MoVNbTe mixed oxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 February 2021 TO 24 June 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

As per OECD Guideline No. 442E

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: As per OECD Guideline No. 442E

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

niobium trivanadium decamolybdenum tellurium dotetracontaoxide

EC Number:

888-364-4

Cas Number:

146569-48-4

Molecular formula:

Mo10V3Nb1Te1O42

IUPAC Name:

niobium trivanadium decamolybdenum tellurium dotetracontaoxide

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: EX.14402.600
- Purity test date: 87.6%
- Solubility and stability of the test substance in the solvent/vehicle: RPMI 1640 media
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: RPMI 1640 media

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design: Frozen stock of cryovial was thawed immediately at 37±1°C in the water bath.
cells were transferred into 15 mL falcon tube with 5 mL culture media, cells were centrifuged at 125g for 10 minutes. Cells pellet were transferred in to a sterile flask with RPMI-1640 supplemented with 10% Fetal Bovine Serum, antibiotics (1% Penicillin-Streptomycin) and 0.05 mM 2-Mercptoethanol
incubated at 37±1°C and 5±1% CO2 for 4 days.
On the day of testing, cells harvested from culture flask were resuspended with fresh culture medium at 2 × 106 cells/mL.
Then, cells are distributed into a 24 well flat-bottom plate with 500 µL (1 × 106 cells/well). The cells were qualified by conducting a reactivity test using the positive controls,
2,4-dinitrochlorobenzene (DNCB) and nickel sulfate (NiSO4) and the negative control, lactic acid (LA), two weeks after thawing. Both DNCB and NiSO4 produced a positive response and LA produced a negative response for both CD86 and CD54 cell surface markers.
A dose finding assay was performed to determine the CV75. The 75% cell viability (CV) of test chemical concentration are compared with RPMI media control.
The test item was prepared on the day of testing. Test item was dissolved or stably dispersed in medium as solvent/vehicle to final concentrations of 500 mg/mL (in medium) or 500 mg/mL (in DMSO).
Starting from the 500 mg/mL (in medium) stock solutions of the test chemicals, the following dilution steps was taken:
For medium as solvent/vehicle: Eleven stock solutions (eleven concentrations) were prepared, by two-fold serial dilutions using the corresponding solvent/vehicle. These stock solutions are then further diluted 50-fold into culture medium (working solutions). The top final concentration in the plate was 5000 µg/mL.

Vehicle / solvent control:

cell culture medium

Negative control:

DL-Lactic acid

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

DNCB was used as the positive control for CD86/CD54 expression measurement at a final single concentration in the plate was 4.0 μg/mL. To obtain a 4.0 μg/mL concentration of DNCB in the plate, a 2 mg/mL stock solution of DNCB in DMSO was prepared and further diluted 250-fold with culture medium to a 8 μg/mL working solution.
RFI values of CD86 for positive control is 412.55, 401.34 and of CD54 for positive control is 303.60, 364.43, for set 1 and set 2 respectively.

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC150, CD86 [442E]

Value:

1.13 µg/mL

Cell viability:

94.75 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC150, CD86 [442E]

Value:

1.4 µg/mL

Cell viability:

94.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC200, CD54 [442E]

Value:

1.51 µg/mL

Cell viability:

93.19 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC200, CD54 [442E]

Value:

2.07 µg/mL

Cell viability:

89.96 %

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Viability of 75 % was observed at 3.3105 µg/mL. All concentrations below showed respective higher viabilities.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: YES
- Acceptance criteria met for positive control: YES
- Acceptance criteria met for variability between replicate measurements: YES
- Range of historical values if different from the ones specified in the test guideline: NO

Any other information on results incl. tables

 

Test Item Concentrations (µg/mL)	RFI Values of test item: Set 1		RFI Values of test item: Set 2
	CD86	CD54	CD86	CD54
3.973	426.39	363.70	350.05	360.46
3.311	392.37	423.64	389.83	344.84
2.759	366.24	350.54	320.97	269.20
2.299	366.36	141.58	345.22	337.42
1.916	282.28	216.51	335.66	106.80
1.597	209.31	233.28	175.13	31.07
1.331	213.97	125.31	140.90	77.93
1.109	141.79	137.05	142.03	193.73

 

 

TABLE 1.       EC150 (CD86), EC200 (CD54) AND PREDICTION OF THE TEST ITEM

Sl. No.	Details	CD86	CD54	EC150 (CD86) µg/mL	EC200 (CD54) µg/mL
1	Set-1	RFI>150	RFI>200	1.13	1.51
2	Set-2	RFI>150	RFI>200	1.40	2.07
3	Prediction	Positive	Positive	-	-

  Refer Appendix 2 and 3

ND: Not Determined, EC: Effective Concentration.

 

TABLE 2.       CV-75 CONCENTRATION AND OVERALL PREDICTION OF THE TEST ITEM

 

CV-75 (µg/mL)	3.3105
Prediction	Positive

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

The data generated with this method may not be sufficient to conclude in isolation on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed (see Chapter 7.4.1).

Conclusions:

Based on the expression levels of CD86 and CD54, the test item in h-CLAT prediction is considered as “Sensitiser”.
The data generated with this method is however not sufficient to conclude in isolation on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed (see Chapter 7.4.1).

Executive summary:

The test item was evaluated in the “In Vitro Skin Sensitization by Human Cell Line Activation Test (h-CLAT) Method” as per the OECD guideline for testing of chemicals, No.: 442E. The measured expression levels of CD86 and CD54 cell surface markers are used for supporting the discrimination between skin sensitisers and non-sensitisers. Study was conducted to determine the cell viability (CV-75), expression level of CD86 and CD54 cell surface markers expression using human monocytic leukaemia cell line THP-1 in the h-CLAT method. Solubility test was conducted by RPMI 1640 media and dimethyl sulphoxide. Test item formed suspension in RPMI 1640 media at 500 mg/mL. On the day of testing, test item was prepared. Eleven concentrations were prepared for dose finding assay, RPMI media was used as solvent/vehicle, the final concentrations in plate was 4.883, 9.766, 19.531, 39.063, 78.125, 156.25, 312.5, 625, 1250, 2500 and 5000µg/mL. The CV75 value of the test item was determined at 3.3105 µg/mL. Based on the CV75 of dose finding assay, eight test concentrations of 1.109, 1.331, 1.597, 1.916, 2.299, 2.759, 3.311 and 3.973µg/mL were selected for CD86/CD54 expression measurement. Upregulation of the biomarkers CD86 and/or CD54 was observed in some of the tested concentration of the test item. EC150 value for CD86 was 1.13 and 1.4 µg/mL for run 1 and 2 respectively. EC200 for CD 54 was 1.51 and 2.07 µg/mL for run 1 and 2 respectively.

Hence, the test item can be consideres as "sensitiser" in the h-CLAT assay. However, the data generated with this method may not be sufficient to conclude in isolation on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA. Since further in vitro studies were not feasible, no conclusion on the sensitising properties via in vitro studies can be made. Hence, a subsequent in vivo study was performed (see Chapter 7.4.1).