Copper diformate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020-05-04 to 2020-05-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD TG 442E

GLP compliance:

yes

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

Copper diformate

EC Number:

208-865-8

EC Name:

Copper diformate

Cas Number:

544-19-4

Molecular formula:

CH2O2.1/2Cu

IUPAC Name:

copper diformate

Test material form:

solid: particulate/powder

Details on test material:

blue in colour

In vitro test system

Details on the study design:

Dose Finding Assay
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control.
The test article was dissolved at 100 mg/mL in saline then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 50-fold in culture medium (working solutions).
The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 7.8-1000 µg/mL.
The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing, ultrasonication (for approximately 4 minutes) and warming at 37°C.

CD86/CD54 Expression Measurement
Eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using the corresponding solvent, and then further diluted 50-fold into the culture medium to give eight working solutions ranging from 0.335 x CV75 to 1.2 x CV75.
The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 11.34-40.62 µg/mL.
The test article was prepared shortly before testing. Preparation was conducted under subdued lighting with the aid of vortex mixing, ultrasonication (for approximately 75 minutes) and warming at 37°C.

METHODS
Specifications
Human monocytic leukemia cell line, THP-1 (ATCC® TIB-202™, an immortalised human monocytic leukemia cell line, used as a surrogate for dendritic cells), was supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.

Identification
The test system was suitably labelled to clearly identify the study number, test article, test article concentration, positive and solvent/vehicle controls.

Cell Culture Maintenance
THP-1 cells were cultured in a humidified incubator set to 37ºC, 5% CO2, in RPMI-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 µg/mL streptomycin. The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 10^6 cells/mL and maintained at a density from 0.1 x 10^6 to 0.8 x 10^6 cells/mL. Cell density did not exceed 1 x 10^6 cells/mL.

Reactivity Check
This was performed using DNCB (CAS no. 97-00-7, =99% purity), nickel sulphate (CAS no. 10101-97-0, =99% purity) and lactic acid (CAS no. 50-21-5, =85% purity) two weeks after thawing. DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.

Plate Preparation
THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 10^6 cells/mL for 48 hours or at a density of 0.1 x 10^6 cells/mL for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with
fresh culture medium at 2 x 10^6 cells/mL. The cells were then distributed into a 24-well flat-bottom plate (500 µL/well) (expression assay) or a 96-well flat-bottomed plate (80 µL/1.6 x 10^5 cells per well) (DRF assay(s)).


Study Design
Dose Finding Assay
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin
(FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL). PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability was calculated according to OECD 442E.
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation accordint to OECD 442E.

CD54 Expression Measurement
One experiment (consisting of two independent runs) was needed to drive a prediction. Each independent run was performed on the same day provided that for each run:
a) Independent fresh stock solutions and working solutions of the test article and antibody solutions were prepared and
b) Independently harvested cells were used (i.e. cells were collected from different culture flasks). The cells may have come from the same passage.
On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 10^6 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 10^6 cells per well).
The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.
After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).
After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.
The stained cells were washed three times with an excess of FACS buffer, resuspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).
The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Data Evaluation - Analysis of Results
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to OECD 442E.
The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was calculated according to OECD 442E

Prediction model, calculation of effective concentration (EC) values and assay acceptance criteria according to OECD 442E.

Results and discussion

Positive control results:

RFI value for DNCB
CD86: EXP 1 = 404
CD86: EXP 2 = 270

CD54: EXP 1 = 563
CD54: EXP 2 = 795

In vitro / in chemico

Results

Results 1

Key result

Parameter:

other: RFI for CD54

Value:

214

Results 2

Key result

Parameter:

other: RFI for CD84

Value:

51

Other effects / acceptance of results:

- All acceptance criteria of the h-CLAT assay parameters were met in each experiment.
- The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.
- In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI =150% and CD54 RFI =200%).
- For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
- For the positive control, RFI values were =150% for CD86 and =200% for CD54, and cell viability was >50% in each independent run.
- For the test article, the cell viability was more than 50% in all tested concentrations in each independent run

Any other information on results incl. tables

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

Concentration (µg/mL)	RFI (CD86)		RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
11.34	112	86	217	419
13.60	86	67	214	542
16.32	126	53	245	508
19.59	121	51	314	688
23.51	123	59	336	648
28.21	118	63	578	794
33.85	154	72	586	1105
40.62	131	77	693	1311
Solvent/vehicle control (culture medium)	100	100	100	100
Positive control (DNCB)	404	270	563	795

In both experiments, the RFI values for CD54 were >200% at all concentrations tested. The test article therefore gave a positive prediction with CD54.

The RFI values for CD84 were all <150%, except at one concentration in the first experiment, however, as the test article was predicted to be positive due to the results with CD54, it was not necessary to conduct a third experiment.

The EC200 value for CD54 calculated by linear regression of endpoint assay data was 9.01 µg/mL.

Expression Assay: MFI and Cell Viability Values

Experiment 1

	MFI (Geo Mean)			Corrected MFI			Viability
Concerntration (µg/ml)	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
11.34	874	651	423	451	228	94.9	93.4	94.3
13.60	777	655	430	347	225	93.1	93.2	93.8
16.32	943	690	433	510	257	91.6	89.1	89.5
19.59	932	774	444	488	330	85.7	82.4	85.6
23.51	956	813	460	496	353	82.9	82.4	82.2
28.21	982	1114	507	475	607	78.6	73.9	73.6
33.85	1153	1146	531	622	615	73.2	68.6	68.7
40.62	1056	1253	525	531	728	71.1	64.6	68.6
Culture medium	821	522	417	404	105	98.2	97.7	96.8
DMSO 0.2%	830	507	393	437	114	98.2	98.5	98.0
DNCB 4µg/ml	2240	1118	476	1764	642	94.0	92.3	93.0

 

 

Experiment 2

	MFI (Geo Mean)			Corrected MFI			Viability
Concerntration (µg/ml)	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
11.34	1220	852	462	758	390	87.8	85.6	87.0
13.60	1044	959	455	589	504	85.3	83.5	84.2
16.32	913	922	450	463	472	83.4	80.5	83.5
19.59	909	1097	457	452	640	79.7	77.2	76.9
23.51	1005	1085	482	523	603	76.5	72.4	71.2
28.21	1054	1239	501	553	738	71.9	65.6	65.6
33.85	1129	1525	497	632	1028	67.3	64.1	64.2
40.62	1200	1740	521	679	1219	75.0	64.6	60.9
Culture medium	1311	524	431	880	93	97.7	96.7	97.5
DMSO 0.2%	1205	530	412	793	118	98.0	98.1	98.0
DNCB 4µg/ml	2602	1395	457	2145	938	92.4	91.8	91.9

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The test article, Copper Diformate, was considered to be positive in the human Cell Line Activation Test.