Tricopper bis(orthophosphate)

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Oct - 25 Oct 2012

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:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 13 April 2004

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

adopted 20 July 2016

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Department of Health of the Government of the United Kingdom, UK

Test material

Test material information

1

Reference substance name:

Tricopper bis(orthophosphate)

EC Number:

232-254-5

EC Name:

Tricopper bis(orthophosphate)

Cas Number:

7798-23-4

Molecular formula:

Cu.2/3H3O4P

IUPAC Name:

tricopper bis(orthophosphate)

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:

MODEL
EpiskinTM Small Model: The purpose of this test is to evaluate the corrosivity potential of the test item using the EPISKINTM in vitro Reconstructed Human Epidermis (RHE) Model after treatment periods of 3, 60 and 240 minutes. The EPISKINTM model is able to distinguish between corrosive and non-corrosive chemicals for all of the chemical types studied, and is also able to distinguish between known R35 (UN packing group I) and R34 (UN packing group II & III) test items.
Description: 0.38 cm2 reconstructed epidermis of normal human keratinocytes
Supplier: SkinEthic Laboratories, Lyon, France
Date received: 23 October 2012
Expiration Date: 11 November 2013
Lot number:13-EKIN-039

Quality controls
- Histological scoring (HE stained vertical paraffin sections): Well diferentiated epidermis consisting of a basal layer, several spinous, and granular layers and a thick stratum corneum, Specification ≥ 19.5, Result 22.3 ± 0.3
- IC 50 determination (MTT test n=14), Specification ≥ 1.5 mg/mL, Result ≥ 2.4 mg/mL
- Statistical analysis - Histology: probability 0.95 that 100% of the batch > 20
- Statistical analysis - IC 50: probability 0.95 that IC50 ≥ 2.3 mg/mL (treshold value)
- Biological Safety: On blood of the same donors the following was verified: absence of HIIV 1 and 2 antibodies, hepatitis C antibodies, hepatitis B antigen, bacteria, fungus and mycoplasm

PRE-TEST
Assessment of Direct Test Item Reduction of MTT
MTT Dye Metabolism, Cell Viability Assay
The MTT assay, a colourimetric 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 formazan dye 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.

Test for Direct MTT Reduction
As specified, a test item may interfere with the MTT endpoint, if it was able to directly reduce MTT and at the same time was present on or in the tissues when the MTT viability test was performed. To identify this possible interference, the test item was checked for the ability to directly reduce MTT according to the procedure below:
20 mg of the test item was added to 2.2 ml of a 0.3 mg/ml MTT solution freshly prepared in assay medium. The solution was incubated in the dark at room temperature for 3 hours. Untreated MTT solution was used as a control. If the MTT solution containing the test item turns blue relative to the control, the test item was presumed to have reduced the MTT.

PRE-INCUBATION (Day 0: tissue arrival)
2.2 mL of maintenance medium, warmed to approximately 37ºC, was pipetted into two wells of the first column of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (2 units per plate). A different 12-well plate was used for each test item, control and time point. The tissues were incubated at 37ºC, 5% CO2 in air overnight.

MAIN TEST
Application of Test Item and Rinsing (Day 1)
2.2 mL of assay medium, warmed to approximately 37ºC, was pipetted into 2 wells of the second and third columns of the 12-well plate. The tissues were transferred into the second column.
Duplicate tissues were treated with the test item for exposure periods of 3, 60 and 240 minutes. Duplicate tissues were treated with the positive and negative control items for an exposure period of 240 minutes. 20 mg of the solid test item was applied topically to the corresponding tissues ensuring uniform coverage of the tissues. 100 μL of 0.9% w/v sodium chloride solution was added for wetting of the test item. Duplicate tissues, treated with 50 μL of 0.9% w/v sodium chloride solution served as negative controls. Duplicate tissues, treated with 50 μL of glacial acetic acid served as positive controls. The treated tissues were kept in a biological safety cabinet at room temperature for the appropriate exposure period.
At the end of each exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing Phosphate Buffered Saline Dulbeccos (PBS) with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of PBS to gently remove any residual test item. Each rinsed tissue was placed into the third column of the 12-well plate until all tissues were rinsed.
2.0 mL of a 0.3 mg/mL MTT solution, freshly prepared in assay medium, was pipetted into 2 wells of the fourth column of each 12 well plate. The tissues were transferred into the MTT filled wells. The tissues were incubated for 3 hours ±5 minutes at 37ºC, 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 taken using the EPISKINTM biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) were placed into labelled 1.5 mL micro tubes containing 500 μL of acidified isopropanol. Each tube was plugged, mixed thoroughly and stored overnight at room temperature, protected from light, to extract formazan crystals out of the MTT-loaded tissues.

Absorbance/Optical Density Measurements (Day 2)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous coloured solution.
For each tissue, duplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 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 540 nm (without a reference filter) using the Anthos 2001 microplate reader.

INTERPRETATION OF RESULTS
Quantitative MTT Assessment (percentage tissue viability)
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution. The relative mean viabilities were calculated in the following way:

Relative mean viability (%) = (mean OD540 of the test item/mean OD540 of negative control) x 100

SCORING SYSTEM:
The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3, 60 and 240-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with 0.9% w/v sodium chloride solution.

QUALITY CONTROL
The results of the assay are considered acceptable if the following assay acceptance criteria are achieved:
Negative Control: The assay establishes the acceptance criterion for an acceptable test if the mean optical density for the negative control treated tissues is ≥0.600 and ≤1.500.
Positive Control: The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues was 0 to 20% relative to the negative control treated tissues following the 240-Minute exposure period.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg, 100 µL of 0.9% (w/v) sodium chloride solution was added for wetting of the test item.

Duration of treatment / exposure:

3, 60 and 240 minutes

Number of replicates:

duplicate tissues

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min experiment

Value:

82.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 min experiment

Value:

63.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 min experiment

Value:

62.3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue. This was taken to indicate the test item did not reduce MTT.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD540 for the negative control treated tissues was 0.812. The negative control acceptance criterion was therefore satisfied
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 2.5% relative to the negative control treated tissues following the 240-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1: Mean OD₅₄₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Period	Mean OD540of duplicate tissues	Relative mean viability (%)
Negative Control Item	240 Minutes	0.812	100
Positive Control Item	240 Minutes	0.020	2.5
Test Item	240 Minutes	0.506	62.3
	60 minutes	0.513	63.2
	3 minutes	0.673	82.9

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive according to OECD 431

Conclusions:

Under the conditions of the conducted test, the test substance did not possess corrosive properties towards reconstructed human epidermis tissue in the Episkin™ model, but no prediction on the skin irritation potential can be made and additional testing should be conducted for classification and labeling purposes.