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Diss Factsheets

Administrative data

Description of key information

Copper diformate has been evaluated in the Keratinosens and h-CLAT in vitro assays for skin sensitisation, both of which returned a positive result. Due to the presence of a metal ion (Cu2+) in the test substance, it was not possible to run the in vitro DRPA assay. While the available in vitro data indicate skin sensitisation potential (CLP Category 1), it is not possible to assign the substance to a sub-Category (i.e. 1A or 1B) on the basis of the available data. Thus, an estimate of the skin sensitisation potential of the substance copper diformate was made using three in silico toxicology models. Where it was possible to obtain a prediction in all three models, there was consensus that copper diformate is predicted to be a non-sensitiser. Following expert review of the model outputs, it is concluded that copper diformate is concluded not to be a skin sensitiser. Consequently, the classification of copper diformate for skin sensitisation in in Category 1A can be excluded.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
Qualifier:
according to guideline
Guideline:
other: OECD TG 442E
GLP compliance:
yes
Type of study:
activation of dendritic cells
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.




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

CD54: EXP 1 = 563
CD54: EXP 2 = 795
Key result
Parameter:
other: RFI for CD54
Value:
214
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

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

 

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.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2020-04-14 to 2020-04-30
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)
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on the study design:
Cell-ine: HaCaT human keratinocytes (immortalised adherent, stably transfected with a selectable plasmid)
Cell Culture: The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37 °C ± 2 °C in a humidified atmosphere containing 5 % CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing GlutaMAX (DMEM) (Gibco, Cat No. 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco, Cat No. 10500) and 5.5 mL Geneticin (Gibco, Cat No. 10131).
Positive control: Cinnamic aldehyde (Sigma, Cat No. 239968) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO
Test Item Solubility: The test item, Copper Diformate, was not found to be soluble in DMSO at 50 mM – 200 mM. Due to the limited solubility of the test item in DMSO, the solubility of Copper Diformate was tested in sterile water. The test item, Copper Diformate, was found to be soluble in sterile water at 200 mM, the highest concentration as recommended by the guideline this test follows.
Preparation of test item: A stock solution of the test item, Copper Diformate, was prepared by weighing the test item into a tared glass container and diluting to 200 mM in sterile water. The solution was sterilised by filtration using a 0.22 µM filter.
For test procedure please see below.
Positive control results:
The results for the positive control, cinnamic aldehyde, are shown in Table 2, Table 4.
The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 µM) in both tests.
The EC1.5 values of the positive control, cinnamic aldehyde, were 6.02 µM.
The average induction in the three replicates for cinnamic aldehyde at 64 µM were 4.62.
Run / experiment:
other: 1
Parameter:
other: Imax
Value:
20.89
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: Imax
Value:
23.93
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Value:
3.71
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Value:
3.31
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: IC30 [µM]
Value:
201.72
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: IC30 [µM]
Value:
219.54
Positive controls validity:
valid
Run / experiment:
other: 1
Parameter:
other: IC50 [µM]
Value:
238.32
Positive controls validity:
valid
Run / experiment:
other: 2
Parameter:
other: IC50 [µM]
Value:
263.03
Positive controls validity:
valid

The results for the test item, Copper Diformate, are shown in Table 1 and Table 3. The Imax for Copper Diformate was 20.89 in Test 1 and 23.93 in Test 2. The Imax for both tests was > 1.5-fold and statistically significant compared to the DMSO control. The EC1.5 was 3.71 µM and 3.31 µM for tests 1 and 2, respectively. The IC30 value was 201.72 µM in Test 1 and 219.54 µM in Test 2 and the IC50 values were 238.32 µM and 263.03 µM in Tests 1 and 2, respectively.

An overall dose-response for luciferase induction was determined.

The EC1.5 values of the positive control, cinnamic aldehyde, were 6.02 µM and 15.20 µM for Test 1 and 2, respectively, which lay within the historical control range for this laboratory (Table 5). The average induction in the three replicates for cinnamic aldehyde at 64 µM were 4.62 and 2.68 for Test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 19.4 % and 12.3 % for Test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Table 1: Results for the test item - Test 1

Test item conc (µM)

 

0.98

1.95

3.91

7.81

15.63

31.25

62.5

125

250

500

1000

2000

 

Mean fold induction

0.89

1.24

1.53

1.83

2.30

2.94

5.13

20.18

20.89

5.76

0.01

0.00

Statistically significant

N/A

N/A

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

N/A

N/A

Viability (%)

123.05

126.01

128.08

134.90

136.60

155.54

169.27

111.92

43.62

6.19

4.67

4.04

Imax

20.89

 

 

 

 

 

 

 

 

 

 

 

EC1.5 (µM)

3.71

 

 

 

 

 

 

 

 

 

 

 

IC30 (µM)

201.72

 

 

 

 

 

 

 

 

 

 

 

IC50 (µM)

238.32

 

 

 

 

 

 

 

 

 

 

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax > 1.5-fold and statistically significant

Yes

Is the cellular viability > 70 % at the EC1.5 determining concentration

Yes

Is the EC1.5 value < 1000 µM

Yes

Is there an apparent overall dose-response for luciferase induction

Yes

KeratinoSens™ prediction

Positive

Table 2: Results for Cinamic Aldehyde and DMSO Control - Test 1

Positive control conc.

4

8

16

32

64

Mean fold induction

1.34

1.65

1.82

2.77

4.62

Statistically Significant

N/A

Yes

Yes

Yes

Yes

Viability (%)

112.37

113.90

115.69

118.47

113.27

Imax

4.62

 

 

 

 

EC1.5 (µM)

6.02

 

 

 

 

IC30 (µM) N/A

N/A

 

 

 

 

IC50 (µM)

N/A

 

 

 

 

DMSO Control

1

2

3

4

5

6

Mean fold induction

0.75

0.89

0.94

0.99

1.25

1.19

Viability (%)

103.31

105.46

97.47

94.24

98.01

101.51

Test Acceptance Criteria

Result

 

Luciferase activity induction obtained with the positive control statistically significant above the threshold of

1.5 in at least one of the test concentrations

Yes

Pass

Average induction of positive control at 64 µM between 2 – 8

Yes (4.62)

Pass

EC1.5 of positive control within two standard deviations of the historical mean (-2.36 to 28.67)

Yes (6.02 µM)

Pass

CV% of blank values < 20%

Yes (19.4 %)

Pass

Table 3: Results for the test item - Test 2

Test item conc (µM)

 

0.98

1.95

3.91

7.81

15.63

31.25

62.5

125

250

500

1000

2000

 

Mean fold induction

0.86

1.29

1.59

1.78

2.05

2.50

3.97

16.36

23.93

6.69

0.01

0.00

Statistically significant

N/A

N/A

Yes

Yes

Yes

Yes

Yes

Yes

Yes

Yes

N/A

N/A

Viability (%)

103.58

115.70

127.11

122.19

135.09

145.09

146.35

124.46

52.46

5.32

3.91

3.28

Imax

23.93

 

 

 

 

 

 

 

 

 

 

 

EC1.5 (µM)

3.31

 

 

 

 

 

 

 

 

 

 

 

IC30 (µM)

219.54

 

 

 

 

 

 

 

 

 

 

 

IC50 (µM)

263.03

 

 

 

 

 

 

 

 

 

 

 

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax > 1.5-fold and statistically significant

Yes

Is the cellular viability > 70 % at the EC1.5 determining concentration

Yes

Is the EC1.5 value < 1000 µM

Yes

Is there an apparent overall dose-response for luciferase induction

Yes

KeratinoSens™ prediction

Positive

 

Table 4:Results for Cinamic Aldehyde and DMSO Control - Test 2

Positive control conc.

4

8

16

32

64

Mean fold induction

1.14

1.32

1.52

2.00

2.68

Statistically Significant

N/A

N/A

Yes

Yes

Yes

Viability (%)

102.25

107.34

102.25

105.69

107.34

Imax

2.68

 

 

 

 

EC1.5 (µM)

15.20

 

 

 

 

IC30 (µM) N/A

N/A

 

 

 

 

IC50 (µM)

N/A

 

 

 

 

 

DMSO Control

1

2

3

4

5

6

Mean fold induction

0.87

0.92

0.99

1.12

1.03

1.07

Viability (%)

91.54

104.05

98.50

100.14

105.22

100.53

 

Test Acceptance Criteria

Result

 

Luciferase activity induction obtained with the positive control statistically significant above the threshold of

1.5 in at least one of the test concentrations

Yes

Pass

Average induction of positive control at 64 µM between 2 – 8

Yes (2.68)

Pass

EC1.5 of positive control within two standard deviations of the historical mean (-2.36 to 28.67)

Yes (15.20)

Pass

CV% of blank values < 20%

Yes (12.3 %)

Pass

 

Table 5: Historical Control Data for Cinnamic Aldehyde

 

Mean

Standard Deviation

Laboratory Historical Data Range (Mean +/-2xSD)

 

EC1.5 (µM)

13.16

7.76

-2.36 to 28.67

 

Data from 106 testing occasions performed from 21 July 2017 until 13 February 2020

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
It was concluded that the test item, Copper Diformate, gave a positive response in the ARENrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item was likely to be a skin sensitizer.
Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
accepted calculation method
Principles of method if other than guideline:
An estimate of the skin sensitisation potential of the substance copper diformate was made using three in silico toxicology models.
Positive control results:
not applicable
Parameter:
other: not applicable
Remarks on result:
other: not applicable
Interpretation of results:
GHS criteria not met
Conclusions:
An estimate of the skin sensitisation potential of the substance copper diformate was made using three in silico toxicology models. Where it was possible to obtain a prediction in all three models, there was consensus that copper diformate is predicted to be a non-sensitiser.
Following expert review of the model outputs, it is concluded that copper diformate is concluded not to be a skin sensitiser. Consequently, the classification of copper diformate for skin sensitisation in in Category 1A can be excluded.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Based on all available data and according to a weight of evidence approach the substance is classified for skin sensitisation Category 1B.