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Description of key information

in-vitro Skin Sensitization:

- LuSens: negative

- h-CLAT: positive

- DPRA: substance is not in the aplicability domain

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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
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).
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.
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.
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).

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
28.03.-30.04.2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
other: OECD Guideline for the Testing of Chemicals, Part 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
adopted 09. October 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
According to Regulation(EC) No 1907/2006 the skin sensitization assessment shall first comprise available human, animal and alternative data before in vivo testing is necessary.
Details on study design:
The solubility of the test item was determined in a non-GLP pre-test in RPMI 1640 (100 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mL and 500 mg/mL) and DMSO (500 mg/mL). The test item was insoluble in DMSO as well as in RPMI 1640 at 300 mg/mL, 400 mg/mL and 500 mg/mL but sufficiently soluble in RPMI1640 at 100 mg/mL (after 25 min rotation) and 200 mg/mL (after 40 min rota-tion). Therefore, RPMI 1640 was used as solvent in the test.

A possible autofluorescence of the test item was determined using a 2475 Multi-λ Fluorescence detector and an excitation wavelength of 488 ± 5nm. The measured autofluorescence of the test item is too low to have an impact on the result of the assay.

Since a reduction of the viability was detected in the pre-test the maximal test item concentration to be tested in the experiments corresponded to 1.2 * CV75 (calculated in the pre-test): 342.7 µg/mL. Therefore, the following eight test item concentrations were tested in each main experiment: 95.6 µg/mL, 114.8 µg/mL, 137.7 µg/mL, 165.3 µg/mL, 198.3 µg/mL, 238.0 µg/mL, 285.6 µg/mL, 342.7 µg/mL.
Positive control results:
As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used. The positive control was valid in a prior reactivity check as well as in the pre-test and the main experiments I and II.
Key result
Parameter:
other: RFI Value [%] for CD54
Remarks:
at test item concentration 114.8 µg/ml
Run / experiment:
1
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
other: valid in prior reactivity check
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI Value [%] for CD54
Remarks:
at test item concentrations 95.6 µg/mL, 114.8 µg/mL, 137.7 µg/mL, 165.3 µg/mL
Run / experiment:
2
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
other: valid in prior reactivity check
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Precipitation of the test item wasn’t visible during lab procedure in the pre-test but in both experiments (I and II) at all concentrations. A different gating process was used to eliminate the precipitate. From this, it follows that the viability values differ between the pre-test and the experiments and the calculated CV75 value is not reflected in the results of the main experiments.
For analysis of the pre-test with the FACS Suite Software the peaks of viable and dead cells were good enough separable to use them for analysis and to use the corresponding viability values for calculation of the CV75.
For analysis of the experiment I and II it wasn´t possible to use the same gates as in the pre-test, because it wasn´t possible to separate the peaks that way. A different gating process was used that enabled to analyze the test item up to a concentration of 165.3 µg/mL. For any higher concentration (198.3 µg/mL – 342.7 µg/mL) the analysis wasn´t possible even with the new gating process due to the high precipitation level.
However, the four lowest concentrations are used (95.6 µg/mL, 114.8 µg/mL, 137.7 µg/mL, 165.3 µg/mL) for analysis of experiment I and II. These concentrations showed viability values above 50% and the test item was positive in both experiments.
In experiment I the RFI of CD86 was not≥150 % at any tested concentration.
The RFI of CD54 was ≥ 200 % at the concentration of 114.8 µg/mL with cell viability ≥ 50 %.
In experiment II the RFI of CD86 was not≥150 % at any tested concentration either.
The RFI of CD54 was ≥ 200 % in the following concentrations: 165.3 µg/mL; 137.7 µg/mL; 114.8 µg/mL; 95.6 µg/mL with cell viability ≥ 50 %.
A calculation of the EC200 (for CD54) for experiment I and II was not possible because no dose-response was evident. In Exp I only one of the RFI values was above 200.
Conclusions:
Under the experimental conditions of this study, the test item, Ammonium hexafluorozirconate, was positive in the h-CLAT.
Executive summary:

This in vitro study was performed to assess the sensitising potential of the test item Ammonium hexafluorozirconate by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. Two experiments with a treatment period of 24 hours were performed. In the experiments, the highest nominal applied concentration (342.7 μg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared and tested. As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium.

As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used. Prior to the study, the cells used in the experiments were checked in a reactivity check and were found to be suitable for the experiments.

Precipitation of the test item wasn’t visible during lab procedure in the pre-test but in both experiments (I and II) at all concentrations. A different gating process was used to eliminate the precipitate. From this, it follows that the viability values differ between the pre-test and the experiments and the calculated CV75 value is not reflected in the results of the main experiments. For analysis of the pre-test with the FACS Suite Software the peaks of viable and dead cells were good enough separable to use them for analysis and to use the corresponding viability values for calculation of the CV75. The cell viabilities of medium and solvent controls were higher than 90 %. The values of the solvent control of RPMI 1640 (CD86 and CD54) and of DMSO (CD86) in experiment II were out of the range of the historical data. For analysis of the experiment I and II it wasn´t possible to use the same gates as in the pre-test, because it wasn´t possible to separate the peaks that way. A different gating process was used that enabled to analyze the test item up to a concentration of 165.3 μg/mL. For any higher concentration (198.3 μg/mL – 342.7 μg/mL) the analysis was not possible even with the new gating process due to the high precipitation level.

However, the four lowest concentration of experiment I and II were used for analysis (95.6 µg/ml, 114.8 µg/ml, 137.7 µg/ml, 165.3 µg/ml).

These concentrations showed viability values above 50% and the test item was positive in both experiments. In experiment I the RFI of CD86 was not ≥ 150 % at any tested concentration. The RFI of CD54 was ≥ 200 % at the concentration of 114.8 μg/mL with cell viability ≥ 50 %. In experiment II the RFI of CD86 was not ≥ 150 % at any tested concentration either. The RFI of CD54 was ≥ 200 % in the following concentrations: 165.3 μg/mL; 137.7 μg/mL; 114.8 μg/mL; 95.6 μg/mL with cell viability ≥ 50 %. A calculation of the EC200 (for CD54) for experiment I and II was not possible because no dose-response was evident. In Exp I only one of the RFI values was above 200.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In-vitro Skin sensitization:

LuSens

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 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).

h-CLAT

This in vitro study was performed to assess the sensitising potential of the test item Ammonium hexafluorozirconate by quantifying changes in the expression level of the two cell surface markers CD86 and CD54, which are associated with the process of activation of monocytes and dendritic cells. Two experiments with a treatment period of 24 hours were performed. In the experiments, the highest nominal applied concentration (342.7 μg/mL) was chosen based on the results obtained in the pre-test. A geometric series (factor 1.2) of 7 dilutions thereof was prepared and tested. As solvent control for the test item, RPMI 1640 was used in a final concentration of 1 % in culture medium.

As positive control, 2,4-dinitrochlorobenzene (DNCB, CAS n. 97-00-7, ≥ 99% purity) was used. Prior to the study, the cells used in the experiments were checked in a reactivity check and were found to be suitable for the experiments.

Precipitation of the test item wasn’t visible during lab procedure in the pre-test but in both experiments (I and II) at all concentrations. A different gating process was used to eliminate the precipitate. From this, it follows that the viability values differ between the pre-test and the experiments and the calculated CV75 value is not reflected in the results of the main experiments. For analysis of the pre-test with the FACS Suite Software the peaks of viable and dead cells were good enough separable to use them for analysis and to use the corresponding viability values for calculation of the CV75. The cell viabilities of medium and solvent controls were higher than 90 %. The values of the solvent control of RPMI 1640 (CD86 and CD54) and of DMSO (CD86) in experiment II were out of the range of the historical data. For analysis of the experiment I and II it wasn´t possible to use the same gates as in the pre-test, because it wasn´t possible to separate the peaks that way. A different gating process was used that enabled to analyze the test item up to a concentration of 165.3 μg/mL. For any higher concentration (198.3 μg/mL – 342.7 μg/mL) the analysis was not possible even with the new gating process due to the high precipitation level.

However, the four lowest concentration of experiment I and II were used for analysis (95.6 µg/ml, 114.8 µg/ml, 137.7 µg/ml, 165.3 µg/ml).

These concentrations showed viability values above 50% and the test item was positive in both experiments. In experiment I the RFI of CD86 was not ≥ 150 % at any tested concentration. The RFI of CD54 was ≥ 200 % at the concentration of 114.8 μg/mL with cell viability ≥ 50 %. In experiment II the RFI of CD86 was not ≥ 150 % at any tested concentration either. The RFI of CD54 was ≥ 200 % in the following concentrations: 165.3 μg/mL; 137.7 μg/mL; 114.8 μg/mL; 95.6 μg/mL with cell viability ≥ 50 %. A calculation of the EC200 (for CD54) for experiment I and II was not possible because no dose-response was evident. In Exp I only one of the RFI values was above 200. Under the experimental conditions of this study, the test item, Ammonium hexafluorozirconate, was positive in the h-CLAT.

DPRA

The Adverse Outcome Pathway (AOP) summarizes the existing knowledge of the chemical and biological mechanisms associated with the skin sensitization from the molecular initiating event through the intermediate events to the adverse allergic contact dermatitis in humans.

The DPRA is proposed to address the molecular initiating event of the AOP by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine.

Unfortunately, the DPRA is not applicable for the testing of metal compounds since they are known to react with proteins with mechanisms other than covalent binding. The test item, ammonium hexafluorozirconate, belongs to the group of metal compounds and therefore is not in the applicability domain of the DPRA.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available in-vitro data raise concerns about a skin sensitizing potential of ammonium hexafluorozirconate. Two in-vitro tests of the AOP for skin sensitization (LuSens, h-CLAT) were performed, whereof one was positive and one negative. For precautionary reasons, ammonium hexafluorozirconate is classified for skin sensitization (Category 1) under Regulation (EC) No 1272/2008.