Tetrasodium enneatitanium icosaoxide hydrate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28.10.2014 - 30.10. 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study was performed by following the recommended method (OECD Guideline for the Testing of Chemicals No. 431 “In Vitro Skin Corrosion: Human Skin Model Test”) and GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 13.4.2004

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate is as an appendix to the study report.

Test material

Test material information

Constituent 1

Reference substance name:

nonatitanium(4+) tetrasodium hydrate icosaoxidandiide

EC Number:

601-472-6

Cas Number:

117314-20-2

Molecular formula:

Sodium form: Na4Ti9O20 × n H2O Sodium/hydrogen form: 50 % Na4Ti9O20 × n H2O, 50 % Na2H2Ti9O20 × n H2O

IUPAC Name:

nonatitanium(4+) tetrasodium hydrate icosaoxidandiide

Test material form:

solid: particulate/powder

Remarks:

powder

Details on test material:

- Substance type: commercial product (pure active substance)
- Physical state: Solid powder
- Storage condition of test material: Ambient temperature, humidity and pressure. Stored in sealed containers in darkness.
- Stability under test conditions: Stable
- Purity: ca 100 %
- Particle size distribution: 2.92% <100μm
- Crystal structure: TiO6-octaedra
- Density: 2.83 x 10^3 kg/m3
- pH value: the pH value of the substance in an aqueous solution is appr. 11

Specific details on test material used for the study:

The substance is alkaline, and when in water the pH is about 11.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

other: The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

Justification for test system used:

The test model incorporates several features, which make it advantageous in the study of potential dermal corrosivity potential. The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium. Test items are applied to the culture surface, at air interface, so that undiluted and/or end use dilutions can be tested directly. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of corrosivity potential of the test item.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 Celsius degrees
- Temperature of post-treatment incubation (if applicable): After rinsing of the test item, the test plates were incubated (37 C, 5% CO2) for 3 hours with MTT. For MTT extraction, 2 mL of Isopropanol was used to completely immerse each insert. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test
item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper. Each tissue was placed into the prepared holding plate until all tissues were rinsed.
- Observable damage in the tissue due to washing: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Absorbency of each sample well was measured using the Anthos 2001 microplate reader
- Wavelength: 562nm (OD562)

DECISION CRITERIA
- The corrosivity potential of the test item was predicted from the relative mean tissue viabilities obtained after the 3 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water. Classification of corrosivity potential was based on relative viabilities for each exposure time
according to the following prediction model:

Mean tissue viability (% negative control) Prediction: Corrosive/Non-Corrosive
3 minute exposure : <50 Corrosive

3 minute exposure : >50
60 minute exposure : <15 Corrosive

3 minute exposure : >50
60 minute exposure : >15 Non-corrosive

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of Potassium Hydroxide
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

3-Minute exposure period
60-Minute exposure period

Duration of post-treatment incubation (if applicable):

After rinsing of the test item, the test plates were incubated (37 C, 5% CO2) for 3 hours with MTT. For MTT extraction, 2 mL of Isopropanol was used to completely immerse each insert. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

Number of replicates:

2x 3-Minute exposure
2x 60-Minute exposure

Test animals

Species:

human

Details on test animals or test system and environmental conditions:

TEST TISSUE:
Tissue: EPIDERM™ Reconstructed Human Epidermis Model Kit
Supplier: MatTek Corporation, Ashland, MA, USA
Date received: 28 October 2014
EpiDermTM Tissues (0.5cm2) lot number : 19690, Kit D
Assay Medium lot number : 102314TMD

Upon receipt of the EpidermTM tissues, the sealed 24-well plate was placed into a refrigerator overnight.

Test system

Type of coverage:

occlusive

Preparation of test site:

other: Not applicable. Skin tissue.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg of test item

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of Potassium Hydroxide
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

Two exposure periods:
- 3 minutes
- 60 minutes

Observation period:

Three hours incubation with MTT (37 C, 5% CO2), after rinsing the tissues from the test material with the help of Dulbecco’s phosphate buffered saline (DPBS). For MTT extraction, 2 mL of Isopropanol was used to completely immerse each insert. The plates were placed into a refrigerator overnight, to allow extraction to proceed.

Number of animals:

Two tissue samples per exposure period.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Rinsing was achieved by filling and emptying each tissue under a constant soft stream of DPBS to gently remove any residual test item. Excess DPBS was removed by blotting the bottom of the tissue insert with tissue paper.
- Time after start of exposure: Three minutes or 60 minutes.

SCORING SYSTEM:
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 and 60-Minute exposure periods, compared to the mean of the negative control tissues (n=2) treated with sterile distilled water.

The relative mean viabilities were calculated in the following way:
Relative mean viability (%) = (mean OD562 of test item / mean OD562 of negative control) x 100

Classification of corrosivity potential was based on relative viabilities for each exposure time
according to the following prediction model:

Mean tissue viability (% negative control) Prediction (Corrosive/Non-Corrosive)
- 3 minute exposure : <50 Corrosive
-3 minute exposure : >50 and 60 minute exposure : <15 Corrosive
- 3 minute exposure : >50 and 60 minute exposure : >15 Non-corrosive

Quality Criteria
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 0.8 or higher.
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 ≤ 30% relative to the negative control treated tissues following the 3-Minute exposure period.

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-Minute exposure

Value:

94.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60-Minute exposure

Value:

99.6

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.
- Colour interference: No. In this case, the true metabolic MTT reduction and the false direct MTT reduction can be differentiated and quantified.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean OD562 for the negative control treated tissues was 1.826 for the 3-Minute exposure period and 2.243 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.

- Acceptance criteria met for positive control: Yes. The relative mean tissue viability for the positive control treated tissues was 8.5 % relative to the negative control treated tissues following the 3-Minute exposure period. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Table 1. Mean OD562 Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	Exposure Time	Mean OD562**	% Viability
Negative Control	3 minute	1.826	100*
	60 minute	2.243	100*
Positive Control	3 minute	0.156	8.5
	60 minute	0.088	3.9
Test Item	3 minute	1.732	94.9
	60 minute	2.235	99.6

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

** = Mean of EpiDerm tissues tested in duplicate

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

According to GHS, the substance is not classified as corrosive to the skin.

Conclusions:

The test item was considered to be non-corrosive to the skin. Therefore, it is not classified as corrosive to skin.

Executive summary:

Introduction

The purpose of this test is to evaluate the corrosivity potential of the test item using the EPIDERM™ Human Skin Model after treatment periods of 3 and 60 minutes.

Corrosion is directly related to cytotoxicity in the EpiDerm tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Methods

Duplicate tissues were treated with the test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the test item was rinsed from each tissue before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL Isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 L samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 562 nm (OD562).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities of the test item treated tissues were: 60 minutes exposure : 99.6 % 3 minutes exposure : 94.9% Quality criteria: The quality criteria required for acceptance of results in the test were satisfied.

Conclusion

The test item was considered to be non-corrosive to the skin.