Dilithium titanate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 24 March 2020 Experimental completion date 27 March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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)

Deviations:

yes

Remarks:

Please see comments below in "Principles of Method if other than guideline"

Principles of method if other than guideline:

In order to determine the potential color interfering properties of the test item, the OECD Guideline 492 proposes absorbance measurement (570 ± 20 nm) of the test item in water and isopropanol. However, in contrast to the MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test, the OECD guideline has not distinguished between intrinsically colored test items and non-intrinsically colored test items.
Non-intrinsically colored test items may become ‘turbid’ but not ‘colored’ in solution and consequently ‘falsely’ measured as colored by way of absorption measurement. Therefore, this study followed the MatTek recommended procedure whereby the non-intrinsically colored Test Item; Lithium titanate; was visually observed for color in water and isopropanol.

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Dilithium titanate

EC Number:

234-759-6

EC Name:

Dilithium titanate

Cas Number:

12031-82-2

Molecular formula:

Li.1/2O3Ti

IUPAC Name:

dilithium(1+) oxotitaniumbis(olate)

Test material form:

solid: bulk

Details on test material:

- Purity: >98%
- Empirical Formula: Li2TiO3
- Molecular mass: 109.8 g/mol

Specific details on test material used for the study:

Identification: Lithium titanate
Chemical Name: Lithium titanium oxide
CAS Number: 12031-82-2
EC Number: 234-759-6
Empirical Formula: Li2TiO3
Molecular Mass: 109.8 g/mol
Batch: SLEA 7084
Sample Number: 2914
Purity: > 98 %
Physical state/Appearance: Off white powder*
Date of Receipt: 12 July 2019
Expiry Date: 23 September 2021
Storage Conditions: Room temperature in the dark
*The appearance of the test item as stated in the Certificate of Analysis was confirmed by the contract laboratory.

Test animals / tissue source

Species:

other: Human cornea model

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

Test System
EpiOcularTM Human Corneal Model (0.6 cm2)
Supplier : MatTek In Vitro Life Science Laboratories, Bratislava - Slovakia
Date received : 24 March 2020
EpiOcularTM Tissues Lot Number : 30651
Assay Medium Lot Number : 032320MSA

MTT-Solution
MTT concentrate and MTT diluent were supplied as an MTT test kit (MTT-100). An MTT solution was prepared when required. MTT concentrate was diluted in MTT diluent (2 mL of concentrate to 8 mL diluent) to produce a 1.0 mg/mL MTT solution and used within 1 hour.

Miscellaneous Assay Reagents
DPBS (without Ca++ Mg++) Lot Number : 2113924
Isopropanol (MTT) extractant) Lot Number :1863832


Preparation and Pre-Incubation of EpiOcularTM Tissues
Upon receipt of the EpiOcularTM tissues, the sealed 24-well plate and the assay medium were placed into the refrigerator (2 to 10 °C) until the equilibration step. The vial containing the MTT concentrate was placed in the freezer (-35 to -10 °C) and the MTT diluent placed in the refrigerator (2 to 10 °C). The positive control, Methyl Acetate, was stored at room temperature, in the dark.

On the day of receipt the equilibration step (15 minutes at room temperature in the 24-well shipping container) was started. An appropriate volume of EpiOcular™ Assay medium was warmed to approximately 37 °C and 1 mL of the medium aliquoted into the appropriate wells of pre-labeled 6-well plates.

Each 24-well shipping container was removed from its plastic bag under sterile conditions and its surface disinfected by wiping with ethanol soaked tissue paper. The sterile gauze was removed and each tissue inspected for air bubbles between the agarose gel and insert. Cultures with air bubbles under the insert covering greater than 50% of the insert area were not used. The tissues were carefully removed from the 24-well shipping container using sterile forceps. Any agarose adhering to the inserts was removed by gentle blotting on sterile filter paper or gauze. The insert was then transferred aseptically into the 6-well plates and pre-incubated at standard culture conditions for 1 hour in Assay Medium. After 1 hour, the Assay Medium was replaced by 1 mL of fresh Assay Medium at 37 °C and the EpiOcular™ tissues was incubated at standard culture conditions overnight (16 to 24 hours).

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg of test item was applied atop duplicate cultures

Duration of treatment / exposure:

Exposure period of 6 hours ±15 minutes at 37 °C, 5% CO2 followed by rinsing, a post treatment immersion and a post treatment incubation.

Duration of post- treatment incubation (in vitro):

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labeled 12-well plate for a 25 ±2 minutes immersion incubation (post‐treatment immersion) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.

Details on study design:

Study Design
Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
A test item may directly reduce MTT, thus mimicking dehydrogenase activity of the cellular mitochondria. Therefore, it was necessary to assess this ability of the test item to directly reduce MTT prior to conducting the assay. This property of the test item is only a problem, if at the time of the MTT test (after the test item has been rinsed off) there is still a sufficient amount 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 the procedure described as follows:

50 mg of the test item was added to 1 mL of MTT solution and incubated at 37 °C, 5% CO2 for 3 hours. A control (50 µL sterile water in MTT solution) was run concurrently. If the MTT solution turned blue/purple, the test item was presumed to have directly reduced the MTT. Blue, dark purple and black test items should be tested on killed controls because it may not be possible to assess their potential to directly reduce MTT.

Assessment of Color Interference with the MTT endpoint
Colored test items or those which become colored after application to the tissues may interfere with the quantitative photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, the test item was checked for its colorant properties.

Test items which absorb light and appear red, yellow, green or blue should be considered as intrinsic colorants. A test item which appears black may absorb light and should be considered as a colorant. Blue, purple and black test items may be directly tested on colorant controls without further tests because it is obvious that they can interfere with the blue/purple MTT product. Such test items should also be tested on killed controls because it may not be possible to assess their potential to directly reduce MTT.

For non-colored test items, tests have to be performed to assess if they become colorants after contact with water or isopropanol. For this purpose 50 mg of the test item was added to 1.0 mL of water in a 6-well plate and the mixture was incubated in the dark at 37 ±1 °C in a humidified atmosphere of 5 ±1% CO2 in air for at least 1 hour. Furthermore, 50 mg of the solid test item was added to 2 mL of isopropanol, the same amount as used for MTT extraction, incubated in 6 well plates, and placed on an orbital plate shaker for 3 hours at room temperature.

Main Test
Application of Test Item and Rinsing
After the overnight incubation, the tissues were pre-wetted with 20 µL of Ca++ Mg++ free DPBS to mimic the wet condition of the human eye. If the Ca++ Mg++ free DPBS was not spread across the tissues, the plate was tapped to assure that the entire tissue surface was wetted. The tissues were incubated at 37 °C, 5% CO2 for 30 ±2 minutes.

50 mg of test item was applied atop duplicate cultures for an exposure period of 6 hours ±15 minutes at 37 °C, 5% CO2 followed by rinsing, a post treatment immersion and a post treatment incubation (described below). 50 μL of the negative and positive controls were similarly applied.

At the end of the test item exposure period, the test item was removed by extensively rinsing the tissues with Ca++ Mg++ free DPBS at room temperature. Three clean beakers (glass or plastic with minimum 150 mL capacity), containing a minimum of 100 mL each of Ca++ Mg++ free DPBS were used per test item or control with each test item or control item utilizing a different set of three beakers. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. The tissues were rinsed two at a time by holding replicate inserts together by their collars using forceps. The test or control items were decanted from the tissue surface onto a clean absorbent paper towel and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed two additional times in the first beaker. The inserts were then rinsed in the second and third beakers of DPBS three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent paper. Decanting was most efficiently performed by rotating the insert to approximately a 45° angle (open end down) and touching the upper lip to the absorbent paper (to break the surface tension).

After rinsing, the tissues were immediately transferred to and immersed in 5 mL of assay medium at room temperature in a pre-labeled 12-well plate for a 25 ±2 minutes immersion incubation (post‐treatment immersion) at room temperature. This incubation in assay medium was intended to remove any test item absorbed into the tissue.

At the end of the post‐treatment immersion, each insert was removed from the assay medium, the medium was decanted off the tissue, the insert was blotted on absorbent paper, and transferred to the appropriate well of the pre-labeled 6-well plate containing 1 mL of assay medium at approximately 37 °C. The tissues were incubated for a period of 18 hours ±15 minutes at 37 °C, 5% CO2 (post treatment incubation).

MTT Assay
At the end of the post-treatment incubation, each insert was removed from the 6-well plate and gently blotted on absorbent paper. The tissues were placed into the 24-well plate containing 0.3 mL of 1.0 mg/mL MTT solution. Once all the tissues were placed into the 24 well plate, the plate was incubated at 37 °C, 5% CO2 in air for 3 hours.

A procedure was used which only extracted from beneath the tissue, since residual test item may remain on the tissue and could contaminate the isopropanol. Inserts were removed from the 24‐well plate after approximately 3 hours. The bottom of the insert was blotted on absorbent paper and then transferred to a pre‐labeled 6 well plate containing 2 mL isopropanol in each well so that no isopropanol flowed into the insert. The plates were sealed with a film sealer (between the plate cover and upper edge of the wells) or a standard plate sealer and stored overnight at 2 to 10 °C in the dark.

Results and discussion

In vitro

Results

Irritation parameter:

other: Percentage cell viability

Run / experiment:

Average value of all runs

Value:

16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

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 water and isopropanol solutions were colorless. It was therefore unnecessary to run color correction tissues.

Test Item, Positive Control Item and Negative Control Item
The relative mean viability of the test item treated tissues was 16.0% after a 6-Hour exposure period and 18 Hour post exposure incubation period.

It was reported that a small amount of test item adhered to the tissue culture surfaces which could not be removed at the end of the exposure period by rinsing.

Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 23.7% relative to the negative control treated tissues. The positive control acceptance criterion was therefore satisfied.
The mean OD570 for the negative control treated tissues was 1.691. The negative control acceptance criterion was therefore satisfied.

The difference in viability between the two relating tissues in each treatment group was <20%. The test item acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Mean OD₅₇₀Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item	OD570of tissues	Mean OD570of duplicate tissues	Individual tissue viability (%)	Relative mean viability (%)	Difference in viability (%)
Negative Control Item	1.607	1.691	95.0	100*	10.0
	1.775		105.0
Positive Control Item	0.424	0.400	25.1	23.7	2.9
	0.375		22.2
Test Item	0.333	0.270	19.7	16.0	7.5
	0.206		12.2

 

OD= Optical Density

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

Applicant's summary and conclusion

Interpretation of results:

other: it can be stated that in this study and under the reported experimental conditions, a prediction on the eye irritating potential of Lithium titanate cannot be made.

Conclusions:

No Prediction can be made and ill-defined exposure duration.

In conclusion, it can be stated that in this study and under the reported experimental conditions, a prediction on the eye irritating potential of Lithium titanate cannot be made.

Executive summary:

Introduction

The purpose of this study was to identify chemicals not requiring classification and labelling for eye irritation or serious eye damage using the EpiOcular™ Eye Irritation Test (EIT) according to the OECD Test Guideline 492 Reconstructed human Cornea-like Epithelium (RhCE) test method.

Method

Duplicate tissues were treated with the test item for an exposure period of 6 hours. At the end of the exposure period each tissue was rinsed before incubating for 18 hours. At the end of the post‑exposure incubation period each tissue was taken for MTT-loading. After MTT-loading each tissue was placed in 2 mL of isopropanol for MTT extraction. 

At the end of the formazan extraction period each well was mixed thoroughly and duplicate 200 mL samples were transferred to the appropriate wells of a pre-labeled 96‑well plate. The optical density (OD) was measured at 570 nm (OD₅₇₀).

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 viability of the test item treated tissues was16.0%.

It was reported that test item had adhered to the tissue culture surfaces which could not be removed at the end of the exposure period by rinsing. The presence of this adhered test item following rinsing may be considered to be extending the time of exposure.

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion

No Prediction can be made and ill-defined exposure duration.

In conclusion, it can be stated that in this study and under the reported experimental conditions, a prediction on the eye irritating potential of Lithium titanate cannot be made.