4-allylanisole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

GLP compliance:

yes

Type of study:

other: activate the Nrf2 transcription factor by using the LuSens cell line.

Justification for non-LLNA method:

This in vitro study is performed to assess the potential of the test item Estragole to activate the Nrf2 transcription factor by using the genetically modified keratinocyte cell-line “LuSens” (Bauch et al. 2012).
It employs the use of a reporter gene for luciferase placed under the control of the antioxi-dant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010). In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two, but a maximum of three independent and valid experiments are performed.

Test material

Test material information

Constituent 1

Reference substance name:

4-allylanisole

EC Number:

205-427-8

EC Name:

4-allylanisole

Cas Number:

140-67-0

Molecular formula:

C10H12O

IUPAC Name:

1-methoxy-4-(prop-2-en-1-yl)benzene

Specific details on test material used for the study:

Appearance :Clear liquid
Composition :4-allylanisole
Purity :99.53 %
Storage :Room Temperature: (20 ± 5 °C), keep away from light

In vitro test system

Details on the study design:

This in vitro study evaluates the potential of the test item Estragole to activate the Nrf2 transcription factor by using the LuSens cell line. This test is 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 assay included a cytotoxicity range finder test (CRFT) and two independent experi-ments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the con-centrations for the two experiments were determined.

Results and discussion

Positive control results:

EGDMA (120 µM) was used as positive control. The viability was above 70 % and a dis-tinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: experiment I

Parameter:

other: Relative Viability of the Cells[%]

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Finally the following test item concentration showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction: Experiment I: 135 µM, 162 µM, 194 µM, 233 µM, 279 µM, 335 µM, 402 µM, 482 µM, 579 µM

Results 2

Key result

Run / experiment:

other: Experiment II

Parameter:

other: Relative Viability of the Cells

Value:

83.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Finally the following test item concentration showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction: Experiment II: 135 µM, 162 µM, 194 µM, 233 µM, 279 µM, 335 µM, 402 µM, 482 µM, 579 µM, 694 µM, 833 µM

Applicant's summary and conclusion

Conclusions:

This in vitro study was performed to investigate the potential of Estragole to activate the Nrf2 transcription factor, by using the LuSens cell line. For this purpose, two independent experiments were performed.
A detailed listing of all measured and calculated values of the assay is given in annex 2 (values of CRFT), annex 3 (values of experiment I), and annex 4 (values of experiment II). In addition, the final results of both experiments are summarised in table 8-a and 8-b and graphically illustrated in figure 8-a to 8-d.
The assay was performed in two independent experiments. 12 concentrations of the test item were evaluated. The exposure time was 48 h. The following nominal concentrations of the test item were investigated in experiment I and II: 135 µM, 162 µM, 194 µM, 233 µM, 279 µM, 335 µM, 402 µM, 482 µM, 579 µM, 694 µM, 833 µM, 1000 µM
None of the real treatment concentrations in both experiments deviated more than 10 % from the nominal concentration. Precipitation of the test item was not visible up to the highest concentration.
EGDMA (120 µM) was used as positive control. The viability was above 70 % and a dis-tinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected.
DL-lactic acid (5000 µM) was used as negative control. The viability was above 70 % and the induction of the luciferase was < 1.5 fold in comparison to the solvent control.
The induction of the luciferase of the growth control (Medium no. 3) was < 1.5 fold.
Since all acceptability criteria of the assay were met (see chapter 9.1) the study is valid.
In experiment I a cytotoxic effect was observed at the concentrations 1000 µM, 833 µM and 694 µM. Those concentrations were excluded from the final evaluation.
In experiment II only the highest test item concentrations induced a cytotoxic effect. Again, this concentration was excluded from the final evaluation.
Finally the following test item concentration showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction:
Experiment I: 135 µM, 162 µM, 194 µM, 233 µM, 279 µM, 335 µM, 402 µM, 482 µM, 579 µM
Experiment II: 135 µM, 162 µM, 194 µM, 233 µM, 279 µM, 335 µM, 402 µM, 482 µM, 579 µM, 694 µM, 833 µM
Concerning the sensitizing potential, a substantial and reproducible dose dependent in-crease in luciferase induction ≥ 1.5 fold in more than 2 non-cytotoxic test item concentra-tions was observed in both experiments.

In conclusion, it can be stated that under the experimental conditions reported, the test item did induce the luciferase activity in the LuSens cells above the threshold.
The recorded data in this study declare that the test item Estragole has the potential to activate the Nrf2 transcription factor.