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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:
weight of evidence
Study period:
03.04.2018-13.04.2018
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)
Version / remarks:
Draft v. 7 July 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Test material form:
solid: particulate/powder

In vitro test system

Details on study design:
The solubility of the test item was determined in a non-GLP pre-test in dimethyl sulfoxide (DMSO) and medium (DMEM). The test item was insoluble in DMSO but soluble in DMEM at the required concentra-tion (200 mM) after 15 min rotation. Therefore, DMEM was used as solvent.

A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the case of a cytotoxic result, the concentrations for experiment I and II should be determined so that at least one of them is in the cytotoxic range. Since a cytotoxic reaction was observed in the CRFT starting with 125.0 µM the following 12 nominal concentrations were chosen for experiment I and II: 16.8 µM, 20.2 µM, 24.2 µM, 29.1 µM, 34.9 µM, 41.9 µM, 50.2 µM, 60.3 µM, 72.3 µM, 86.8 µM, 104.2 µM, 125.0 µM.

The LuSens cell line was specially designed for this test system by the BASF SE (Ludwigshafen, Germany). It employs the use of a reporter gene for luciferase placed under the control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany) carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the RWTH, Aachen (laboratory of PD Dr. Wruck).

Results and discussion

Positive control results:
In difference to the OECD 442D draft guideline, not Ethylene glycol dimethylacrylate (EGDMA) was used as positive control, but p-Phenylenediamine. This is uncritical since the guideline indicates that other suitable positive controls, preferentially providing EC 1.5 values in the mid-range, may be used if historical data are available to derive comparable run acceptance criteria.
The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.
Found in experiment I: Positive control: Fold induction: 5.0; Relative viability: 83.4 %
Found in experiment I: Positive control: Fold induction: 5.8; Relative viability: 78.3 %
This luciferase induction is well within the historical data range of the positive control.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Luciferase induction
Run / experiment:
1
Value:
< 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Luciferase induction
Run / experiment:
2
Value:
< 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
Each valid experiment (i.e. meeting all acceptance criteria, according to the procedure described above) is interpreted as follows:
A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70 %) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid experiments.

A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed.
A negative result obtained with test chemicals that do not form a stable dispersion and/or were not test-ed up to 2000 µM (or 2000 µg/mL for test chemicals with no defined molecular weight) and for which no cytotoxicity is observed in any of the tested concentration should be considered as inconclusive.

In order to come to a conclusion on the skin sensitization hazard of a substance, a minimum of two valid and independent experiments needs to indicate a positive or negative result according to the above-described criteria. If the first two experiments come to the same result (i.e. either being negative or be-ing positive) no further testing is required. In case that the first two experiments give discordant results (i.e. one is negative and the other is positive), a third independent experiment needs to be conducted to complete the study. The skin sensitizing potential (corresponding to the potential to activate the Nrf2 transcription factor) of a test substance is determined by the result of the majority of the repetitions of an experiment. If two of two or two of three experiments are negative/positive, the substance is consid-ered as negative/positive.

The luciferase induction was not above 1.5 fold in more than 2 consecutive non-cytotoxic test item con-centrations in experiment I and II.

Therefore, the test item Ammonium hexafluorozirconate is considered not to have the potential to acti-vate the Nrf2 transcription factor (sensitizing potential) under the conditions of the LuSens test.

Applicant's summary and conclusion

Conclusions:
The test item, Ammonium hexafluorozirconate, was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).
Executive summary:

This in vitro study was performed to investigate the potential of Ammonium hexafluorozirconate to activate the Nrf2 transcription factor (sensitizing potential), by using the LuSens cell line.

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:

16.8 μM, 20.2 μM, 24.2 μM, 29.1 μM, 34.9 μM, 41.9 μM, 50.2 μM, 60.3 μM, 72.3 μM, 86.8 μM, 104.2 μM, 125.0 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.

p-Phenylenediamine (80 μM) was used as positive control. The viability was above 70 % and a distinct increase in luciferase induction above 2.5 fold in comparison to the solvent control was detected. This luciferase induction is well within the historical data range of the positive control. 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 and well within the historical data range of the negative 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 the study is valid.

In experiment I a cytotoxic effect was observed in the test item concentrations 86.8 μM, 104.2 μM and 125.0 μM. In experiment II, again a cytotoxic effect was observed in the test item concentrations 104.2 μM and 125.0 μM. Those concentrations were excluded from the evaluation of the luciferase induction. Finally the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction:

Experiment I: 16.8 μM, 20.2 μM, 24.2 μM, 29.1 μM, 34.9 μM, 41.9 μM, 50.2 μM, 60.3 μM, 72.3 μM

Experiment II: 16.8 μM, 20.2 μM, 24.2 μM, 29.1 μM, 34.9 μM, 41.9 μM, 50.2 μM, 60.3 μM, 72.3 μM, 86.8 μM

In all tested non-cytotoxic concentrations of the test item no increase ≥ 1.5 fold in luciferase induction was measured. Therefore, both experiments are clearly negative.

In conclusion, it can be stated that under the experimental conditions of this study, the test item, Ammonium hexafluorozirconate, was negative in the LuSens assay and is therefore considered not having the potential to activate the Nrf2 transcription factor (no sensitizing potential).