Barium fluoride, Strontium fluoride, europium doped

Endpoint summary

Description of key information

A valid KeratinoSens assay was performed according to OECD guideline 442D and GLP principles. CH02886 was suspended in dimethyl sulfoxide at 40 mg/mL, final test concentrations in the assay were 0.20 – 400 µg/mL (2-fold dilution series). No precipitate was observed at any dose level tested. Two independent experiments were performed. 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 either experiment. The maximum luciferase activity induction (I_max) was 0.97-fold and 1.20-fold in experiment 1 and 2, respectively. CH02886 is classified as negative in the KeratinoSens^TMassay since only negative results (<1.5-fold induction) were observed at test concentrations up to 400 µg/mL.

A valid U-SENS Assay was performed according to OECD guideline 442E and GLP principles. CH02886 was suspended in complete medium at 0.4 mg/mL. In the first experiment the stock was diluted to six test concentrations between 1 and 200 μg/mL. In the second and third experiment, a narrower dose-response analysis was performed (test concentrations ranging from 90 to 200 µg/mL). No precipitate was observed at any dose level tested. CH02886 showed no toxicity (No CV₇₀value) in any of the experiments. In the first experiment the test item showed a biologically relevant, induction of the CD86 activity (EC150 values of 117 µg/mL). However, as the induction was only observed at the highest non-toxic concentration, this was regarded as an inconclusive result. In the second experiment no biologically relevant induction of the CD86 activity (No EC₁₅₀value) was measured at any of the test concentrations and the individual run conclusion was therefore negative. In the third experiment as well, no biologically relevant induction of the CD86 activity (No EC₁₅₀value) was measured at any of the test concentrations and the individual run conclusion was negative. Based on these results, CH02886 was classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed in 2 out of 3 experiments at all test concentrations with a cell viability of >70% compared to the vehicle control.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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

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

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.

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).

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

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.

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.

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.

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.

Reference 2

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

Qualifier:

according to guideline

Guideline:

other: OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)

Version / remarks:

25 June 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

Background of the test system:
The existing knowledge of the chemical and biological mechanisms associated with skin sensitization has been summarized in the form of an Adverse Outcome Pathway (AOP). The third key event in this AOP is the activation of dendritic cells (DC), typically assessed by expression of specific cell surface markers, chemokines and cytokines.
The U-SENS™ method is proposed to address this third key event by quantifying the change in the expression of a cell surface marker associated with the process of activation of monocytes and DC (i.e. CD86), in the human histiocytic lymphoma cell line U937, following exposure to sensitisers. The measured expression levels of CD86 cell surface marker in the cell line U937 is then used for supporting the discrimination between skin sensitisers and non-sensitisers.
The design of this study is based on the following study guideline:
• OECD 442E – Annex II ‘In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)’ (25 June 2018).

Controls:
Lactic Acid (LA, RS471) is used as negative control. On the treatment day, a solution at
10 mg/mL was prepared in RPMI medium. This solution was diluted 1:25 in order to obtain a 0.4 mg/mL stock solution (final dose level 200 µg/mL).
2,4,6-Trinitrobenzenesulfonic acid (TNBS; RS599) was provided as 1 M solution. On the treatment day a 10 mg/mL solution was prepared in RPMI. This solution was diluted 1:100 in order to obtain a 0.1 mg/mL stock solution (final dose level 50 µg/mL).

Dose formulation:
No correction was made for the composition/purity of the test item.
A solubility test was performed. The test item was either dissolved or suspended in complete medium and DMSO (Hybrimax, Sigma, Zwijndrecht, The Netherlands) to a final concentration of 50 mg/mL in RPMI and DMSO or 0.4 mg/mL in RPMI. The test item formed a non-homogenous suspension in complete medium at 50 mg/mL. In DMSO the test item formed non-homogenous suspension at 50 mg/mL. In complete medium at 0.4 mg/mL the test item formed a homogenous suspension (glassy pink). Complete medium was selected as solvent for the main assay, since this is the first solvent option.
 
In the main experiments the test item was suspended in complete medium at 0.4 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL in the 96-well plate in experiment 1 and of 200, 180, 160, 140, 120, 100 and 90 µg/mL in the 96-well plate in experiment 2 and 3.
No precipitation was observed at the end of the incubation period in the 96-well plates.
Test item concentrations were used within 3.5 hours after preparation.

Test System:
Test System U937 human monocytes.
Justification Inducible CD86-expressing cells
Source ATCC (American Type Culture Collection, Virginia, USA).
ATCC no.: CRL-1593.2TM.
Stock cultures of these cells are stored in the freezer (-150°C). Cells were used after an acclimatisation period of approximately 8 days after thawing and were not sub-cultured more than 21 times. Once a year the cell line is checked for infection with a mycoplasma detection test.
Each batch of cells received from a supplier are submitted to a qualification process to guarantee their suitability (spontaneous CD86 level) for the test by comparison with the historical data or data from the literature.

Cell Culture
Cell culture medium
Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).

Environmental conditions
All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 71 – 95%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.6 – 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, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Experimental Design
1. Plating of Cells
Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.
2. Number of experiments
Three valid experiments were conducted per test item to demonstrate reproducibility of the results and conclusion. Initially, experiment 1 and 3 did not pass all the acceptability criteria and therefore these part of the study were repeated.
3. Treatment of Cells
Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL.
In the second and third experiment cells were treated with seven selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second and third experiment were 90, 100, 120, 140, 160, 180 and 200 µg/mL.
In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.
4. Precipitate evaluation
After 45 ± 3 hours of exposure, wells were checked for precipitate.
5. Cell antibodies staining for IgG1 and CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control (#555748; BD, Amsterdam, The Netherlands)
- Human CD86 specific mouse IgG1 (#555657; BD, Amsterdam, The Netherlands)
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.
 
Flow cytometry method
1. Acquisition
Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
2. Analysis
All analysis parameters were set on the RPMI wells for I gG1 and remained unchanged, for the analysis of all the other wells. The P1 region was adjusted if necessary in a SSC (X-axis) and FSC (Y-axis) plot.
The P2 region was defined for the PI negative cells among P1 in a histogram with counts (Y-axis) and PI fluorescence (X-axis). The amount of cytotoxicity were analyzed as percentage of cells in P2. The P2 region was then plotted in a Dot-plot as fluorescence (X-axis) and SSC (Y-axis) and a quadrant was placed according acceptability criterion b. The percentage of cells in the UR quadrant was used to calculate the stimulation index.
3. Color Interferences
On IgG1 analysis
There is colour interference in the IgG1 evaluation when the X Median of the FITC-fluorescence in the UL Quad is 50% higher than the X Median fluorescence of the vehicle control IgG1 well (IgG1 X Median S.I. ≥ 150%).

ACCEPTABILITY CRITERIA
• At the end of the incubation treatment period, the mean viability of the triplicate untreated (RPMI) U937 cells is > 90%
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25%.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5%.
• No drift in CD86 expression is observed. A drift is defined by i) the corrected %CD86+ value of the untreated control replicate 3 is less than 50% of the mean of the corrected %CD86+ value of untreated control replicates 1 and 2; and ii) the corrected %CD86+ value of the negative control replicate 3 is less than 50% of mean of the corrected %CD86+ value of negative control replicates 1 and 2.
• The positive control (TNBS) is considered as valid if at least two out of the three wells are positive (CD86 S.I. ≥ 150%) and non-cytotoxic (cell viability ≥ 70%).
• Negative control LA is considered as valid if at least 2 out of 3 LA wells are negative (CD86-IgG1 SI < 150%) and non-cytotoxic (cell viability ≥ 70%).

INTERPRETATION
• For CD86 expression measurement, each test chemical is tested in at least two independent runs (performed on a different day) to derive a single prediction (NEGATIVE or POSITIVE).
• The individual conclusion of an U-SENS™ run is considered Negative (hereinafter referred to as N) if the S.I. of CD86 is less than 150% at all non-cytotoxic concentrations (cell viability ≥ 70%) and if no interference (cytotoxicity, solubility or colour) is observed. Solubility interference is defined as crystals or drops observed under the microscope at 45 ± 3h post treatment (before the cell staining). Colour interference is defined as a shift of the FITC-labelled IgG1 dot-plot (IgG1 FL1 X Median S.I. ≥ 150%).
• In all other cases: S.I. of CD86 higher or equal to 150% and/or interferences observed, the individual conclusion of an U-SENS™ run is considered Positive (hereinafter referred to as P).
• An U-SENS™ prediction is considered NEGATIVE if at least two independent runs are negative (N). If the first two runs are both negative (N), the U-SENS™ prediction is considered NEGATIVE and a third run does not need to be conducted.
• An U-SENS™ prediction is considered POSITIVE if at least two independent runs are positive (P). If the first two runs are both positive (P), the U-SENS™ prediction is considered POSITIVE and a third run does not need to be conducted.
• There is an exception if, in the first run, the S.I. of CD86 is higher or equal to 150% at the highest non-cytotoxic concentration only. The run is then concluded NO CONCLUSION (NC), and additional concentrations (between the highest non cytotoxicity concentration and the lowest cytotoxicity concentration) should be tested in additional runs. A run NC conducts automatically to the need of at least 2 more runs, and to a fourth run in case runs 2 and 3 are not concordant (N and/or P independently). Follow up runs will be considered positive even if only one non cytotoxic concentration gives a CD86 equal or above 150%, since the dose setting has been adjusted for the specific test chemical. The final prediction will be based on the majority result of the three or four individual runs (i.e. 2 out of 3 or 2 out of 4).

Positive control results:

Experiment 1
The positive control (TNBS) showed a S.I. ≥ 293% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 102% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 2
The positive control (TNBS) showed a S.I. ≥ 399% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 104% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 3
The positive control (TNBS) showed a S.I. ≥ 372% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 98% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Run / experiment:

other: Experiment 1

Parameter:

other: CV70

Remarks:

µg/ml

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • CH02886 showed no toxicity, the viability of the cells was higher than 70% at all test concentrations

Run / experiment:

other: Experiment 1

Parameter:

other: EC150

Remarks:

µg/ml

Value:

117

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 117 µg/mL

Run / experiment:

other: Experiment 2

Parameter:

other: CV70

Remarks:

µg/ml

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • CH02886 showed no toxicity, the viability of the cells was higher than 70% at all test concentrations

Run / experiment:

other: Experiment 2

Parameter:

other: EC150

Remarks:

µg/ml

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with CH02886

Run / experiment:

other: Experiment 3

Parameter:

other: CV70

Remarks:

µg/ml

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • CH02886 showed no toxicity, the viability of the cells was higher than 70% at all test concentrations

Run / experiment:

other: Experiment 3

Parameter:

other: EC150

Remarks:

µg/ml

Value:

200

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: • No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with CH02886

Other effects / acceptance of results:

Experiment 1
• No precipitation was observed at the end of the incubation period in the 96-well plates.
• CH02886 showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
• A biologically relevant increase in the expression of CD86 was observed after treatment with the test item, the EC150 is 117 µg/mL.
• The test item showed no colour interference.

Experiment 2
• No precipitation was observed at the end of the incubation period in the 96-well plates.
• CH02886 showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
• No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with CH02886. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
• The test item showed no colour interference.

Experiment 3
• No precipitation was observed at the end of the incubation period in the 96-well plates.
• CH02886 showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
• No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with CH02886. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
• The test item showed no colour interference.

All tests passed the acceptance criteria:
• At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (99% in experiment 1, 2 and 3).
• The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and
≤ 25% in all experiments.
• At least two out of three IgG1 values of untreated U937 cells fell within the range of
≥ 0.6% and < 1.5% in all experiments.
• No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

CH02886 showed no toxicity (No CV₇₀value) in all experiments. In the first experiment the test item showed a biologically relevant, induction of the CD86 activity (EC₁₅₀values of
117µg/mL). The individual run conclusion was inconclusive as the induction was only observed at the highest non-toxic concentration. In the second experiment no biologically relevant induction of the CD86 activity (NoEC₁₅₀value) was measured at any of the test concentrations and the individual run conclusion was negative. As the results of the first and second experiment were not sufficient to reach a conclusion, an additional third experiment was conducted. In the third experimentno biologically relevant induction of the CD86 activity (NoEC₁₅₀value) was measured at any of the test concentrations and the individual run conclusion was negative. CH02886is classified as negative in the U-Sens™ assaysince negative results (< 150% increase) were observed in 2 out of 3 experiments at all test concentrations with a cell viability of >70% compared to the vehicle control.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, CH02886 is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described.

Executive summary:

The objective of this study is to evaluate the ability of CH02886 to increase the expression levels of CD86 cell surface marker in the U937 cell line in the U937 cell line activation Test (U-Sens™) assay.

The study procedures described in this report were based on the most recent OECD 442E guideline (June 2018).

Batch /EJ of CH02886 was a white moist solid. CH02886 was suspended in complete medium at 0.4 mg/mL. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL). In the second and third experiment, a more narrow dose-response analysis was performed (90, 100, 120, 140, 160, 180 and 200 µg/mL)to investigate the increase in expression of experiment 1 in more detail. No precipitate was observed at any dose level tested. Three independent experiments were performed.

All experiments passed the acceptance criteria:

·     At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90%.

·     The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in all experiments.

·     At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in all experiments.

·     No drift in CD86 expression was observed in the untreated controls (RPMI) and negative (LA) controls.

In all experiments the positive (TNBS) and negative (LA) control were considered valid. Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

CH02886 showed no toxicity (No CV₇₀value) in any of the experiments. In the first experiment the test item showed a biologically relevant, induction of the CD86 activity (EC₁₅₀values of 117 µg/mL). The conclusion was inconclusive as the induction was only observed at the highest non-toxic concentration. 
In the second experiment no biologically relevant induction of the CD86 activity (No EC₁₅₀ value) was measured at any of the test concentrations and the individual run conclusion was negative. As the results of the first and second experiment were not sufficient to reach a conclusion, an additional third experiment was conducted. In the third experimentno biologically relevant induction of the CD86 activity (No EC₁₅₀ value) was measured at any of the test concentrations and the individual run conclusion was negative.
CH02886 is classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed in 2 out of 3 experiments at all test concentrations with a cell viability of >70% compared to the vehicle control.

In conclusion, CH02886 is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

As CH02886 is an inorganic substance, no DEREK (QSAR) assessment could be performed. Furthermore it is scientifically not justified to perform a DPRA. CH02886 was tested in a KeratinoSens assay and based on the results it is concluded that the substance cannot activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. The negative outcome of a U-SENS Assay indicates that the substance is not expected to activate dendritic cells, as shown by the lack of increase in the expression levels of CD86 cell surface marker in the U937 cell line upon exposure to CH02886.

As both available in vitro tests did not indicate skin sensitizing properties of CH02886, it is considered scientifically justifiable not to classify CH02886 for skin sensitizing properties.

Justification for classification or non-classification

A strategy was followed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and as presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

 

It was considered not scientifically justifiable to perform a DEREK assessment and a DPRA

assay, based on the fact that CH02886 contains metals for which these two methods are not applicable. The results of a KeratinoSens^TMassay performed with CH02886 demonstrated that the substance is not able to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes. The negative outcome of a U-SENS™ Assay indicates that the substance is not expected to activate dendritic cells, as shown by the lack of increase in the expression levels of CD86 cell surface marker in the U937 cell line upon exposure to CH02886.

 

As both available in vitro tests did not indicate skin sensitizing properties of CH02886, it is considered scientifically justifiable not to classify CH02886 for skin sensitizing properties.