Titanium zirconium oxide

Endpoint summary

Description of key information

The test item was not irritating to skin in the In Vitro Skin Irritation Test (Reconstructed Human Epidermis Model) according to OECD Guideline 439.

The test item was not irritating to eye in the In Vitro Eye Irritation Test (Reconstructed human Cornea-like Epithelium (RhCE) Model) according to OECD Guideline 492.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 6, 2017 - December 8, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 28, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: SkinEthic Skin Irritation Test (42 bis) Standard Operating Procedure (SOP): Using the Reconstructed Human Epidermis (RHE) model, INVITTOX

Version / remarks:

Version 2.1 (2009)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Episkin/SkinEthic Laboratories, Lyon, France

Source strain:

not specified

Justification for test system used:

The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number(s): 17-RHE-124
- Date of initiation of testing: December 6, 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The test item, negative and positive control were removed immediately by gently rinsing with a minimum volume of 25 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours +/- 5 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD > 0.7
- Barrier function: 4.0 h <= ET50 <= 10.0 h
- Morphology: Number of cell layers >= 4. Absence of significant histological abnormalities. Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum.

NUMBER OF REPLICATE TISSUES: in triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues
- N. of replicates : 1
- Method of calculation used: A direct interaction with the MTT was detected if the solution clearly turned blue or purple.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after exposure and post-treatment incubation is less than or equal to 50%. Since the in vitro skin irritation test according to OECD 439 cannot resolve between UN GHS Categories 1 and 2, further information on skin corrosion is required to decide on its final classification.
- The test substance is considered as non-irritant to skin if the viability after exposure and post-treatment is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount applied: 16 mg +/- 2 mg

NEGATIVE CONTROL
- Amount applied: 16 µL +/- 0.5 µL
- Concentration (if solution): 16 µL +/- 0.5 µL

POSITIVE CONTROL
- Amount applied: 16 µL +/- 0.5 µL

Duration of treatment / exposure:

42 minutes +/- 1 minute

Duration of post-treatment incubation (if applicable):

42 hours +/- 1 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 1

Value:

90.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 2

Value:

94.8

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue 3

Value:

89.5

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 μL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 μL of deionized water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met.

Following treatment with the test item, the tissue viability was 91.5% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 12, 2017 - December 14, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

October 9, 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; For use with MatTek Corporation's Reconstructed Human EpiOcular Model; MatTek Corporation

Version / remarks:

June 29, 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 164: Ocular Irritation Assay for Chemicals using EpiOcular™ EIT

Version / remarks:

September 14, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability :
The reconstructed human comea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye Epiocular™-model closely mimics the biochemical and physiological properties ofthe human eye, i.e. the comea.
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: The Epiocular™-model is a nonkeratinized epithelium prepared from normal human keratinocytes. lt models the cornea epithelium with progressively stratified, but not cornified cells. lt is used for eye irritation testing.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 50 mg

The test item was applied neat to the tissues.

Duration of treatment / exposure:

6 hours +/- 15 minutes

Duration of post- treatment incubation (in vitro):

18 hours +/- 15 min

Number of animals or in vitro replicates:

two tissues

Details on study design:

- Details of the test procedure used
- RhCE tissue construct used, including batch number: EpiOcular™ Tissue (OCL-200, OCL-212), Lot No. 27017
- Doses of test chemical and control substances used: 50 mg test item, 50 µL negative control, 50 µL positive control
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods: 6 hours (+/- 15 min) at 37°C, 25 min (+/- 2 min) at room temperature, 18 hours (+/- 15 min) at 37°C
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: A pre-test for direct MTT-reducing capacity of the test item was conducted and the test item was checked for its colorant properties.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2 tissues (test item, positive control, negative control), 2 tissues to assess direct MTT reduction and staining test items, respectively
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm, ELx800, BioTek Instruments GmbH
- Description of the method used to quantify MTT formazan: To extract the MTT, the plates was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The extract solution was mixed and 2 x 200 μL were transfe1Ted into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength.
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: If the test item-treated tissue viability is >60.0% relative to negative control-treated tissue viability, the test item is labeled non-irritant (UN GHS No Category). If the test item-treated tissue viability is less than or equal to 60.0% relative to negative control-treated tissue viability, the test item is labeled irritant (UN GHS Category 1 or Category 2).

- Acceptability of the Test:
- The negative control OD is >0.8 and <2.5
- The mean relative viability of the positive control is below 50% of the negative control viability.
- The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals).

Irritation parameter:

other: Cell viability (%)

Run / experiment:

Tissue 1

Value:

93

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Irritation parameter:

other: Cell viability (%)

Run / experiment:

Tissue 2

Value:

107

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions ofthe present study, the test item did not show an eye hazard potential. The test item is labeled non-irritant (UN GHS: No Category).

Executive summary:

The objective of the present study was to investigate the potential of the test item to induce eye irritation in an in vitro human cornea model.

The test item was applied topically to a reconstructed human cornea-like epithelium model (Epiocular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the Epiocular™-model were treated with the test item, the negative or the positive control for 6 hours (± 15 minutes). 50 mg of the test item and 50 μL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues.

After treatment with the negative control (sterile deionized water) the mean OD was 1.509 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 47.0% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 85.7% and, thus, higher than 60%, i.e. according to OECD 492 the test item is labeled non-irritant (UN GHS: No Category).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the data provided, the test item is not classified and labelled for skin and eye irritation according to Regulation (EC) No 1272/2008.