 2, respectively). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (IMAX) was 0.87 fold and 1.37 fold in experiment 1 and 2 respectively. The test substance was classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5 -fold luciferase induction) were observed at all test concentrations. Under study conditions, the results were determined to be inconclusive (Westerink, 2017).