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Toxicological information

Skin irritation / corrosion

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Administrative data

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)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
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

Reference
Name:
Unnamed
Type:
Constituent
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

In vitro test system

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

Results and discussion

In vitro

Resultsopen allclose all
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.

Applicant's summary and conclusion

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