Copper Chelate Complex

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - June 2020

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- odour: odourless
- color: turquoise (blue-green)
- expiration date: 28 February 2021

Specific details on test material used for the study:

- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: no data

Details on animal used as source of test system:

EpiDerm™ Model (EPI-200-SCT) (Source: MatTek, Bratislava, Slovakia, Batch No.: 30859, Expiry date: 24 April 2020) units consist of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis (0.63 cm2). It’s 3D structure consisting of organized and proliferative basal cells, spinous and granular layers, and cornified epidermal layers are mitotically and metabolically active. Its use for skin corrosivity testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EpiDerm™ Model (EPI-200-SCT) has been validated for corrosivity testing in an international validation study [2] and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 431); therefore, it was considered to be suitable for this study.

Vehicle:

water

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- The plates with the treated epidermis units were incubated for the exposure time of 3 minutes and 1 hours at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere.
- The plates with the treated epidermis units were incubated for the exposure time of 25 minutes (± 0.5 minute) at room temperature (23.5-23.8°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
- After the incubation times the EpiDerm™ units were removed and rinsed thoroughly with DPBS solution (= insert was filled and emptied 20 times in a constant soft stream of DPBS in a glass beaker filled with at least 100 mL DPBS solution) to remove all of the test item from the epidermal surface. The rest of the DPBS was removed from the epidermal surface using a pipette (without touching the epidermis).

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number 298-93-1))
- Wavelength: 570 nm
- Spectrophotometer: standard apparatus

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-corrosive to skin if the relative tissue viability after 3-minute treatment with a test item is above 50%.and 1 hour >= 15%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

The test item was applied in this original form; no formulation was required. 25 mg test item was applied to the epidermal surfaces in each exposure time point and 25 μL of distilled water was added for wetting of the test item (to increase tissue surface contact).

Duration of treatment / exposure:

3 and 60 minutes

Duration of post-treatment incubation (if applicable):

not applicable

Number of replicates:

In this assay, two replicates for the test item were used. Two negative controls and two positive controls were also run. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific colour optical density (OD) evaluation.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

100.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

86.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

Following exposure to the 0 test item the mean cell viability after 3 minutes of treatment was 100.8 %, the mean cell viability after 1 hour of treatment was 86.8% compared to the negative control. These are above the threshold values in both cases, therefore the test item was considered to be non-corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was found to be non-corrosive in this in-vitro test.

Executive summary:

An in vitro skin corrosivity test was conducted on the test item in a reconstructed human epidermis model according to OECD test guideline 431 and EU-method. B40. Following exposure to the test item the mean cell viability after 3 minutes of treatment was 100.8 %, the mean cell viability after 1 hour of treatment was 86.8% compared to the negative control. These are above the threshold values in both cases, thereforethe test item was considered to be non-corrosive to skin under the conditions of this assay. The experiment met the validity criteria, and therefore the study was considered to be valid. In conclusion, under the conditions of this in vitro EpiDerm™ SCT Model corrosivity assay, the results indicate that the test item is non-corrosive.