Barium fluoride, Strontium fluoride, europium doped

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

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

Guideline:

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

Version / remarks:

adopted February, 2015

Deviations:

yes

Remarks:

One of the luminescence readings for the solvent control was excluded from the analysis, since the variation was above the acceptability criteria of 20% (29%). Based on evaluation of the data with the Dixon’s Q-test this value was a clear outlier.

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Barium fluoride, Strontium fluoride, europium doped

EC Number:

947-973-6

Molecular formula:

Ba0.9-0.95. F2. Sr0.05-0.10 . Eu0.001-0.003

IUPAC Name:

Barium fluoride, Strontium fluoride, europium doped

In vitro test system

Details on the study design:

Vehicle
The vehicle of the test item, i.e. dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

Positive Control (RS582)
Ethylene dimethacrylate glycol. Ethylene dimethacrylate glycol was used as positive control since Charles River Laboratories Den Bosch has a historical database for this sensitizer. Moreover Ethylene dimethacrylate glycol is like cinnamic acid a weak sensitizer but gave more stable results than cinnamic acid. Laboratory proficiency was shown in Charles River Laboratories Den Bosch project 510593.

Cell Culture
Basic medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
Maintenance medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 µg/mL).
Exposure medium:
Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
Environmental conditions:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 65 – 94 %), containing 5.0 ± 0.5% CO2 (actual range 5.0 ± 0.0%) in air in the dark at 37.0 ± 1.0°C (actual range 35.6 – 37.2°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Subculturing
Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (P+25).

Experimental Design
Plating of Cells
For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+9 in experiment 1 and P+11 in experiment 2.
Treatment of Cells
The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2. In total 2 valid experiments were performed.
Luciferase Activity Measurement
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
Cytotoxicity Assessment
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma, Zwijndrecht, The Netherlands) and cells were incubated for 3 - 4 hours at 37°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma, Zwijndrecht, The Netherlands) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
b) The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test (2). If the variability is still higher, the results should be discarded.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

Results and discussion

Positive control results:

• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was between 5 and 125 µM (104 µM and 15 µM in experiment 1 and 2, respectively) and within two standard deviations of the historical mean. A dose response was observed and the induction at 250 µM was higher than 2-fold (2.21-fold and 4.62-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.2% and 12% in experiment 1 and 2, respectively).

In vitro / in chemico

Results

Results 1

Run / experiment:

other: Experiment 1 and 2

Parameter:

other: Cell viability (%)

Value:

70

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No IC30 and IC50 could be determined because cell viability was >70% at all test concentrations

Results 2

Run / experiment:

other: Experiment 1

Parameter:

other: Luminescence activity induction

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations. The Imax was 0.97 and therefore no EC1.5 could be calculated.

Results 3

Run / experiment:

other: Experiment 2

Parameter:

other: Luminescence activity induction

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations. The Imax was 1.20 and therefore no EC1.5 could be calculated.

Other effects / acceptance of results:

In both experiments, no precipitation was observed at the start and end of the incubation period in the 96-well plates.

Any other information on results incl. tables

CH02886 showed no toxicity (no IC₃₀and IC₅₀value) and no biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 0.97-fold and
1.20-fold in experiment 1 and 2 respectively. CH02886is classified as negative in the KeratinoSens^TMassaysince only negative results (<1.5-fold induction) were observed at test concentrations up to 400µg/mL.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

The study should be used as part of a weight of evidence approach

Conclusions:

In conclusion, CH02886 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of CH02886to activate theantioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensÔassay.

The study procedures described in this report were based on the most recent OECD guideline 442D (February 2015).

Batch/EJof CH02886 was awhite moist solid. CH02886 was suspended in dimethyl sulfoxide at 40 mg/mL. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of0.20 – 400 µg/mL (2-fold dilution series). The highest test concentration corresponded to thehighest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

·       The luciferase activity induction obtained with the positive control,Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration. 

·       The EC_1.5of the positive control was between 5 and 125 µM (104 µM and 15 µM in experiment 1 and 2, respectively) and within two standard deviations of the historical mean. A dose response was observed and the induction at 250 µM was higher than 2-fold (2.21-fold and 4.62-fold in experiment 1 and 2, respectively).

·       Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (9.2% and 12% in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

CH02886 showed no toxicity (no IC₃₀and IC₅₀value) and no biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 0.97-fold and
1.20-fold in experiment 1 and 2, respectively. CH02886is classified as negative in the KeratinoSens^TMassaysince only negative results (<1.5-fold induction) were observed at test concentrations up to 400 µg/mL.

In conclusion, CH02886 is classified as negative(noactivation of theantioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.