Copper dichromium tetraoxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental work started on 26 July 2017 and was completed on 08 August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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:

Adopted 28 July 2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Copper dichromium tetraoxide

EC Number:

234-634-6

EC Name:

Copper dichromium tetraoxide

Cas Number:

12018-10-9

Molecular formula:

Cr2CuO4

IUPAC Name:

dichromium(3+) copper(2+) tetraoxidandiide

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: Copper dichromium tetraoxide
CAS Number: 12018-10-9
Sample: CuCr2O4
Batch: EX.14502.6
Purity: 88.5%
Physical state/Appearance: Dark blue powder
Expiry Date: 01 January 2023
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted human epidermis

Cell type:

non-transformed keratinocytes

Justification for test system used:

Following a full validation study the EpiSkin reconstructed human epidermis model showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation when the endpoint is measured by MTT reduction and for being used as a replacement for the Draize Skin Irritation Test for the purpose of distinguishing between Irritating and Non-Irritating test items.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Reconstructed Human Epidermis Model Kit
- Date received: 25 July 2017
- EpiSkin Tissues (0.38cm2) lot number: 17-EKIN-030
- Maintenance Medium lot number: 17-MAIN3-030
- Assay Medium lot number: 17-ESSC-029

PRE-TEST PROCEDURE

- Assessment of Direct Test Item Reduction of MTT:
MTT Salt Metabolism, Cell Viability Assay: The MTT assay, a colorimetric method of determining cell viability, is based on reduction of the yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt by mitochondrial succinate dehydrogenase in viable cells. One limitation of the assay is possible interference of the test item with MTT. A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. This property of the test item is only a problem, if at the time of the MTT test (after rinsing) there are still sufficient amounts of the test item present on or in the tissues. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified by using killed tissues to act as controls.

Test for Direct MTT Reduction: As specified, a test item may interfere with the MTT endpoint, if it is able to directly reduce MTT and at the same time is present on or in the tissues when the MTT viability test is performed. To identify this possible interference, the test item is checked for the ability to directly reduce MTT according to the following procedure: 10 mg of the test item was added to 2 mL of a 0.3 mg/mL MTT solution freshly prepared in assay medium. The solution was incubated in the dark at 37 °C, 5% CO2 in air for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue/purple, the test item is presumed to have reduced the MTT and the determination of skin irritation potential would be performed in parallel on viable and water-killed tissues for quantitative correction of the results.

- Assessment of Color Interference with the MTT endpoint: A test item may interfere with the MTT endpoint if it is colored. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 μL of test item was added to 90 μL of sterile water. After mixing for 15 minutes on a
plate shaker a visual assessment of the color was made. The test item was found to produce a colored solution which may interfere with the MTT endpoint. Therefore, color correction tissues were incorporated into the test to correct for this possibility. These tissues were treated identically to the tissues of the main test with the exception of being placed into assay medium for three hours post-exposure instead of MTT. Three tissues were dosed with the test item and three remained untreated to act as negative controls.

- Pre-incubation (Day 0: Tissue Arrival):
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

MAIN TEST

- Application of Test Item and Rinsing (Day 1): 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. The test item was applied topically to the corresponding tissues ensuring uniform covering. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes. At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 mL of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

- MTT Loading/Formazan Extraction (Day 3): Following the 42-Hour post-exposure incubation period each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the maintenance medium. 1.6 mL of the maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer at -14 to -30 ºC for possible inflammatory mediator determination. 2 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12-well plates. The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-Hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labeled 1.5 mL micro tubes containing 500 μL of acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed thoroughly on a vortex mixer. The tubes were refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of formazan crystals out of the MTT-loaded tissues.

- Absorbance/Optical Density Measurements (Day 6): At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored solution. For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. 200 μL of acidified isopropanol alone was added to the two wells designated as ‘blanks’. The optical density was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

- Amount of test material applied: Approximately 10 mg (26.3 mg/cm2)
- Amount of DPBS applied (negative control): 10 μL
- Amount of SDS 5% w/v applied (positive control): 10 μL
- Preparation of Negative and Positive Control Items and MTT: The negative control item, DPBS, was used as supplied. The positive control item, SDS, was prepared as a 5% w/v aqueous solution. A 3 mg/mL MTT stock solution was prepared in DPBS. The stock solution was diluted to 0.3 mg/mL with assay medium when required. A 0.04 N solution of hydrochloric acid in isopropanol was prepared when required. See 'Any other information on materials and methods' for further details on reference items.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

3 replicates treated with test item, 3 replicates treated with negative control and 3 replicates treated with positive control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test item

Value:

112.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- Direct MTT Reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Assessment of Color Interference with the MTT endpoint: The solution containing the test item was a green color, therefore additional color correction tissues were incorporated into the testing procedure.  However, the results obtained showed a negligible amount of color interference occurred.  It was therefore considered unnecessary to use the results of the color correction tissues for quantitative correction of results or for reporting purposes.
- Test Item, Positive Control Item and Negative Control Item: The initial test was repeated due to a failure to meet the assay acceptance criteria.  The individual and mean OD570 values, standard deviations and tissue viabilities for the test item, negative control item and positive control item are given in Table 1.  The mean viabilities and standard deviations of the test item and positive control, relative to the negative control are also given in Table 1.  The relative mean viability of the test item treated tissues was 112.4% after a 15-Minute exposure period and 42-Hour post-exposure incubation period. It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.
- Quality Criteria: The relative mean tissue viability for the positive control treated tissues was 10.4% relative to the negative control treated tissues and the standard deviation value of the viability was 1.9%. The positive control acceptance criteria were therefore satisfied. The mean OD570 for the negative control treated tissues was 0.661 and the standard deviation value of the viability was 0.009%. The negative control acceptance criteria were therefore satisfied. The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 3.4%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

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

Item	OD570 of tissues	Mean OD570   of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.671	0.661	0.009	101.5	100*	1.3
	0.656			99.2
	0.656			99.2
Positive Control Item	0.081	0.069	0.013	12.3	10.4	1.9
	0.070			8.5
	0.070			10.6
Test Item	0.721	0.743	0.023	109.1	112.4	3.4
	0.743			112.4
	0.766			115.9

OD =  Optical Density

SD =  Standard deviation

∗ =  The mean viability of the negative control tissues is set at 100%

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was classified as non-irritant. The following classification criteria apply: EU DSD & CLP Not classified for Irritation; UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

The study was conducted to GLP and according to OECD Guideline for the Testing of Chemicals No. 439 (Adopted 28 July 2015) and Method B.46. in vitro skin irritation: Reconstructed Human Epidermis Model Test as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009. The test item was classified as non-irritant.  The following classification criteria apply: EU DSD & CLP Not classified for Irritation; UN GHS Not classified for Irritation (category 3 can not be determined).