Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-21 - 2018-04-25

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

EC Number:

942-252-2

IUPAC Name:

Reaction products of stearic acid with 2-aminoethanol, maleic anhydride and sodium sulphite

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

In vitro test system

Details on the study design:

The test item was first tested in two independent runs using cells from a different passage number. A third run was performed since non-analyzable results were obtained in the first run due to the high cytotoxicity of the test item. The plates were processed as described below in the § Method.

- Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium). Magnetic stirring for 10 minutes was used in order to improve the solubility of the test item.

- Method for a run of KeratinoSens assay
* Cell seeding for testing
. Cells were grown using general culture procedures up to 80-90% confluence,
. the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
. cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 µL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
. after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.
* Treatment
. After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 µL of treatment medium,
. from the Master plate 4x, a volume of 50 µL was added to each well of the three white assay plates and 50 µL to the transparent plate for the cytotoxicity evaluation,
. all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
. the plates were then incubated for 48 (± 2) hours at 37°C, 5% CO2, 90% humidity.
* Endpoint measurements
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate: After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.
Luminescence flash signal to evaluate induction signal - white plates: After incubation, the supernatants from the white assay plates were discarded, the cells were washed once with D-PBS, a volume of 20 µL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking, the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
50 µL of the luciferase substrate was added to each well,
1 second after this addition, the luciferase signal was integrated for 2 seconds.
Absorbance signal to evaluate the cytotoxicity - transparent plate: For the cell viability assay plate, the medium was replaced by 200 µL of treatment medium, a volume of 27 µL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate, the plates were covered with a sealing membrane and returned at 37°C in the incubator in humidified atmosphere for 4 hours (± 10 minutes), at the end of the incubation period, the medium was removed and a volume of 200 µL of a 10% SDS solution was added to each well, the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells, after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.

Acceptance criteria :
Each run was considered valid if the following criteria were met:
. the positive control results should be positive, thus the gene induction should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations,
. the average EC1.5 value for the positive control should be within two standard deviations of the historical mean. In addition, the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8. If the latter criterion was not fulfilled, the dose-response of Cinnamic Aldehyde was carefully checked, and the run was accepted if there was a clear dose response with increasing luciferase activity at increasing concentrations for the positive control,
. the average coefficient of variation of the luminescence reading in the negative control wells of the triplicate plates should be < 20%.

Evaluation criteria of the test item:
The results of each run are analyzed individually and if the test item is classified as positive in two runs, the final outcome is considered positive. If the test item is classified as negative in two runs, the final outcome is negative. In case, the first two runs were not concordant, a third run was performed and the final outcome was that of the two concordant runs.

The test item is considered as positive if the following four conditions are all met in two of two or in two of three runs, otherwise the KeratinoSens prediction is considered as negative:
. the Imax is > 1.5-fold and statistically significantly different as compared to the negative control (as determined by a two-tailed, unpaired Student’s T-test),
. at the lowest concentration with a gene induction > 1.5-fold (i.e. at the EC1.5 determining value), the cell viability is > 70%,
. the EC1.5 value is < 1000 µM (or < 200 µg/mL for test item without MW),
. there is an apparent overall dose-response for luciferase induction (or a reproducible biphasic response).

Results and discussion

Positive control results:

All acceptance criteria were fulfilled for the positive control in each run.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: First run

Parameter:

other: IC50

Value:

2.24

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Since none of the tested concentrations achieved a cell viability > 70%, the results obtained in this first run were considered as not analyzable.

Results 2

Key result

Run / experiment:

other: Second and third run

Parameter:

other: Imax

Value:

1.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assay when tested up to 2.5 μM, highest concentration limited by the high cytotoxicity of the test item.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no data

DEMONSTRATION OF TECHNICAL PROFICIENCY: no data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Any other information on results incl. tables

SOLUBILITY TEST

In the solubility test, the test item was found not soluble in DMSO at 200µM,whereas it was found soluble in water for injections at 200µM, following a 10-minute of magnetic stirring step. Therefore, water for injections was the vehicle selected for the preparation of the test item formulations.

KERATINOSENS RUN

 First run

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          slight to strongprecipitate were observed in test item-treated wells atconcentrations≥ 62.5 µMat the end of the 48-hour treatment period,

.          a high decrease in cell viability (i.e.cell viability < 70%) was noted from the lowest concentration of 0.98 µM. The corresponding IC₃₀was < 0.98 µM and the IC₅₀was calculated to be 2.24 µM.

 

Since none of the tested concentrations achieved a cell viability > 70%, the results obtained in this first run were considered as not analyzable.

Second run

Due to the high cytotoxicity observed in the first run, a lower range of concentrations was used in the second run in order to determine if a potential gene induction occurred at non cytotoxic concentration. This run was therefore performedusing the following concentrations:0.001, 0.002, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25 and 2.50 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, at any tested concentrations,

.          a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations≥0.63 µM, andthe corresponding IC₃₀wascalculated to be 0.40 µM.No IC₅₀was calculated since the cell viability was > 50% in this run,

.          nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the I_maxvalue was<1.5 (i.e.1.20).

 

The evaluation criteria for a negative response were met in this second run.

 Third run

Due to results not analyzable obtained in the first run and since two concordant runs are necessary to evaluate the potential of the test itemtoactivate the Nrf2 transcription factor, a third run was performed in the same experimental conditions and using the same concentrations as those tested in the second run.

At these tested concentrations:

.          noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, at any tested concentrations,

.          a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations≥1.25 µM, andthe corresponding IC₃₀was 0.62µM. Again, no IC₅₀was calculated since the cell viability was > 50% in this third run,

.          nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the I_maxvalue was again<1.5 (i.e. 1.00).

 

The evaluation criteria for a negative response were also met in this third run.

Thegeometric mean IC₃₀of the two analyzable runs was calculated to be 0.50 µM.

 

The evaluation criteria for a negative response were met in both analyzable runs, the final outcome is therefore negative.This negative result can be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitization potential.

 

 

Applicant's summary and conclusion

Interpretation of results:

other: negative

Conclusions:

The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assay when tested up to 2.5 µM, highest concentration limited by the high cytotoxicity of the test item. Therefore, under the experimental conditions of this study, the test item SOPROMINE 1686 was considered to have no potential to activate the Nrf2 transcription factor.

Executive summary:

The objective of this study was to evaluate the potential of the test item, SOPROMINE 1686, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment. The design of this study was based on the guideline No. 442D and the study was performedin compliance with Citoxlab France standard operating procedures and with the OECD Principles of Good Laboratory Practice. The KeratinoSens is an in vitro test which quantifies luciferase gene induction in an immortalized adherent human keratinocyte cell line (HaCAT cell line) as a measure of the activation of the Keap1-Nrf2-ARE pathway.

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37°C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37°C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence.In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Three independent runs were performed as part of the study.

All acceptance criteria were fulfilled for the positive and negative controls in each run; all runs were therefore considered as validated.

 

First run

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          slight to strongprecipitate were observed in test item-treated wells atconcentrations≥62.5 µMat the end of the 48-hour treatment period,

.          a high decrease in cell viability (i.e.cell viability < 70%) was noted from the lowest concentration of 0.98 µM. The corresponding IC₃₀was < 0.98 µM and the IC₅₀was calculated to be 2.24 µM.

 

Since none of the tested concentrations achieved a cell viability > 70%, the results obtained in this first run were considered as not analyzable and two additional runs were necessaryto evaluate the potential of the test item toactivate the Nrf2 transcription factor.

 

Second and third run

Due to the high cytotoxicity observed in the first run, a lower range of concentrations was used in the second and third runs in order to determine if a potential gene induction occurred at non cytotoxic concentration. These runs were therefore performedusing the following concentrations:0.001, 0.002, 0.005, 0.01, 0.02, 0.04, 0.08, 0.16, 0.31, 0.63, 1.25 and 2.50 µM in culture medium containing 1% DMSO and 1% water for injections.

At these tested concentrations:

.          noprecipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, at any tested concentrations and in either run,

.          a decrease in cell viability (i.e.cell viability < 70%) was noted at concentrations≥0.63 µM in the second run and≥1.25 µM in the third run, andthe corresponding IC₃₀values werecalculated to be 0.40 µM and0.62µM, in the second and third runs, respectively.No IC₅₀was calculated since the cell viability was > 50% in these runs,

.          nostatistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations in any of the runs. Moreover, the I_maxvalue was<1.5 (i.e.1.20 and 1.00 in the second and third runs, respectively).

 

The evaluation criteria for a negative response were met in these two analyzable runs and thegeometric mean IC₃₀was calculated to be 0.50 µM.Therefore, the final outcome is negative.This negative result can be used to support the discrimination between skin sensitizers and non‑sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitization potential.

The test item SOPROMINE 1686 was considered as negative in the KeratinoSens assaywhen tested up to 2.5 µM, highest concentration limited by the high cytotoxicity of the test item. Therefore, under the experimental conditions of this study, the test item SOPROMINE 1686 was considered to have no potential to activate the Nrf2 transcription factor.