Zirconium dihydride

Endpoint summary

Description of key information

Skin and eye irritation/corrosion were studied with the test item Zirconium 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 ZrH2 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 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 ZrH2 is identified as non-corrosive to the skin.

b. Irritation (OECD 439) - Down

The potential of the test item ZrH2 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™ following the oECD guideline 439.

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 blank subtraction, was 91%.

Based on the results obtained, the test item ZrH2 is classified as non-irritant to the skin, UN GHS No Category.

2) Eye damage/irritation

a. Eye damage (OECD 437) - Top

Under the conditions of this study, the test item ZrH2 showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 3.21.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.

b. Irritation (OECD 492) - Down

ZrH2 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 97.8 %. This value is above the threshold for eye irritation potential (≤ 60%).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

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)

Qualifier:

according to guideline

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. QA statement)

Specific details on test material used for the study:

- Identity ZrH2
- Alternative names Zirconium Hydride S
- 453330000 ZrH2 Typ S - Lager
- Batch no. 74031
- Expiry date 25 February 2018
- Storage conditions room temperature
- RTC number 15067

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 048
- Arrived at RTC on 29 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 079
- AssayMedium SkinEthic; batches: 16ESSC045 and 16ESSC051

* 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.

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. 108

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

60 minutes (OD-blank background substraction)

Value:

ca. 104

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

240 minutes (OD-blank background substraction)

Value:

ca. 102

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

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 2.872, 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, at any treatment time. 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 results obtained from the additional controls, only the OD-blank background subtraction was performed.
Note: Intra-replicate variability was acceptable with a difference of viability between the two replicates lower than 30%, for all treatment times.

Interpretation of results:

other: Based on the results obtained, the test item ZrH2 is identified as non-corrosive to the skin.

Conclusions:

The potential of the test item ZrH2 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 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 ZrH2 is identified as non-corrosive to the skin.

Executive summary:

The potential of the test item ZrH2 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.

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 2.872, 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, no relevant colouring ability of the test item was noted, at any treatment time. 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 results obtained from the additional controls, only the blank subtraction was performed. Intra-replicate variability was acceptable with a difference of viability between the two

replicates lower than 30%, for all treatment times.

--> Based on the results obtained, the test item ZrH2 is identified as non-corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

June 1st, 2017 5signed protocol) - December 1st, 2017

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

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. QA statement)

Specific details on test material used for the study:

- Identity: ZrH2
- Alternative name: Zirconium Hydride S - 453330000 ZrH2 Typ S - Lager
- Batch no.: 74031
- Expiry date: 25 February 2018
- Storage conditions: room temperature
- RTC number: 15067

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:

- ZrH2: 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

Value:

ca. 91

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

mean value: 100% viability

Positive controls validity:

valid

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 theMTT 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 2.842, 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 reduce MTT, 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 1%, while the NSMTT value was -1%. Based on this result, only the
OD-blank background subtraction was performed and the mean cell viability was 91%, when
compared to the negative control. Acceptable intra-replicate variability was obtained (SD of
% viability = 5.8 lower than 18).

Interpretation of results:

GHS criteria not met

Conclusions:

The potential of the test item ZrH2 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™ following the oECD guideline 439.
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 blank subtraction, was 91%.
Based on the results obtained, the test item ZrH2 is classified as non-irritant to the skin, UN GHS No Category.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)