Barium zirconium trioxide

Endpoint summary

Description of key information

The human skin sensitisation potential of Barium Zirconate was assessed using the validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. In this study, Barium Zirconate was classified as Negative using the KeratinoSens prediction model.

In addition, the skin sensitisation potential of Barium Zirconate was assessed using a further In Vitro human Cell Line Activation Test (h-CLAT) method according to OECD Test Guideline 442E.

For Barium Zirconate, no cytotoxicity was elicited by the test item at the concentrations tested in the assay. So, Barium Zirconate has been classified as negative as per the prediction model. Due to solubility limitations of the test item, the concentrations used in this test were the lowest permittable based upon the test criteria.

Both in-vitro tests suggest that there is no skin sensitisation potential associated with barium zirconate.

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:

key study

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)

Deviations:

no

Principles of method if other than guideline:

The detailed method followed in this study is described in XCellR8 SOP L0057 ‘Skin Sensitisation Key Event 2 (KeratinoSensTM) following guideline OECD TG442D’, and is based on the KeratinoSensTM assay, adapted to fully animal product-free conditions by XCellR8 and validated in-house (studies 14XC004, 15XC001, 16XCO04) using the 10 proficiency chemicals and 11 reference chemicals described in TG 442D. Luciferase measurements and MTT viability testing were performed at the end of the appropriate exposure period (48h).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

Supplier batch/lot number 17/1791
CAS number 12009-21-1
Purity 99.17 %
Expiry Date 27SEP2020
Physical state: Powder (white)
Concentrations tested (µg/ml) 0.488, 0.977, 1.953, 3.906, 7.813, 15.625, 31.250, 62.500, 125.00, 250.000, 500.000, 1000.000

Details on the study design:

Method of administration of test item:
Per plate, a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

Method of administration of reference items:
Per plate, a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).

Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h prior to endpoint measurements

Number of repetitions
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=9 overall) and 2 x 96-well plates for MTT (n=6 overall). The validity of each repetition was assessed following acceptance criteria

Positive control results:

Test results are acceptable if:
The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction produced by this control is above the threshold of 1.5 in at least one of the tested concentrations and this induction is statistically significant compared to the solvent (negative) control (p<0.05).
The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets:
o Average induction in the three replicates for cinnamic aldehyde at 32 µM is within the XCellR8 historical range (currently 1.6 and 3)
o EC1.5 value for cinnamic aldehyde is within the XCellR8 historical range (currently 6 µM and 39 µM).
Note: At least one of these criteria must be met, otherwise the run is discarded unless there is sufficient reason not to do this as determined by the Study Director.
If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
CV% of blank values < 20%


Criteria Result PASS or FAIL
Positive Control (PC) (Cinnamic aldehyde) induction >1.5-fold in at least one concentration Yes (4/5) PASS
Average induction of PC at 32µM is [1.6-3.0] Yes (2.973) PASS
EC1.5 value is [6-39µM] Yes (9.950µM) PASS
CV% of blank values < 20% Yes (13.4393) PASS

Key result

Run / experiment:

other: Mean of three runs

Parameter:

other: Two parameters: EC1.5 and the IMAX value

Remarks:

The EC1.5 value is the Effective Concentration (EC) of test item that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. The IMAX value is the maximum induction observed within the concentration range tested

Vehicle controls validity:

valid

Remarks:

1% DMSO in cell culture medium

Negative controls validity:

valid

Remarks:

1% DMSO

Positive controls validity:

valid

Remarks:

1 Cinnamic Aldehyde in DMSO

Remarks on result:

no indication of skin sensitisation

Remarks:

The test item did not induce statistically significant luciferase induction >1.5 in any of the 3 repetitions. The IMAX value is the maximum induction observed within the concentration range tested. Although the KeratinoSensTM test is not validated to predict potency, the IMAX value can provide a useful tool for preliminary comparison of sensitisation potential between test items. The maximum induction was observed at a test concentration of 7.813 µM, which showed an IMAX value of 1.077 in repetition 1; the IMAX in repetition 2 was 0.755 at a concentration of 500.000 µM and in repetition 3 the IMAX was 1.019 at 62.500 µM in repetition 3. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls

 

Determination criteria for the skin sensitisation potential ofthe test item
	REP1	REP2	REP3
Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold:	No	No	No
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	N/A	N/A	N/A
Is p value < 0.05 for at least one concentration that yielded>1.5-fold induction with viability above 70%	N/A	N/A	N/A
Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)	N/A	N/A	N/A
Does the test item induce the luciferase in a dose-dependent manner	N/A	N/A	N/A
Classification	Negative	Negative	Negative

Interpretation of results:

GHS criteria not met

Conclusions:

The human skin sensitisation potential of Barium Zirconate was assessed using the validated in vitro method: the KeratinoSensTM test to determine keratinocyte activation. The method was adapted to animal product-free conditions by XCellR8 and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitivity and specificity values.
In this study, Barium Zirconate was classified as Negative using the KeratinoSens prediction model.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 442E: human Cell Line Activation Test (h-CLAT)

Version / remarks:

To determine the In Vitro skin sensitisation potential of Barium Zirconate using the human Cell Line Activation Test (h-CLAT) method as detailed in OECD TG 442E (adopted 25JUN18) and also in EURL ECVAM DB-ALM Protocol No. 158 human Cell Line Activation Test (h-CLAT); Skin Sensitisation and Allergic Contact Dermatitis (Issued: 02 Mar 2017).

Principles of method if other than guideline:

Skin sensitisers have been reported to induce the expression of cell membrane markers associated with Dendritic Cell (DC) activation. The h-CLAT method is an in vitro assay that quantifies these changes in cell surface marker expression (i.e. CD86 and CD54) on a human monocytic leukaemia cell line, THP-1 cells (a cell line that mimics DCs), following 24-hour exposure to the test item. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement is also conducted concurrently to assess whether upregulation of surface maker expression occurs at sub-cytotoxic concentrations. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control are calculated and used in the prediction model, to support the discrimination between sensitisers and non-sensitisers.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Supplier:Thermograde Process Technology
Test Item Name:Barium Zirconate
Supplier Batch/Lot Number:17/1791
CAS Number 12009-21-1
Purity 99.17 %
Expiry Date 27SEP2020
Physical State:Powder (white)
Storage Conditions:Room Temp.
Solubility:1 mg/ml DMSO
Based on the information supplied by the sponsor, the test item was stable, and test item formulations were prepared and used on the day of dosing.

Details on the study design:

Concentrations Tested:
CV75 (viability) assay (µg/ml) 0.02, 0.03, 0.06, 0.13, 0.25, 0.50, 1.00, 2.00

Concentrations Tested:
CD54/CD86 expression assay (µg/ml) 0.56, 0.67, 0.81, 0.97, 1.16, 1.39, 1.67, 2.00

Solvent used DMSO

Positive control results:

Positive Control - CV75: Supplier:Sigma-Aldrich
Reference Item name:2,4-dinitrochlorobenzene (DNCB)
Lot number BCBP8259V
Purity ≥99%
Expiry 06FEB21
CAS number 97-00-7
Concentration tested 8µg/ml (final)
Solvent Dimethyl sulphoxide (DMSO; Neat)
Storage Conditions Prepared and used on the day of testing then discarded.
Study test item code PC (CV75)

Positive Control - CD54 and CD86 Expression:Supplier:Sigma-Aldrich
Reference Item name:Nickel Sulphate
Lot number SZB2960V
Purity 99%
Expiry 15JUN27
CAS number 10101-97-0
Concentration tested 100µg/ml (final)
Solvent RPMI (culture medium)
Storage Conditions Prepared and used on the day of testing then discarded.
Study test item code PC (CD54 and CD86 Expression)

CD54/CD86 Expression: Run acceptance criteria
• Cell viability of medium and DMSO controls should be greater than 90%
• In the positive control (Nickel Sulphate; 100µg/ml), RFI values of both CD86 and CD54 should be over the positive criteria (CD86 ≥ 150 and CD54 ≥ 200) and cell viability should be > 50%
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150 and CD54 ≥ 200)
• For both medium and DMSO controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%

Acceptance criteria for all controls and the test item were met in both runs for the CV75 determination and for the measurement of CD54 and CD86 expression.

Key result

Parameter:

other: CV75 dose value

Remarks on result:

no indication of skin sensitisation

Remarks:

The CV75 dose informs the dosing range selected for the CD54/86 expression assay. However, because no CV75 value could be determined for the test item as no cytotoxicity was elicited at the concentrations tested, and therefore, the top dose for the CD54/86 expression assay (main test) was determined from the highest soluble concentration of the test item in the solvent.

Interpretation of results:

GHS criteria not met

Conclusions:

For Barium Zirconate no CV75 was derived because there was no cytotoxic effect elicited by the test item at the concentrations tested. In the main test, neither the expression thresholds for CD54 or CD86 were crossed and therefore Barium Zirconate was classified as Negative as per the prediction model

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)