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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Experimental start date: 05 April 2018. Experimental completion date: 27 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:
equivalent or similar to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: aqueous solution
Details on test material:
Identification: FSM-005W
CAS Number: 2601-33-4
Batch: 170321
Purity: 19.9% aqueous solution of FSM-005W (solid)
Physical state/Appearance: clear colorless liquid
Expiry Date: 20 November 2018

Specific details on test material used for the study:
Name: FSM-005W
Synonyms: Ink jet additive J-19
1-Tetradecanaminium, N-(carboxymethyl)-N,N-dimethyl-, inner salt
CAS No.: 2601-33-4
Batch No.: 170321
Container: One transparent plastic flask
Description: Colorless liquid
Storage conditions: In the refrigerator set at 5 °C. Protected from light
Molecular Weight: 299.50 g/mol
Log P: -1.65
Specfic requirements (handling conditions): None
Purity: 19.9%
Correction factor: 5.025
Date of receipt: 23 March 2018
Expiry (or re-test date): 20 Novemenber 2018

In vitro test system

Details on study design:
Test System
KeratinoSens Cells: the cell line KeratinoSens is stably transfected wit a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin/G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: this cell line was provide by Givaudan
Batch: D1
Storage conditions: at -80 °C
Mycoplasm: absence of mycoplasm was confirmed.

Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle among DMSO, water for injections ro treatment culture medium.
Since the test item was found soluble in DMSO at 200 mM, this stock formulation was diluted in treatment culture medium to the final concentration of 2000 µM. Then, a visual inspection of the sample was performed to evaluate the presence of absence of precipitate/emulsion.

Method for a run of KeratioSens 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-supended in Maintenance medium No. 2. and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 10^4 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 10^4 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 the test item addition.

Treatment
After 24-hours 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 treatment plate for the cytotoxicity evaluation.
All plates were covered by a sealing membrane to avoid evaporation of volatile test items and to aviod 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 precipitae - transparent plate
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each weel by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates
After incubation, the supernatants from the white assau 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) minuttes at room temperature and under orbital shaking.
The plates containing the passive lysis buffer were then placed in the luminometet 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 intergrated for 2 seconds.

Absorbance signal to evaluate the cytoxicity - 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 witha 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 adsorption of each well was detemined at 60 nm using the plate reader.

Results and discussion

In vitro / in chemico

Results
Parameter:
other: Imax
Run / experiment:
Mean
Value:
1.41
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
SOLUBILITY TEST
In the solubility test, the test item was found soluble in DMSO at 200 mM, Therefore, this vehicle was selected for the preparation of the test item stock formulations.
No precipitate or emulsion was observed once the test item stock formulation was diluted in the treatment culture medium to a final concentration of 2000 µM. However, a orange-yellow coloration was observed

KERATINOSENS RUN
First run
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax of 25.63). However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for the positive control, this was considered not to have any impact on the validity of the results of this run.
This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO.
At these tested concentrations:
no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, a yellow coloration was observed at concentrations 1000 µM,
a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 7.81 µM,
the corresponding IC30 and IC50 were calculated to be 6.63 and 11.48 µM, respectively,
no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤1.5 (i.e. 1.38).

Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
This run was performed using the following concentrations: 0.46, 0.64, 0.91, 1.28, 1.81, 2.55, 3.6, 5, 7, 10, 14 and 20 PM in culture medium containing 1% DMSO.
At these tested concentrations:
no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period,
a decrease in cell viability (i.e. cell viability < 70%) was noted at 20 PM,
the corresponding IC30 was 16.80 µM, no IC50 was calculated since the cell viability was > 50% in this run,
no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was ≤ 1.5 (i.e. 1.44).

Any other information on results incl. tables

Viability values, induction values, Imax, IC30, IC50 and EC1.5 values obtained after treatment with the test item in each run as well as the mean and SD values.

Evaluation of the viability (%) of cultures treated with the test item for each run

Concentrations (µM)
FSM-005W 0.98 1.95 3.91 7.81 15.63 31.25 62.5 125 250 500 1000 2000
Viability (%) in Run 1 90 90 80 66 32 -1 -1 -1 -1 -1 -1 0
Viability (%) in Run 2 - - - - - - - - - - - -
Viability (%) in Run 3 - - - - - - - - - - - -
Mean viability (%) 90 90 80 66 32 -1 -1 -1 -1 -1 -1 0
Geometric Mean (%) 90 90 80 66 32 n.c. n.c. n.c. n.c. n.c. n.c. n.c.
SD

Concentrations (µM)
FSM-005W 0.46 0.64 0.91 1.28 1.81 2.55 3.6 5 7 10 14 20
Viability (%) in Run 1 91 94 95 96 94 95 90 93 87 82 77 61
Viability (%) in Run 2 - - - - - - - - - - - -
Viability (%) in Run 3 - - - - - - - - - - - -
Mean viability (%) 91 94 95 96 94 95 90 93 87 82 77 61
Geometric Mean (%) 91 94 95 96 94 95 90 93 87 82 77 61
SD

Gene induction values Imax, IC30, IC50 and EC1.5 values, mean and SD values obtained after treatment with the test item in each run

Concentrations (µM)
FSM-005W 0.98 1.95 3.91 7.81 15.63 31.25 62.5 125 250 500 1000 2000
Induction values in Run 1 1.2 1.2 1.1 1.3 1.4 0 0 0 0 0 0 0
Induction values in Run 2 - - - - - - - - - - - -
Induction values in Run 3 - - - - - - - - - - - -
Mean induction 1.2 1.2 1.1 1.3 1.4 0 0 0 0 0 0 0

Imax and EC1.5 results
FSM-005W Imax EC1.5 (µM) EC150 (µM) EC30 (µM)
Run 1 1.38 - 11.48 6.63
Run 2 - - - -
Run 3 - - - -
Mean n.c n.r n.r n.r
Geometric Mean n.r - n.c n.c
SD n.c - n.c n.c

Concentrations (µM)
FSM-005W 0.98 1.95 3.91 7.81 15.63 31.25 62.5 125 250 500 1000 2000
Induction values in Run 1 1.1 0.8 1 1.1 1 1.1 1 1.2 1.1 1.4 1.3 1.4
Induction values in Run 2 - - - - - - - - - - - -
Induction values in Run 3 - - - - - - - - - - - -
Mean induction 1.1 0.8 1 1.1 1 1.1 1 1.2 1.1 1.4 1.3 1.4
SD

Imax and EC1.5 results
FSM-005W Imax EC1.5 (µM) EC150 (µM) EC30 (µM)
Run 1 1.38 - 11.48 6.63
Run 2 1.44 - - 16.8
Run 3 - - - -
Mean 1.41 n.r n.r n.r
Geometric Mean n.r n.c n.c 10.56
SD 0.04 n.c n.c 7.18

-: no data available

n.c: not calculated

n.r: not requested by the OECD guideline

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item, FSM-005W, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.
Executive summary:

SUMMARY

The objective of this study was to evaluate the potential of the test item, FSM-005W, 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

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-lfrom the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

Methods

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. Two independent runs were performed.

Results

The test item was diluted in DMSO at 200 mM.

First run

All acceptance criteria were fulfilled for the positive and negative controls. The run was thereforeconsidered to be valid. The criterion "the average induction (Imax) in the three replicate plates for the positive control at 64 µM should be between 2 and 8" was not fulfilled (i.e. Imax of 25.63), However, since a clear dose-response with increasing luciferase activity at increasing concentrations was obtained for thepositive control, this was considered not to have any impact on the validity of the results of this run.

This run was performed using the following concentrations: 0.98, 1.95, 3.91, 7.81 , 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM in culture medium containing 1% DMSO

At these tested concentrations:

no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, a yellow coloration was observed at concentrations 1000 µM,

a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations 7.81 µM, the corresponding IC30 and IC50 were calculated to be 6.63 and 11.48 µM, respectively,

no statistically significant gene-fold induction above the threshold of 1.5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was 1.5 (i.e. 1.38).

Second run

All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.

This run was performed using the following concentrations: 0.46, 064, 0.91, 1.28, 1.81, 2.55, 3.6, 5, 7, 10,14 and 20 µM in culture medium containing 1% DMSO.

At these tested concentrations:

no precipitate/emulsion was observed in any test item-treated wells at the end of the 48-hour treatment period, 

a decrease in cell viability (i.e. cell viability < 70%) was noted at 20 µM,

the corresponding IC30 was 16.80 µM, no IC50 was calculated since the cell viability was > 50% in this run,

no statistically significant gene-fold induction above the threshold of 1 5 was noted in comparison to the negative control at any tested concentrations. Moreover, the Imax value was 1 5 (i.e. 1 .44).

The evaluation criteria for a negative response are met in both runs, the final outcome is therefore negative.

Conclusion

Under the experimental conditions of this study, the test item, FSM-005W, was negative in the KeratinoSens assay and therefore was considered to have no potential to activate the Nrf2 transcription factor.