Dilithium titanate

Endpoint summary

Description of key information

The corrosive and irritating properties of dilithium titanate to the skin were tested by means of In vitro Membrane Barrier Test for Skin Corrosion (OECD TG 435) and EPISKIN reconstructed human epidermis model (OECD TG 439) respectively. It was concluded that the test item was non-corrosive and non-irritating to the skin.

 

The serious eye damage and the irritating potential of dilithium titanate was assessed based on Reconstructed Human Cornea Like Epithelium (RhCE) test method EpiOcularTM (OECD 492) and The Bovine Corneal Opacity and Permeability (BCOP) Assay (OECD 437), as well as on data available on a structurally related substance. It was concluded that the substance does not cause serious eye damage or irritation to the eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 16 October 2019 Experimental completion date 21 October 2019

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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
Date of Receipt: 12 July 2019
Expiry Date: 23 September 2021
Physical state/Appearance: Off white powder
Purity: >98%
Storage Conditions: Room temperature in the dark

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EPISKIN™ Reconstructed Human Epidermis Model Kit
Supplier: EpiSkin Laboratories, Lyon, France
Date received: 15 October 2019
EpiSkinTM Tissues (0.38cm^2) lot number: 19-EKIN-042
Maintenance Medium lot number: 19-MAIN3-046
Assay Medium lot number: 19-ESSC-043

Vehicle:

unchanged (no vehicle)

Details on test system:

Pre-Test Procedure
Assessment of Direct Test Item Reduction of MTT
MTT Salt Metabolism, Cell Viability Assay
The MTT assay, a colorimetric 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/purple formazan salt 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 by using killed tissues to act as controls.
Test for Direct MTT Reduction
As specified, 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 is 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.
Assessment of Color Interference with the MTT endpoint
A test item may interfere with the MTT endpoint if it is colored or becomes colored when in contact with water. 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 color was made.
Pre-incubation (Day 0: Tissue Arrival)
Before removal from the transport plate each tissue was inspected for any air bubbles between the agarose gel and the insert:
Tissues Satisfactory: Yes
Temperature Indicator Color Satisfactory: Yes
Agar Medium Color Satisfactory: Yes
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air approximately 24 hours.

Main Test
Application of Test Item and Rinsing (Day 1)
2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the second column of 3 wells of the 12-well plate.
Triplicate tissues were treated with the test item for an exposure period of 15 minutes. 5 μL of sterile distilled water was topically applied to the epidermal surface in order to improve contact between the test item and the epidermis. Approximately 10 mg (26.3 mg/cm^2) of the neat test item was then applied topically to the epidermal surface ensuring uniform covering. Triplicate tissues treated with 10 μL of DPBS served as the negative controls and triplicate tissues treated with 10 μL of SDS 5% w/v served as the positive controls. To ensure satisfactory contact with the positive control item the SDS solution was spread over the entire surface of the epidermis using a pipette tip (taking particular care to cover the center). After a 7-Minute contact time the SDS solution was re-spread with a pipette tip to maintain the distribution of the SDS for the remainder of the contact period (re-spreading is not required for the negative control or test item). The plates were kept in the biological safety cabinet at room temperature for 15 minutes.
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. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
MTT Loading/Formazan Extraction (Day 3)
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 (-35 to -10 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 2 to 10 °C until Day 6 of the experiment (approximately 75 hours), allowing the extraction of formazan crystals out of the MTT-loaded tissues.
Absorbance/Optical Density Measurements (Day 6)
At the end of the formazan extraction period each tube was mixed thoroughly on a vortex mixer to produce a homogenous colored 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 (OD570) was measured (quantitative viability analysis) at 570 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

10 mg (26.3 mg/cm^2) of the neat test item
10 μL of DPBS served as the negative controls
10 μL of SDS 5% w/v served as the positive controls.

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

42 hours

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean

Value:

109.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

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 solution containing the test item was 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 109.2% after a 15-Minute exposure period and 42-Hour post-exposure incubation period. This value is above the threshold for irritancy of ≤ 50%. Therefore the test item is considered not to possess an irritant response.
It was considered unnecessary to perform IL-1α analysis as the results of the MTT test were unequivocal.
Acceptance Criteria
The relative mean tissue viability for the positive control treated tissues was 7.9% relative to the negative control treated tissues and the standard deviation value of the viability was 3.0%. The positive control acceptance criteria were therefore satisfied.
The mean OD570 for the negative control treated tissues was 0.962 and the standard deviation value of the viability was 4.5%. The negative control acceptance criteria were therefore satisfied.
The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 2.1%. The test item acceptance criterion was therefore satisfied.
The control absorbance values were within the historically established boundaries.

Item	OD570of tissues	Mean OD570of triplicate tissues	± SD of OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	1.012	0.962	0.044	105.2	100∗	4.5
	0.934			97.1
	0.939			97.6
Positive Control Item	0.061	0.076	0.029	6.3	7.9	3.0
	0.109			11.3
	0.058			6.0
Test Item	1.070	1.050	0.020	111.3	109.2	2.1
	1.030			107.1
	1.051			109.3

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, in the study and under the experimental conditions reported, Lithium Titanate is non-irritant to the skin. The following classifications apply:
EU CLP Not classified for Irritation.
UN GHS Not classified for Irritation (category 3 can not be determined).

Executive summary:

Introduction

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The principle of the assay is based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test item by means of the colorimetric MTT reduction assay. Cell viability was measured by enzymatic reduction of the yellow MTT tetrazolium salt (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to a blue/purple formazan salt (within the mitochondria of viable cells) in the test item treated tissues relative to the negative controls.

Method

Triplicate tissues were treated with the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labeled micro tubes and stored in a freezer for possible inflammatory mediator determination. After MTT-loading a total biopsy of each epidermis was made and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues.

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

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 was 109.2% after the 15-Minute exposure period and 42-Hours post-exposure incubation period.

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

Conclusion

In conclusion, in the study and under the experimental conditions reported, Lithium Titanate is non-irritant to the skin. The following classifications apply:

EU CLP Not classified for Irritation.

UN GHS Not classified for Irritation (category 3 can not be determined).

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 03 September 2019 Experimental completion date 05 September 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 435 (In Vitro Membrane Barrier Test Method for Skin Corrosion)

Deviations:

yes

Remarks:

Please see any other information on materials and methods section

GLP compliance:

yes (incl. QA statement)

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 Number: 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

Test system:

artificial membrane barrier model

Details on animal used as source of test system:

Corrositex®
Supplier: InVitro International, Irvine, USA
Corrositex® lot number: CT040119
Corrositex® expiry date: April 2021

Vehicle:

unchanged (no vehicle)

Details on test system:

Qualification Screen
The qualification screen was used to determine whether the assay was suitable for the test item. For the qualification screen 100 mg of the test item was added to the "Qualify" test tube. If the test item failed to produce a color or physical change in the qualifying test within 5 minutes, it could not be analyzed with the CORROSITEX® assay.

Categorization Screen
The categorization screen was used to enable the test item to be measured against the appropriate classification scale. Test items having high acid/alkaline reserves are defined as ‘Category 1’ items, while those with low acid/alkaline reserves are defined as ‘Category 2’ items.
The screen was performed by adding 100 mg of the test item to each tube (A and B). Each tube was agitated and the resulting colors observed. The categorization kit and color chart provided by InVitro International were used to determine the category.

Biobarrier Preparation
The membrane discs were prepared the day prior to the assay and were refrigerated at 2 to 8 °C overnight before use. Biobarriers are stable for 7 days if wrapped and stored at 2 to 8 °C.
A scintillation vial containing the biobarrier matrix powder was placed in a water bath on a hot plate set at 68 to 70 °C, with the stir switch set to maintain a steady rate for the stir-bar. The entire contents of the biobarrier diluent vial were added slowly to the biobarrier matrix powder to ensure complete and uniform solubilization. The solution was warmed to 68 to 70 °C for 20 minutes to solubilize the biobarrier matrix. Immediately after solubilization a volume of 200 μL of the biobarrier matrix was carefully pipetted onto the membrane discs whilst avoiding air bubbles which would affect the integrity of the solidified biobarrier.

Assay
The assay was used to assess the corrosive potential of the test item and to determine the appropriate Packing Group criteria for the test item.
One tray of seven pre-filled Chemical Detection System vials was removed from the kit box. The CDS vials were stored at room temperature (17 to 25 °C) before use. Vials 1 to 4 were used for test item replicate testing. The vial labeled “+” was used for the positive control sample, the vial labeled “-” was used for the negative control sample and the vial labeled “C” served as the color control.
A membrane disc was placed on top of the first vial. 500 mg of the test item was evenly applied onto the top of the membrane disc, and the timer was started. The vial was observed for 3 minutes for any change in the CDS. No color change was observed within 3 minutes, therefore the remaining three discs were similarly treated. A 1 minute interval was left between each treatment. The vials were observed continuously for the first 10 minutes, and at approximate 5 minute intervals for 60 minutes (Category 2 test items) or until breakthrough of the test item had occurred.
The positive and negative control vials were prepared as above and received 500 μL of the relevant reference item. The vials were monitored either until breakthrough had occurred or for 60 minutes.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

500 mg of the test item.
500 μL of the postive and negative control

Duration of treatment / exposure:

Vials were monitored either until breakthrough had occurred or for 60 minutes.

Number of replicates:

4

Irritation / corrosion parameter:

penetration time (in minutes)

Value:

> 60

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Qualification Screen
The test item produced an immediate color change and therefore qualified for use in the CORROSITEX® assay.

The positive and negative control items were within the study acceptance criteria.
The negative control did not induce a change in the colour of the CDS reagent after 60 minutes. The positive control sulphoric acid induced a change in the colour of the CDS reagent after 53 seconds and is in the acceptable historical range of breakthrough times. These results assure the validity of the test.

Assay

		Test Item Breakthrough Time
	Vial 1	Vial 2	Vial 3	Vial 4
Start Time (hr:min:sec)	00:05:00	00:13:00	00:17:00	00:21:00
Detection Time (hr:min:sec)	>60 minutes	>60 minutes	>60 minutes	>60 minutes
Breakthrough Time (hr:min:sec)	No breakthrough	No breakthrough	No breakthrough	No breakthrough

Positive/Negative Controls Quality Criteria

	Chemical	Concentration (%)	Breakthrough Time (hr:min:sec)
Positive Control	Sulphuric Acid	95 to 98	00:00:53
Negative Control	Citric Acid	10	No breakthrough observed

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this valid study and under the reported experimental conditions, the test item is NON-CORROSIVE to skin.

Executive summary:

Introduction

This in vitro study was performed to assess the corrosive potential of Lithium titanate by means of the In vitro Membrane Barrier Test for Skin Corrosion (OECD TG 435) using the Corrositex™ test kit.

Following an initial determination of the compatibility of the test item with the test system (qualify test) and a test system-specific categorisation (categorisation test), approx. 500 mg of the test item were applied on quadruplicate bio-barriers for the classification test. The bio-barriers were placed atop vials containing the chemical detection system (CDS). The time needed to possibly corrode the bio-barrier and to induce a distinct change in the colour of the CDS reagent was monitored for each of the vials.

No change of colour of the CDS reagent after treatment of the bio-barriers with the test item was observed after 60 minutes. Thus, according to the classification criteria given in chapter 3.7 the test item does not require classification as skin corrosive.

The test item as well as the negative and the positive controls fulfilled the acceptance criteria for the test system.

Results

Test item qualified: YES

Test item category: 2

Mean Breakthrough time: No breakthrough observed

Conclusion

In conclusion, it can be stated that in this valid study and under the reported experimental conditions, the test item is NON-CORROSIVE to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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)

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.

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).

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.

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.

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%

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.

 

 

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Experimental start date 29 October 2019 Experimental completion date 12 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 Number: SLEA 7084
Sample Number: 2914
Purity: > 98%
Physical state/Appearance*: Off white powder
Expiry Date: 23 September 2021
Date of Receipt: 12 July 2019
Storage Conditions: Room temperature in the dark

Species:

cattle

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival

Vehicle:

other: sodium chloride 0.9% w/v

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL of the test item preparation or control items were applied to the appropriate corneas

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Corneas that have opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one-hour equilibration period were discarded.
Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

Treatment of Corneas
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed at least 3 times with fresh complete EMEM containing phenol red, since residual test material could not be removed. The corneas were finally rinsed with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. No tissue peeling and non-uniform opacity patterns were observed.

Application of Sodium Fluorescein
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed from both chambers. The posterior chamber was filled with fresh EMEM first. Then the anterior compartment was filled with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.

Permeability Determinations
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.

Irritation parameter:

in vitro irritation score

Run / experiment:

Mean

Value:

17.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Corneal Opacity and Permeability Measurement
After exposure to the negative control an increase of opacity or permeability of the corneas was not observed.
Exposure to the positive control resulted in clear opacity and distinctive permeability of the corneas.
Relative to the negative control, the test item caused a slight increase of the corneal opacity and permeability.

Corneal Epithelium Condition
The corneas treated with the test item had small areas of test item adhered to the cornea which could not be removed via washing post treatment. The results of the opacity measurements are therefore confounded by the presence test item material. Since the residual material could not be removed by non-invasive methods it is concluded that the in vitro eye irritancy potential of Lithium titanate cannot be accurately assessed in the bovine corneal opacity and permeability assay. Due to these technical limitations, an accurate classification/category cannot be given.
The corneas treated with the negative control item were clear post treatment. The three corneas treated with the positive control item were cloudy post treatment.

Criteria for an Acceptable Test
The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity				Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	4	5	1		0.005
	3	4	6	2		0.004
	6	4	7	3		0.006
				2.0*		0.005¨		2.1
Positive Control	10	5	83	78	76.0	1.880	1.875
	11	3	69	66	64.0	1.865	1.860
	12	4	89	85	83.0	1.141	1.136
					74.3·		1.624·	98.7
Test Item	5	5	19	14	12.0	0.131	0.126
	3	3	24	21	19.0	0.430	0.425
	4	4	17	13	11.0	0.096	0.091
					14.0·		0.214·	17.2

OD= Optical density           * = Mean of the post-treatment -pre‑treatment values             ¨= Mean permeability                     ·= Mean corrected value

Corneal Epithelium Condition Post treatment

Treatment	Cornea Number	Observation Post Treatment
Negative Control	2	Clear
	3	Clear
	6	Clear
Positive Control	10	Cloudy
	11	Cloudy
	12	Cloudy
Test Item	14	Small amount of test item adhered to slightly cloudy cornea, could not be removed via washing
	15	Small amount of test item adhered to slightly cloudy cornea, could not be removed via washing
	16	Small amount of test item adhered to slightly cloudy cornea, could not be removed via washing

Interpretation of results:

other: Due to technical limitations (residual test material), an accurate classification/category cannot be given

Conclusions:

The in vitro eye irritancy potential of Lithium titanate cannot be accurately assessed in the bovine corneal opacity and permeability assay. Due to technical limitations (residual test material), the study cannot be performed with Lithium titanate according to the guideline OECD 437 and an accurate classification/category cannot be given. Further testing in another suitable method is required.

Executive summary:

Introduction

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS). Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

Method

The test item was applied at a concentration of 20% w/v in sodium chloride 0.9% w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS	UN GHS / EU CLP
≤ 3	No Category
>3; ≤ 55	No prediction can be made
> 55	Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	17.2
Negative Control	2.1
Positive Control	98.7

Conclusion

The in vitro eye irritancy potential of Lithium titanate cannot be accurately assessed in the bovine corneal opacity and permeability assay. Due to technical limitations (residual test material), the study cannot be performed with Lithium titanate according to the guideline OECD 437 and an accurate classification/category cannot be given. Further testing in another suitable method is required.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

In order to assess the serious eye damage / eye irritation potential of the substance, an in-vitro (OECD TG 492) and an ex-vivo (OECD TG 437) studies were conducted. Due to technical limitations (residual test item adhering to the surface) in both cases, no prediction on the eye irritating potential of the substance could be made based on those studies alone.

Since, for Annex VII information requirements, no in-vivo testing is foreseen, alternative information needs to be used to conclude on the eye hazard potential of the substance.

The substance does not have an extreme pH (<=2 or >=11,5) and is not corrosive or irritating to skin. Besides, both in-vitro and in-vivo studies are available on a structurally related substance: tetralithium pentatitanium dodecaoxide (Li₄Ti₅O₁₂, CAS 12031-95-7). Since the two substances have similar physico-chemical and toxicological properties, it was considered fit to rely on data available for tetralithium pentatitanium dodecaoxide (CAS 12031-95-7) in order to conclude on the eye irritation or serious eye damage potential of lithium titanate (CAS 12031-82-2). 

The GLP ex-vivo study (OECD TG 438, Klimisch 1, 2012) on tetralithium pentatitanium dodecaoxide did not allow a prediction to be made due to technical limitations (residual test item adhering to the surface). The GLP in-vivo study (OECD TG 405, Klimisch 1, 2012) lead to the conclusion that the substance was not irritating to the eye. 

Based on the aforementionned considerations, it is concluded that dilithium titanate (CAS 12031-82-2) does not meet the CLP classification criteria (Regulation (EC) No 1272/2008) for serious eye damage or eye irritation.

Taking into account the REACH requirements for Annex VII registrations, ECHA Guidance R.7a (V6.0, 2017, p. 214) and animal welfare considerations, and the fact that a reliable conclusion could be made based on available data, it is concluded that it is not necessary to conduct further in-vivo testing on the registered substance. 

Justification for classification or non-classification

Dilithium titanate was found to be non-corrosive and non-irritating to the skin. A classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is, therefore, not justified.

Dilitium titanate was found to not cause serious damage or irritation to the eye. A classification according to CLP classification criteria (Regulation (EC) No 1272/2008) is, therefore, not justified.