Dimolybdenum trizinc nonaoxide

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dimolybdenum trizinc nonaoxide

EC Number:

245-322-4

EC Name:

Dimolybdenum trizinc nonaoxide

Cas Number:

22914-58-5

Molecular formula:

Mo2O9Zn3

IUPAC Name:

dimolybdenum trizinc nonaoxide

impurity 1

Reference substance name:

Molybdenum zinc tetraoxide

EC Number:

237-377-8

EC Name:

Molybdenum zinc tetraoxide

Cas Number:

13767-32-3

Molecular formula:

MoO4Zn

IUPAC Name:

molybdenum zinc oxide

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: adult human-derived epidermal keratinocytes

Source strain:

not specified

Details on animal used as source of test system:

The EPISKIN model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-Day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.

Justification for test system used:

Standard as per OECD Guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s): 18-EKIN-032
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: 07 August 2018 (expiry date 13 August 2018)
- Date of initiation of testing: Pre-incubation commenced on day of tissue arrival.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 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.
- Observable damage in the tissue due to washing: None specified
- Modifications to validated SOP: Not applicable

MTT LOADING/FORMAZAN EXTRACTION
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 EPISKIN 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.

ADSORBANCE/OPTICAL DENSITY
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-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.

NUMBER OF REPLICATE TISSUES: Triplicate tissues treated with the test item for an exposure period of 15 minutes.

MTT INTERFERANCE
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 was 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. A test item may interfere with the MTT endpoint if it is coloured. The MTT assay is affected only if the test item is present in the tissues when the MTT viability assay is performed. 10 mg 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 colour was made.

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability is - The test substance is considered to be a non-irritant to skin if the relative mean tissue viability is >50%.

Control samples:

yes, concurrent negative control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Approximately 10 mg (26.3 mg/cm2).

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): DPBS used as supplied

POSITIVE CONTROL
- Amount(s) applied (volume or weight): SDS
- Concentration (if solution): Applied at 0.3 mg/mL in assay medium

Duration of treatment / exposure:

Tissues were exposed for 15 minutes ahead of rinsing

Duration of post-treatment incubation (if applicable):

42 hours post exposure incubation

Number of replicates:

Tissues were tested in triplicate

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

107

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:

- OTHER EFFECTS:
- Visible damage on test system: Not specified
- 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.
- Colour interference with MTT: The solution containing the test item was colourless. It was therefore unnecessary to run colour correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.704 and the standard deviation value of the viability was 5.5%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 5.8% relative to the negative control treated tissues and the standard deviation value of the viability was 1.6%. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 6.5%. The test item acceptance criterion was therefore satisfied.
- Range of historical values if different from the ones specified in the test guideline: Not applicable.

Any other information on results incl. tables

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	0.660	0.704	0.039	93.8	100	5.5
	0.718			102.0
	0.734			104.3
Positive Control Item	0.052	0.041	0.011	7.4	5.8	1.6
	0.030			4.3
	0.040			5.7
Test Item	0.784	0.753	0.046	111.4	107.0	6.5
	0.700			99.4
	0.774			109.9

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance did not induce a relative mean tissue viability of