Copper diformate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-07-02 to 2019-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

29 July 2016

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EU Method B.40bis (In Vitro Skin Corrosion: Reconstructed Human Epidermis Test Method (RHE))

Version / remarks:

30 May 2008

GLP compliance:

yes (incl. QA statement)

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 color

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue lot number(s): 30804
- Delivery date: 02 July 2019
- Date of initiation of testing: 02 July 2019

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure and post-treatment incubation: 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps:
3-Minute exposure: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) for approximately 40 seconds, to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed. They were then blotted and transferred to the 24-well plate prepared for MTT-loading. The plate was incubated (37 °C, 5% CO2) for 3 hours. Once the 60-Minute exposure period was complete, the same rinsing and MTT-loading procedure was repeated.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Optical Density (OD): OD was read in a microplate reader (Labtech LT-4500 microplate reader and LT-com analysis software)
- Filter bandwidth: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: mean OD of two tissues is >=0.8 <=2.8

NUMBER OF REPLICATE TISSUES: 2


CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
-Test for direct MTT reduction:
25 mg of the test item was added to 1 mL of a freshly prepared 1.0 mg/mL MTT solution. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. Untreated MTT solution was tested concurrently to act as a control. The MTT solution containing the test item turned a green color as opposed to a blue/purple colour. A blue/purple color is considered to be a positive and therefore indicative of a direct MTT reducer. The green colored MTT solution was considered to be inconclusive and therefore, as a precaution, an additional procedure using freeze-killed tissues was performed. There was a possibility that if the test item could not be totally rinsed off the tissues, any residual test item present on or in the tissue may directly reduce MTT and could have given rise to a false negative result. Therefore, the determination of skin corrosion potential was performed in parallel on viable and freeze-killed tissues.
Freeze-killed tissues: Freeze-killed tissues were prepared prior to the study by placing untreated EPIDERMTM tissues in an empty 12-well plate and storing in a freezer (-35 to -10 °C) for a minimum of 24 hours. Before use each tissue was thawed by placing in 0.9 mL of assay medium for approximately 1 hour at room temperature. In addition to the normal test procedure, the test item was applied to two freeze-killed tissues per exposure period. In addition, two freeze-killed tissues per exposure period remained untreated. The untreated freeze-killed control normally show a small amount of MTT reduction due to residual reducing enzymes within the killed tissues.
-Assessment of Color Interference with the MTT Endpoint:
25 mg of test item was added to 300 µL of sterile water. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 60 minutes. A visual assessment of the color was then made.
The test item was found to produce a colored solution which may interfere with the MTT assay. Therefore, viable color correction tissues were incorporated into the test to correct for this possibility. These tissues were treated the same as the main test with the exception of being placed into assay medium for 3 hours post-exposure instead of MTT. Two tissues were dosed with the test item and two remained untreated to act as negative controls for the 3 minute exposure group. The same was also performed for the 60 minute exposure group.
-Double Correction Check:
A third set of controls was also used to prevent a double correction from a colored test item that also reduces MTT using killed tissues.
Two freeze-killed tissues were dosed with the test item and two freeze-killed tissues remained untreated to act as the negative control for the 3 minute exposure, with the same number being used for the 60 minute exposure group. These tissues were incubated with assay medium instead of MTT post-exposure.


PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- For the test item the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.
- Classification of corrosivity potential is based on relative viabilities for both exposure times
- The test substance is considered to be corrosive to skin if relative mean tissue viability is <50% after 3 min exposure or >= 50% after 3 min exposure AND < 15% after 60 min exposure.
- The test substance is considered to be non-corrosive to skin if relative mean tissue viability is >= 50% after 3 min exposure AND >= 15% after 60 min exposure.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

other: Assessment of Color Interference with the MTT endpoint

Duration of treatment / exposure:

3 min and 60 min

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Remarks:

3 min exposure

Run / experiment:

mean of 2 replicates

Value:

84.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to the skin

Results 2

Irritation / corrosion parameter:

% tissue viability

Remarks:

60 min exposure

Run / experiment:

mean of 2 replicates

Value:

31.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive to the skin

Any other information on results incl. tables

During the assessment of direct reduction of MTT the MTT solution containing the test item turned a green color as opposed to a blue/purple colour. A blue/purple color is considered to be a positive and therefore indicative of a direct MTT reducer. The green colored MTT solution was considered to be inconclusive and therefore, as a precaution, an additional procedure using freeze-killed tissues was performed. However, the results obtained showed that no or negligible interference due to direct reduction of MTT occurred. It was therefore considered unnecessary to use the results of the freeze-killed tissues for quantitative correction of results.

The solution containing the test item turned a blue color, therefore additional viable color correction tissues were incorporated into the testing procedure. However, the results obtained showed that no color interference occurred. It was therefore considered unnecessary to use the results of the viable color correction tissues for quantitative correction of results.

As it was unnecessary to use the results of the viable color correction tissues for quantitative correction it was therefore also unnecessary to use the results of the killed color correction tissues as no double correction for color interference would have occurred.

Table 1: Mean OD570 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Tissue		Exposure Period	Mean OD570 of individual tissues	Mean OD570 of duplicate tissues	Standard Deviation	Coefficient of Variation (%)	Relative Mean Viability (%)
Negative Control		3 Minutes	1.898	1.775	0.174	9.8	100*
			1.652
		60 Minutes	1.832	1.788	0.063	3.5
			1.743
Positive Control		3 Minutes	0.096	0.085	0.016	na	4.8
			0.074
		60 Minutes	0.079	0.067	0.018	na	3.7
			0.054
Test Item		3 Minutes	1.644	1.499	0.206	13.7	84.4
			1.353
		60 Minutes	0.603	0.561	0.060	10.7	31.3
			0.518

OD = Optical density

* = The mean percentage viability of the negative control tissue is set at 100%

na = Not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.