Copper dioleate

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11. May- 13. July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

Version 439, 28. July 2015

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

dated 24. Aug. 2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Copper dioleate

EC Number:

214-307-4

EC Name:

Copper dioleate

Cas Number:

1120-44-1

Molecular formula:

C18H34O2.1/2Cu

IUPAC Name:

copper(2+) bis((9Z)-octadec-9-enoate)

Test material form:

solid

Remarks:

paste

Details on test material:

Solid green paste. Batch number 21092015

Specific details on test material used for the study:

Name: Copper dioleate
Batch no.: 21092015
Appearance: Green, solid/paste
Composition: Cu-dioleate.
Purity: > 99 %, IR.
Homogeneity: homogeneous
Expiry date: 21. Sep. 2018
Storage: Room Temperature (20 ± 5°C)

In vitro test system

Test system:

human skin model

Remarks:

EpiDermTM

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EpiDermTM tissue consists of human-derived epidermal keratinocytes

Source strain:

other: Human derived keratinocytes

Details on animal used as source of test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have
been cultured to form a multi-layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially
prepared cell culture inserts.

Vehicle:

unchanged (no vehicle)

Details on test system:

The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have
been cultured to form a multi-layered, highly differentiated model of the human epidermis.
It consists of organized basal, spinous and granular layers, and a multi-layered stratum
corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially
prepared cell culture inserts. EpiDermTM tissues were procured from MatTek In Vitro Life Science Laboratories, Bratislava.

MTT assay:
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (=MTT), which can be reduced
to a blue formazan.
A MTT stock solution of 5 mg/mL in DPBS buffer was prepared and stored in aliquots of
2 mL in the freezer (– 20 ± 5 °C).
2 mL of the stock solution were thawed and diluted with 8 mL of medium. This MTTsolution
with the resulting concentration of 1 mg/mL was used in the test.
For the pre-test (testing the ability of direct formazan reduction), the concentrate was
thawed and diluted with serum-free MEM directly before use.
For the main tests, the concentrate was thawed and diluted with assay medium directly
before use.

The following instruments and devices were used in the performance of the study.
Autoclave, 3870 ELV-B
Stop watches
96-well-plate photometer, Anthos Reader
Precision scales, Sartorius CPA8201
Precision scales, Mettler Toledo PB 5001-S
Analytical scales, Mettler Toledo XS 205 DU
Incubation chamber Binder, adjustable to 37 °C, 5% CO2
Table water bath, neoLab
Glass thermometer
Adjustable pipettes with sterile tips
Clean bench, category 2 (Axo Safe, MARS 12000)
Forceps, sterile
Orbital shakers, GFL 3005
Nylon mesh circles 8 mm diameter (200 μm pore (EPI-MESH))
Pipetting device, Accu Jet
Freezer
Weighing funnels

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

Tissue Amount
1.exp Add test 2. exp 3. exp
1 24.4 mg 26.4 mg 25.7 mg 25.4 mg
2 26.7 mg -- 25.8 mg 25.9 mg
3 26.0 mg -- 25.4 mg 25.9 mg

Duration of treatment / exposure:

three tissues of the human skin model EpiDermTM were treated with Copper dioleate for 60 min.
The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

other:

Remarks:

% photometric absorbance

Run / experiment:

3 experiments

Value:

63.2

Negative controls validity:

valid

Remarks:

In all experiments, the optical density of the negative control was well within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8

Positive controls validity:

valid

Remarks:

The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system

Remarks on result:

positive indication of irritation

Remarks:

In the first experiment the mean value lead to the classification “non-irritant to skin”, in the second experiment the result was “skin irritant” and in the third experiment the result was again “non-irritant to skin”. Therefore, Copper dioleate cannot be classified with the Human Skin Model Test because of the discrepant results of the three experiments.

Other effects / acceptance of results:

All validity criteria were met.
Values for negative control and for positive control were within the range of historical data of the test .

Criterion: OD of negative control
Demanded: ≥ 0.8 and ≤ 2.8
1. Experiment 1.4
2. Experiment 1.9
3. Experiment 1.8

Criterion: % Formazan production of positive control
Demanded: ≥ 20% of negative control
1. Experiment 3.6%
2. Experiment 3.5%
3. Experiment 3.4%

Criterion: SD of mean viability of the tissue replicates(%)
Demanded: ≤ 18%
1. Experiment
3.0% (negative control)
0.5% (positive control)
8.7% (test item)

2. Experiment
4.3% (negative control)
0.2% (positive control)
1.6% (test item)

3. Experiment
7.1% (negative control)
0.2% (positive control)
3.6% (test item)

Any other information on results incl. tables

All validity criteria were met.

Values for negative control and for positive control were within the range of historical data

of the test facility .

The first experiment was insufficient for classification of the test item, because the three

replicates lead to a discrepant classification, the mean value would lead to “non-irritant to

skin”. Therefore a second experiment was performed. In the second experiment the test

item showed skin irritation potential. This was a deviating value compared to the first experiment.

Therefore a third experiment was performed. The result was “non-irritant to

skin”.

Due to the equivocal results of the three experiments, no prediction can be made and the

test item cannot be classified for skin irritation with this test method.

Experiment 1              %Formazanproduction

 

Designation	Copperdioleate	PositiveControl
% Formazanproduction(tissue1)	62.1%	3.9%
% Formazanproduction(tissue2)	55.7%	3.1%
% Formazanproduction(tissue3)	44.9%	3.8%
%Formazan production (mean)	54.2%	3.6%
±SD ofmean Formazan production (%)	8.7%	0.5%

 

Designation	Copperdioleate	PositiveControl
% Formazanproduction(tissue1)	37.6%	3.3%
% Formazanproduction(tissue2)	37.0%	3.6%
% Formazanproduction(tissue3)	34.6%	3.6%
%Formazan production (mean)	36.4%	3.5%
±SD ofmean Formazan production (%)	1.6%	0.2%

 

Experiment 2              %Formazanproduction

Designation	Copperdioleate	PositiveControl
% Formazanproduction(tissue1)	37.6%	3.3%
% Formazanproduction(tissue2)	37.0%	3.6%
% Formazanproduction(tissue3)	34.6%	3.6%
%Formazan production (mean)	36.4%	3.5%
±SD ofmean Formazan production (%)	1.6%	0.2%

Experiment 3              %Formazanproduction

Designation	Copperdioleate	PositiveControl
% Formazanproduction(tissue1)	61.6%	3.7%
% Formazanproduction(tissue2)	60.6%	3.3%
% Formazanproduction(tissue3)	67.3%	3.3%
%Formazan production (mean)	63.2%	3.4%
±SD ofmean Formazan production (%)	3.6%	0.2%

 

Applicant's summary and conclusion

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The results were equivocal. They showed test substance to be irritant and non irritant. Invivo study may be neccessery to verify. However considering to avoid undue animal testing a precautionary classification will be applied.

Executive summary:

Three experiments plus an additional test on colour binding capacity of the test item were

performed. The first experiment was not sufficient for classification, because the three replicates

lead to different classification results, the mean value was above the threshold for

skin irritation. The second experiment showed a discrepant result compared to the first

experiment, the mean value was below the threshold for skin irritation. Therefore a third

experiment was performed. In the third experiment the mean value was again above the

threshold for skin irritation.

After the treatment with the test item, the relative absorbance values were reduced to

54.2 % (1. experiment), 36.4% (2. experiment) and 63.2% (3. experiment).

Therefore in the first experiment the mean value lead to the classification “non-irritant to

skin”, in the second experiment the result was “skin irritant” and in the third experiment the

result was again “non-irritant to skin”.

Therefore, Copper dioleate cannot be classified with the Human Skin Model Test because

of the discrepant results of the three experiments.

In all experiments, the optical density of the negative control was well within the required

acceptability criterion of 0.8 ≤ mean OD ≤ 2.8.

The positive control has met the acceptance criterion too, for thus ensuring the validity of

the test system.

Variation within replicates was within the accepted range for negative control, positive control

and test item (required: ≤ 18%).