Registration Dossier

Administrative data

Description of key information

Skin and eye irritation/corrosion were studied with the test item Titanium hydride. No study was currently available, therefore studies were launched in an external lab (RTC, Italy/LAUS, Germany) following the top-down approach.

1) Skin corrosion/irritation

a. Corrosivity (OECD 431) - Top

The potential of the test item TiH2 to be corrosive to the skin was investigated through an in vitro skin corrosion study (OECD guideline N°431), using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.

The blank, negative and positive controls gave acceptable results, at all treatment times, thus the study was accepted as valid.

The mean cell viability of the test item treated tissues was higher than 35% at all treatment times. Nevertheless, it has to be noted that the result presented was obtained after an appropriate subtraction due to the fact that the test item to water lead to a dark grey solution, indicating a colouring potential of the test item. In conclusion, the test item TiH2 is identified as non-corrosive to the skin.

b. Irritation (OECD 439) - Down

The potential of the test item TiH2 to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The blank, negative and positive controls gave acceptable results and the study was accepted as valid. The mean cell viability of the test item treated tissues was 88%. Based on the results obtained, the test item TiH2 is classified as non-irritant to the skin, UN GHS No Category (for member states that do not adopt optional category 3). In this case also appropriate substraction on the results have been made due to the coloring potential of the test item.

2) Eye damage/irritation

a. Eye damage (OECD 437) - Top

Following OECD Guideline no. 437 (BCOP), the test item TiH2 showed effects on the cornea of the bovine eye. However, the calculated IVIS (in vitro irritancy score) is 5.49. Therefore, as the substance has an IVIS between > 3 and ≤ 55 induces effects on the cornea, it cannot be classified in an UN GHS Category for eye damage.

b. Irritation (OECD 492) - Down

TiH2 is considered non-eye irritant in the test condition of the EpiOcularTM Eye Irritation Test (OECD guideline N°492). After treatment with the test item, the mean value of relative tissue viability was reduced to 97.3 %. This value is above the threshold for eye irritation potential (≤ 60%) and is therefore, not considered as irritant.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Protocol signed (October 2016) - Final report signed (April 2017)
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
Adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
- Identity TiH2
- Alternative names Titanium Hydride VM
- 454013000 TiH2 Typ VM - Lager
- Batch no. 75727
- Expiry date 21 June 2018
- Storage conditions room temperature
- RTC number 15068
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Vehicle:
unchanged (no vehicle)
Remarks:
Only for solid chemicals, the epidermis surface will be moistened with 100 ± 5 μL of 0.9% NaCl solution before application of the test item.
Details on test system:
SKIN DISC PREPARATION
*EPISKIN(TM)
- Commercial Name EPISKIN™ - 0.38 cm2
- Supplier SkinEthic Laboratories (4, A. Fleming – 69366 Lyon – France)
- Batch 16 EKIN 045
- Arrived at RTC on 08 November 2016

Functional controls:
- Quality controls: histology scoring, magnitude of viability and barrier function (IC50 determination).
- Biological safety: absence of HIV1 and 2, Hepatitis B and C antigens, absence of bacteria, fungi and mycoplasma.
(A certificate of analysis can be found in the main report)

Preparation of the Test System:
- Examination at arrival
Temperature indicator: pale grey (suitable for use)
pH indicator: orange (suitable for use)
- Preparation and pre-treatment incubation period
The test system was shipped on Monday and received on Wednesday. According to the supplier procedure, at arrival, plates were opened under a sterile airflow and each insert, containing the epidermal tissue, was carefully taken out and placed in a 12-well plate in which each well had previously been filled with 2mL/well SkinEthicMaintenanceMedium. Culture plates were placed in the incubator at 37°C, 5% CO2 and saturated humidity for approximately 24 hours.

Media:
- MaintenanceMedium SkinEthic; batch: 16MAIN3 075
- AssayMedium SkinEthic; batches: 16ESSC045 and 16ESSC048

* Experimental procedure
- Preliminary test

Direct MTT reduction test (Step 1)
Non-specific reduction of MTT was evaluated as follows: two mL of MTT ready-to-use solution (0.3mg/mL) was incubated with 20mg of test item at 37°C, 5% CO2 and saturated humidity for 3 hours, simulating test conditions. Observation of blue or purple appearance of the solution at the end of the incubation time was carried out.

Colouring potential test (Step 2)
Chemicals’ colouring potential was assessed for potential interaction with the test system. 10mg of test item was added to 90 µL of distilled water (Baxter; batch no. 15I0211) in a transparent tube and the resulting solution/suspension mixed by using a vortex for 15 minutes. At the end of the incubation time, colouring of the solution/suspension evident to the unaided eye and measured by spectral analysis at 595 nm, was evaluated.

- Main Assay
Treatment
In theMain Assay, alive tissues were treated with the test item, positive and negative controls.

Exposure period
Exposure times of 3, 60±5 and 240±5 minutes were allowed in a ventilated cabinet at room temperature.

Washing
At the end of the exposure, each tissue was rinsed with approximately 25mL of sterile PBS, filling and empting the tissue insert. The excess liquid was carefully removed and the sample transferred in new wells pre-filled with 2mL/well of maintenance medium.

MTT staining
Each tissue insert was incubated with 2mL/well of MTT ready-to-use solution, with the exception of tissues used for the unspecific colouring potential control. Plateswere incubated for 3 hours ± 5 minutes at 37°C, 5% CO2 and saturated humidity. At the end of the incubation period, tissues were placed on absorbent paper to dry. A total biopsy was carried out by means of a biopsy punch to allow biopsies of the same dimensions.
The epidermis were separated from the collagen matrix and both placed in a microtube prefilled with 500 µL of acidic isopropanol. Tubes were mixed by vortexing and preserved overnight at room temperature to allow formazan extraction. At the end of the extraction period, debris were eliminated by short centrifugation of the tubes (14000 rpm for 2 minutes) and aliquots of 200 µL from each sample were read in duplicate for their absorbance at
595 nm. Six aliquots (200 µL) of acidic isopropanol were analysed and used as blank. On the day of spectrophotometer analysis, quality control solutions of MTT formazan were prepared and analysed in order to ensure that the MTT formazan calibration curve was still appropriate.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Amount per well: 20 mg of the test item during 3, 60 and 240 minutes (2 replicates)
- Positive control: 50 µL 240 minutes (2 replicates)
- Negative controls: 50 µL during 3, 60 and 240 minutes (2 replicates)
Duration of treatment / exposure:
Exposure times of 3, 60±5 and 240±5 minutes were allowed in a ventilated cabinet at room temperature.
Duration of post-treatment incubation (if applicable):
Amount per well: 20 mg of the test item during 3, 60 and 240 minutes (2 replicates)
- Positive control: 50 µL 240 minutes (2 replicates)
- Negative controls: 50 µL during 3, 60 and 240 minutes (2 replicates)
Number of replicates:
2
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minutes (OD-blank background substraction)
Value:
ca. 78
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minutes (OD-blank background substraction)
Value:
ca. 66
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
240 minutes (OD-blank background substraction)
Value:
ca. 88
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Preliminary test
Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. Due to the dark colour of the substance, it was not possible to clearly evaluate the actual ability of the
test item of reducing MTT. Dark precipitate was noted. In a second step, the test item was assayed for the ability of colouring water per se. A dark grey solution, with an OD value of 2.876, was obtained indicating a colouring potential of the test item in contact with water. Based on these results, additional controls were added in the main phase for the evaluation of MTT non specific reduction and of non specific colouring potential.

- Main Assay
A Main Assay was performed. For each treatment time, the mean Optical Density of Blank Controls was lower than the maximum acceptable value (0.1). All negative control mean OD values gave the expected baseline value and variability, in agreement with guideline indications. According to the method, each negative control mean value is considered the baseline value for the concurrent treatment series, thus they represent 100% of cell viability.
Positive control results indicated an appropriate cell death with an acceptable relative cell viability (1% of the negative control value). Based on the stated criteria, the study was accepted as valid. Following treatment of alive tissues without MTT, no relevant colouring ability of the test item was noted. For each treatment time, the calculated percent value, over the concurrent negative control (NSCliving), was as follows:
Treatment time (minutes): 3, 60, 240 - NSCliving% 16, 13, 18 respectively

No relevant interaction was recorded between the test item and MTT at any treatment time. The test item did not induce cell death in any replicate, at any treatment time. Based on the resuts obtained from the additional controls, for each treatment time, the percent viability value was also subtraced from the concurrent NSCliving.
Interpretation of results:
GHS criteria not met
Conclusions:
The potential of the test item TiH2 to be corrosive to the skin was investigated through an in vitro skin corrosion study (OECD guideline N°431), using a commercial reconstructed human epidermis (RhE) model named EPISKIN™.
The blank, negative and positive controls gave acceptable results, at all treatment times, thus the study was accepted as valid.
The mean cell viability of the test item treated tissues, after the appropriate subtraction, was higher than 35% at all treatment times. Based on the results obtained, the test item TiH2 is identified as non-corrosive to the skin.
Executive summary:

The potential of the test item TiH2 to be corrosive to the skin was investigated through an in vitro skin corrosion study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 431. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 3, 60 and 240 minutes. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system, being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In particular, the test item was assayed for the ability of reducing MTT and colouring water per se. After addition of the test item to water, a dark grey solution was observed, with an OD value of 2.876, indicating a colouring potential of the test item. Due to the dark colour of the substance, it was not possible to clearly evaluate the actual ability of the test item of reducing MTT. Based on these results, additional controls both for colour potential and MTT unspecific reduction were added in the main phase.

In the Main Assay, for each treatment time, the test item was applied as supplied in two replicates at the treatment level of 20mg/epidermis unit, each measuring 0.38cm2 (treatment level: 52.6 mg/cm2). Positive and negative controls (Glacial acetic acid and Physiological saline, respectively) were concurrently tested, in the same number of replicates and test conditions at the treatment level of 50 μL/epidermis unit. Positive control was included only at the longest treatment time of 240 minutes, while a negative control was included at each treatment time.

In theMain Assay, the negative controls gave the expected baseline value (Optical Density values ≥ 0.6 and ≤ 1.5) and variability (difference of viability between the two replicates lower than 30%), at each treatment time, in agreement with the guideline indications. For each treatment time, the concurrent negative control mean value is considered the baseline value

of the treatment series and thus represents 100% of cell viability.

The positive control caused the expected cell death (1% of cell viability, when compared to the negative control).

Based on the stated criteria, the assay was regarded as valid.

Following treatment of alive tissue without MTT, colouring ability of the test item was noted. For each treatment time, the calculated percent value, above the concurrent negative control (NSCliving), was as follows:

 Treatment time (minutes)  Mean cell viability (%)
 3  78
 60  66
 240

 88

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 9th, 2017 (signed protocol) - December 1st, 2017
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
Adopted on 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
-Identity: TiH2
-Alternative name: Titanium Hydride Powder VM (454013000 TiH2 Typ VM - Lager)
-Batch no.: 75727
-Expiry date: 21 June 2018
-Storage conditions: room temperature
-RTC number: 15068
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: Adult donor - 17-EKIN-032
Vehicle:
unchanged (no vehicle)
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
-TiH2: 20+/-2mg
Duration of treatment / exposure:
15 min followed by a 42 +/- 1 hour recovery period
Number of replicates:
Main assay: three replicates
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
alive tissue with appropriate background substractions
Value:
ca. 88
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
mean value: 100% viability
Positive controls valid:
yes
Remarks:
mean value: 3% viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- Preliminary test
Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system.

* In a first step, the test item was assayed for the ability of reducing MTT per se. Dark grey suspension, with black precipitate, was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT.
* In a second step, the test item was assayed for the ability of colouring water per se. A dark grey suspension was observed; spectrophotometric analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 2.810, indicating that the test item has a potential interfering ability. Based on the results obtained, additional controls were added in the Main Assay for the evaluation of Non Specific Colouring potential (NSCliving) and Non Specific MTT reduction (NSMTT). Since the test item was able both to stain tissue and reduceMTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was performed.
- Main Assay
A Main Assay was performed. The mean Optical Density of Blank Controls was 0.041, lower than the maximum acceptable value (0.1). The negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability (Standard Deviation of the % viability
lower or equal to 18), in agreement with guideline indications. According to the method, the mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. Positive control results indicated an appropriate cell death with an acceptable relative cell viability (3% of the negative control value). Variability between replicates gave also the expected value (SD of%viability = 0.3). Based on the stated criteria, mean viability, expressed
as percentage of the negative control, lower or equal to 40% and standard deviation of % viability equal or lower than 18, the study was accepted as valid.
The NSCliving value was 0%, while the NSMTT value was 7%. Based on this result, correction for non specific MTT reduction and OD-blank background were performed and the mean cell viability was 88%, when compared to the negative control. Acceptable intra-replicate variability was obtained (SD of % viability = 5.6 lower than 18).
Interpretation of results:
GHS criteria not met
Conclusions:
The potential of the test item TiH2 to be irritant to the skin was investigated through an in vitro skin irritation study, using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The blank, negative and positive controls gave acceptable results and the study was accepted as valid.
The mean cell viability of the test item treated tissues, after the appropriate subtractions, was 88%. Based on the results obtained, the test item TiH2 is classified as non-irritant to the skin, UN GHS No Category (for member states that do not adopt optional category 3).
Executive summary:

Study summary:

The potential of the test item TiH2 to be irritant to the skin was investigated through an in vitro skin irritation study using a commercial reconstructed human epidermis (RhE) model named EPISKIN™. The experimental procedures are based on the OECD Guideline for testing of chemicals no. 439. The test item, as well as controls, were tested for their ability to impair cell viability after an exposure period of 15 minutes followed by a 42 ± 1 hour recovery period. The final endpoint of the assay is the colorimetric measurement of MTT reduction (blue formazan salt) in the test system being this reaction an index of cell viability. The test item was tested as supplied by the Sponsor.

Before the Main Assay, a preliminary test was carried out to evaluate the compatibility of the test item with the test system. In a first step, the test item was assayed for the ability of reducing MTT per se. A dark grey suspension, with black precipitate was noted in the MTT solution at the end of the incubation period, indicating that the test item could direct interact with MTT. In a second step, the test item was assayed for the ability of colouring water per se.

A dark grey suspension was observed; spectrophotometric analysis of the test item in water, to evaluate the ability of the test chemical to absorb light at 595 nm, was performed. The value obtained for the Optical Density (OD) was 2.810, indicating that the test item has a potential interfering ability. Based on these results, additional controls were added in the Main Assay.

In the Main Assay, the test item was applied as supplied in three replicates at the treatment level of 20±2mg/epidermis unit, each measuring 0.38cm2 (treatment level: 53mg/cm2). Positive and negative controls [a 5% (w/v) sodium dodecyl sulphate solution in water and Dulbecco’s phosphate buffered saline (D-PBS), respectively] were concurrently tested, in the same number of replicates and test conditions at the treatment level of 20 μL/epidermis unit. In order to verify if the test item results had to be corrected, the non specific colour (NSCliving) was evaluated using two alive treated tissues without MTT staining and compared with the D-PBS control. Moreover, non specific MTT reduction (NSMTT) was evaluated using two killed tissues and compared with negative control performed with alive tissues. Since the test item is able both to stain tissue and reduce MTT, to avoid a possible double correction for colour interference, a third control for Non Specific Colour in killed tissue (NSCkilled) was

performed.

In the Main Assay, the negative control gave the expected baseline value (Optical Density values of the three replicates higher than 0.6) and variability [Standard Deviation (SD) of % viability lower or equal to 18], in agreement with the guideline indications. According to the method, the negative control mean value is considered the baseline value of the experiment and thus represents 100% of cell viability. The positive control caused the expected cell death (3% of cell viability when compared to the negative control) and variability (SD of % viability equal to 0.3). Based on the stated criteria (mean viability ≤ 40% and SD of % viability ≤ 18), the assay was regarded as valid. The NSCliving value was 0%, while the NSMTT value was 7%, thus correction for non specific MTT reduction and OD-blank background were performed. The test item did not induce cell death in any replicate, the mean cell viability after the appriopriate subtractions was 88% when compared to the negative control. Intra-replicate variability was acceptable with a SD of % viability values equal to 5.6 (lower than 18, as stated in the Study Protocol).

Based on the results obtained, the test item TiH2 is classified as non-irritant to the skin.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 2017 (Protocol signed) to October 2017 (Final report)
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
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)
Version / remarks:
Edition adopted 26 Jul. 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 14 Feb 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Name TiH2
Batch no. 78562
Appearance black to grey powder
Composition Titanium Hydride Powder VM (Ti total min 94 %)
Purity Min 94 %
Homogeneity homogen metal hydride
Expiry date Feb. 2019
Storage Room Temperature (20 ± 5°C)
Species:
cattle
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. Within 1 hour 5 minutes, the eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container.
Vehicle:
unchanged (no vehicle)
Remarks:
The test item is a solid substance. It was tested directly, without dilution or preparation of a solution. No 20% solution or suspension of the test item was tested, because this procedure is more close to reality.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
- Replicate 1: 499.2 mg
- Replicate 2: 608.5 mg
- Replicate 3: 482.8 mg
Duration of treatment / exposure:
The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item.
Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid with a microtiter plate photometer at 492 nm.
Duration of post- treatment incubation (in vitro):
Exposure time on the corneas was 4 hours at 32 ± 1 °C.
Number of animals or in vitro replicates:
Three replicates - Test item and positive and negative controls.
Details on study design:
* Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

* Experimental Parameters
Date of treatment 26. Jun. 2017
Incubation time 4 h
Negative control HBSS
Positive control Imidazole, 20 % solution in HBSS

* Method Description
The experimental procedure was performed under the fume hood. After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative and positive control solution and a defined amount of test item were applied to each replicate.

* Evaluation
- Calculation of Opacity
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

Opacity= ( I0/I)-b/a

a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and
Medium, here: Io= 1055.62
I = the measured illuminance (unit: LUX)

- Calculation of Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

- Calculation of IVIS (In Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)

The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results

Irritation parameter:
cornea opacity score
Run / experiment:
Mean opacity difference corrected of the test item (4 hours exposure)
Value:
ca. 5.49
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
cornea opacity score
Run / experiment:
Mean Opacity Difference of the negative controls (4 hours exposure)
Value:
ca. 2.45
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
cornea opacity score
Run / experiment:
Mean Opacity Difference Corrected of the positive controls ( 4 hours exposure)
Value:
ca. 106.79
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
* In vitro Irritancy Score (IVIS)
- Negative control HBSS:
Mean IVIS: 2.45
Relative Standard Deviation IVIS: 34.35%
- Test item TiH2
Mean IVIS: 5.49
Relative Standard Deviation IVIS: 103.32%
- Positive control 20% imidazole solution:
Mean IVIS: 106.79
Relative Standard Deviation IVIS: 5.48%
* Optical density at 492 nm of Negative Control, Test Item and Positive Control
Corrected mean of the 3 replicates:
Negative Control: /
Test Item: 0.0267
Positive Control: 1.8741
Interpretation of results:
study cannot be used for classification
Conclusions:
Following OECD Guideline no. 437 (BCOP), the test item TiH2 showed effects on the cornea of the bovine eye. However, the calculated IVIS (in vitro irritancy score) is 5.49. Therefore, as the substance has an IVIS between > 3 and ≤ 55 induces effects on the cornea, it cannot be classified in an UN GHS Category for eye damage.
Executive summary:

BCOP Study (OECD 437) with Titanium hydride

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item TiH2 was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 2.45. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 106.79.

Under the conditions of this study, the test item TiH2 showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 5.49.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 2017 (Study plan) to February 2018 (Final report)
Reliability:
1 (reliable without restriction)
Reference:
Composition 0
Qualifier:
according to
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:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
Name TiH2
Batch no. 78562
Appearance powder, grey to black
Composition Titanium Hydride Powder VM (Ti total min 94%)
Purity Min 94%
Homogeneity homogen metal hydride
Expiry date Feb. 2019
Storage Room Temperature (20 ± 5°C)
Species:
human
Strain:
other: human-derived keratinocytes
Remarks:
EpiOcularTM tissues were procured from MatTek In Vitro Life Science Laboratories, Mylnské Nivy 73, 82105 Bratislava, Slovakia.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
Tissue 1 --> 53.6 mg (Main test) and 48.9 mg (Additional test)
Tissue 2 --> 50.3 mg (Main test) and 52.2 mg (Additional test)
Duration of post- treatment incubation (in vitro):
Main test: 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
Number of animals or in vitro replicates:
Two replicates: tissue 1 and tissue 2
Details on study design:
Main test:

* Preparations
On the day of the start of the experiment, the MTT concentrate was thawed. The MTT concentrate was diluted with assay medium directly before use.
The assay medium was warmed in the water bath to 37 ± 1°C.
6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.

* Exposure and Post-Treatment
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes (main test) and 30 minutes (additional test)..
After that, 50 µL of the controls and a defined amount of the test item (see table 7.2-a) were applied in duplicate in one- minute- intervals.
At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours 5 minutes (main test) and 18 hours 10 minutes (additional test) at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
After the post-treatment incubation, the MTT Assay was performed.

* MTT Assay and Extraction
A 24-well-plate was prepared with 300 µL freshly prepared MTT solution in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solu-tion. The plate was incubated for 190 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity. For the additional test the tissues were incubated in assay medium in-stead of MTT medium for 180 minutes at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol was flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then shaken for 2 hours at room temperature, protected from light.

* Measurement
The inserts were removed from the 6-well plate and discarded. The content of each well was thoroughly mixed in order to achieve homogenisation.
From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were also pipetted. The plate was read in a plate spectrophotometer at 570 nm.

Irritation parameter:
cornea opacity score
Run / experiment:
Tissue 1
Value:
ca. 1.843
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
1.890
Positive controls valid:
yes
Remarks:
0.558
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
Tissue 2
Value:
ca. 1.819
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
1.861
Positive controls valid:
yes
Remarks:
0.633
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.9 (> 0.8 and < 2.5).
The positive control induced a decrease in tissue viability as compared to the negative control to 31.7 %. Variation within the replicates was acceptable (< 20%).
For these reasons, the result of the test is considered valid.

Pre-Tests

*  Assessment of Direct Reduction of MTT by the Test Item

The test item was tested for the ability of direct MTT reduction. To test for this ability,

48.3 mg of the solid test item were added to 1 mL of MTT solution in a 6-well plate and the mixture was incubated in the dark at 37 ± 1 °C, 5.0 ± 1 % CO2and 80 – 100 % relative humidity for 3 hours. 1 mL of MTT solution plus 50 µL of H2O demin. was used as negative control.

The MTT solution did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

*  Assessment of Coloured or Staining Test Items

53.3 mg of the test item were added to 2 mL isopropanol, incubated in 6-well plates on an orbital shaker for 3 hours at room temperature.As the extract solution was turbid, it was centrifuged (30 seconds at 16 000* g).

Then, two 200 µL aliquots of the supernatant and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm.

After subtraction of OD for isopropanol, the OD of the test item solution was > 3.5 (>0.08). The test item was possibly interacting with the photometrical measurement and an additional test on colourant controls had to be performed.

The additional test was performed in order to evaluate the amount of colour bound to the tissues. The test item was applied to two additional tissues (= colourant controls) and the test was performed in the same way as described for the main test, but no MTT assay was performed: instead of 300 µL MTT solution, 300 µL assay medium was used. The bound colour was extracted and the absorbance of the isopropanol extract was measured in the same fashion as in the MTT assay for coloured test items (without piercing the tissues).

As the colourant control result was ≤ 50 % of the viable negative control, a data correction procedure could be performed.

Interpretation of results:
GHS criteria not met
Conclusions:
TiH2 is considered non-eye irritant in the test condition of the EpiOcularTM Eye Irritation Test (OECD guideline N°492). After treatment with the test item, the mean value of relative tissue viability was reduced to 97.3 %. This value is above the threshold for eye irritation potential (≤ 60%).
Executive summary:

* Title of Study:                    Determination of Eye Irritation Potential of TiH2 using the EpiOcularTMReconstructed human Cornea-like Epithelium (RhCE) test method following OECD 492

 * Findings and Results:

 

One valid experiment was performed.

 

The test itemTiH2was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

As the test item showed intense coloring in the pre-test, there was the risk to influence the photometric measurement. Thereforean additional test for intensely coloured test items was performed. But the result of the additional test showed, that the test item colour did not critically influence the result of the study.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD>0.8 and < 2.5, OD was 1.9. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 31.7 % (< 50%).

Variation within tissue replicates was acceptable (< 20%).

 

After treatment with the test item, the mean value of relative tissue viability was 97.3 %.

This value is above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant.

 

 

Under the conditions of the test,TiH2 is considered non- eye irritant in the EpiOcularTMEye Irritation Test.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification