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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July 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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The objective of this study was to evaluate the potential of the test item, ISOMETHOL, 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 reactivity of the test item was evaluated in vitro : it is an alternative method in order to avoid animal study

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-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Batch 44034
Description: Cream to light grey powder
Expiry date: 30 November 2019
Storage conditions: Controlled room temperature (15-25 ºC, below 70 RH%).

In vitro test system

Details on study design:
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. Three independent runs were performed.

Results and discussion

Positive control results:
Name : Cinnamic Aldehyde (CA)
Synonym : trans-Cinnamaldehyde

For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of five concentrations. Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM. All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
2.19
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5
Run / experiment:
1
Value:
658.31
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
1.26
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: IC30
Run / experiment:
2
Value:
898.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: Imax
Run / experiment:
3
Value:
1.52
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
First run
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid.
The evaluation criteria for a positive response are met in this run.

Second run
All acceptance criteria were met for the positive and negative controls, this run was therefore considered to be valid.
The evaluation criteria for a negative response are met in this run.

Third run
Since non-concordant results were obtained in the first two runs, a third run was performed.
All acceptance criteria were fulfilled for the positive and negative controls. The run was therefore considered to be valid. In this run, an outlier luminescence value was removed from the data analysis of the negative control wells (the second replicate of the third plate), therefore 17 negative control values were taken into consideration for the analysis instead of 18.
The evaluation criteria for a negative response are met in this run.

Applicant's summary and conclusion

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

For each run, the test item was prepared in pre-warmed water for injections at 100 mM. The formulation was then heated at 60°C for up to 1h30 and sonicated for 10 minutes prior being filtered through a 0.22 μm filter.

Each run was considered to be valid since all acceptance criteria were fulfilled for the positive and negative controls. In the third run, an outlier luminescence value was removed from the data analysis of the negative control wells (the second replicate of the third plate), therefore 17 negative control values were taken into consideration for the analysis instead of 18.

First run

This run was performed using the following concentrations: 0.49, 0.98, 1.95, 3.91, 7.81, 15.63, 31.3, 63, 125, 250, 500 and 1000 μM in culture medium containing 1% DMSO and 1% water.

At these tested concentrations:

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

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), therefore no IC30 or IC50 was calculated,

. a statistically gene-fold induction above the threshold of 1.5 was noted at concentration of 1000 μM,

. the Imax was 2.19 and the calculated EC1.5 was 658.31 μM.

The evaluation criteria for a positive response are met in this run.

Second run

Due to results obtained in the first run (i.e. statistically significant gene-fold induction > 1.5 at the highest tested concentration only), a narrower range of concentrations was used in the second run (i.e. dilution factor of 1.41): 22.83, 32.20, 45.40, 64.01, 90.25, 127.26, 179.4, 253, 357, 503, 709 and 1000 μM in culture medium containing 1% DMSO and 1% water.

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 1000 μM, the corresponding IC30 was 898.70 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.

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

Third run

Since non-concordant results were obtained in the first two runs, a third run was performed.

The same concentrations as those used in the second run were used in this third run and the following results were obtained:

At these tested concentrations:

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

. no noteworthy decrease in cell viability was noted (i.e. cell viability > 70%), therefore no IC30 or IC50 was calculated,

. 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 1.52.

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

No geometric mean IC30 or IC50 was calculated since the cell viability was > 70% in two out of three runs.

The evaluation criteria for a negative response are met in two out of three 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 sensitisation potential.