Barium zirconium trioxide

Skin sensitisation

Administrative data

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

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)

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

Test material

Test material information

Constituent 1

Reference substance name:

Barium zirconium trioxide

EC Number:

234-546-8

EC Name:

Barium zirconium trioxide

Cas Number:

12009-21-1

Molecular formula:

BaO3Zr

IUPAC Name:

barium(2+);oxygen(2-);zirconium(4+)

Test material form:

solid: particulate/powder

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

In vitro test system

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

Results and discussion

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

In vitro / in chemico

Results

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

Any other information on results incl. tables

 

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

Applicant's summary and conclusion

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.