Zirconium oxide, hafnium and ytterbium doped

Endpoint summary

Description of key information

The potential of zirconium oxide, hafnium and ytterbium doped (target substance) to induce skin irritation (OECD 439, GLP) and eye irritation (OECD 492, GLP) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-08 to 2018-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Ytterbium stabilized Zirconium and Hafnium Oxide
EC No.: 945-888-9
Batch No.: 7170502
Physical State: solid
Colour: white
Purity: > 95%
Expiry Date: 26 October 2022
Storage conditions: room temperature, in a tightly closed container in a dry place

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™-Standard Model (EPI-200™)
- Tissue batch number(s): 25867

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1
- Observable damage in the tissue due to washing: None

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 5 mg/mL
- Incubation time: 60 min
- Wavelength: 570 nm
- Filter bandwidth: +/- 30 nm

NUMBER OF REPLICATE TISSUES: 3 per dose group

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : Killed tissues
- N. of replicates : 2

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than equal to 50%

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS solution

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3 per dose group, 3 dose groups

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Mean of three replicates

Value:

92.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: No reduction observed
- Colour interference with MTT: No interference observed


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

- The mean absolute OD₅₇₀of the three negative control tissues was ≥0.8 and ≤ 2.8 (1.907)

- The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.1%)

- Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.2% - 5.3%)

Table 1: Mean Relative Tissue Viability for the test item

	Negative Control	Positive Control	Test item
Mean Relative Tissue Viability [%]	100.0	3.1	92.8

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.

Executive summary:

In a primary skin irritation study conducted according to the guideline OECD 439, 25 mg of zirconium oxide, hafnium and ytterbium doped (purity > 95 %) was topically applied to the EpiDerm tissue for 60 min followed by a 42 h post-incubation period. Irritant potential of the test item was predicted from the relative mean tissue viabilities obtained compared to the corresponding negative control tissues concurrently treated with DPBS. The controls confirmed the validity of the study. The mean relative tissue viability (% negative control) was observed to be > 50% (92.8%). Based on these results, the test item is classified as a non-irritant under the UN GHS Criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-12-08 to 2018-03-19

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)

Version / remarks:

Adopted, 28 July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Details on test animals or tissues and environmental conditions:

- Justification of the test method: This in vitro method is recommended to identify chemicals that do not require classification for eye irritation or serious eye damage according to UN GHS (UN GHS “No Category”) and consequently require no further testing within a tiered testing strategy in contrast from those requiring classification and labelling (UN GHS categories 1 and 2). Therefore, it can be used for regulatory purposes as an initial step in the bottom-up approach or as one of the last steps in a top-down approach to test eye irritation/corrosion potential. It is not intended to differentiate between UN GHS “Category 1” (serious eye damage) and UN GHS “Category 2” (eye irritation) which would require additional testing. Ocular irritation potential is predicted by the relative viability of the tissue after a single exposure to the test substance. Relative viability is determined by measuring the MTT dye to formazan conversion by the EpiOcular™ tissue construct after topical exposure to the test substance.

- Description of the cell system used: The test was carried out with the EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium that is morphologically similar to that found in a human cornea. The EpiOcular™ RhCE tissue construct consists of at least 3 viable layers of cells and a non-keratinized surface, showing a cornea-like structure analogous to that found in vivo.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

Duration of treatment / exposure:

6 h

Duration of post- treatment incubation (in vitro):

18 h

Number of animals or in vitro replicates:

2 tissues per dose group. 3 dose groups.

Details on study design:

- RhCE tissue construct used, including batch number : EpiOcular™ tissues on agarose (Lot No.: 27018, 27019)
- Pre-experiments:
- To check the non-specific MTT-reducing capability of the test item 50 mg of the test item was mixed per 1 mL MTT medium and incubated for 3 h in a humidified incubator at 37+/- 1 °C, 5.0% CO2 / 95% air. The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues if the mean relative tissue viability of the test item treated tissues (TM) is above the 60% threshold value.
- For quantitative correction of results, two killed tissues were treated with 50 mg of the test item (KT) and one tissue was treated with 50 µL of the negative control (Aqua dest.; KU). NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:
NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100

- To check the colouring potential of the test item, 50 mg of the test item was mixed per 1 mL Aqua dest. or per 2 mL isopropanol each in a 6-well plate. The water solution was incubated for at least 1 h in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air. The isopropanol solution was shaken on a plate shaker for 2 to 3 h. After the respective incubation periods, 2 x 200 µL aliquots per test solution were transferred into a 96-well plate as well as using 200 µL Aqua dest. and isopropanol as respective blanks and OD was measured in a range of 570 ± 30 nm without reference wavelength in a plate spectrophotometer.

- For test items which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colouring in killed tissues (NSCkilled) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 50 mg of the test item (TKT). For the MTT-staining of the test item treated with tissues, incubation was performed in medium without MTT. The non-specific colour of additional killed tissues (NSCkilled) was then calculated according to the following formula:
NSCkilled [%] = [ODTKT/ODNK]*100
- The true tissue viabilitywas then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSCliving plus NSCkilled.

- Experimental Procedure:
- Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. The EpiOcular™ tissues were then transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h at 37 +/- 1 °C, 5.0% CO2 / 95% air. Then the tissues were transferred into new 6-well plates containing 1 mL fresh assay medium per well and pre-incubated in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air for 16 - 24 h.
- After the overnight incubation the tissues were pre-treated with 20 µL of DPBS and incubated for 30 ± 2 min in a humidified incubator at 37 +/- 1 °C, 5.0% CO2 / 95% air to mimic the wet conditions of the human eye.
- Afterwards, the tissues were treated with each dose group in duplicate, starting with the negative and positive control. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air until the 6 ± 0.25 h of the first dosed tissue was over. At the end of the exposure period the test item and control substances were removed by extensively rinsing the tissue with DPBS. Excess DPBS was removed by decanting the insert and blotting bottom with blotting paper. After rinsing the inserts were transferred to and immersed in a prepared 12-well “post-soak plate“, containing 2 mL fresh pre-warmed assay medium per well and incubated for 25 ± 2 min at room temperature. Afterwards, the inserts were removed from the assay medium, the medium was decanted off the tissue and the tissues were blotted on blotting paper. The inserts were transferred to a new 6-well plate (post-treatment plate) containing 1 mL pre-warmed assay medium. The tissues were incubated for 18 ± 0.25 h at 37 ± 1°C, 5.0% CO2 / 95% air.
- After this incubation period excess medium was removed by blotting bottom on absorbent paper before the inserts were transferred in a prepared 24-well “MTT assay plate” containing 0.3 mL pre-warmed MTT medium and further incubated for 3 h ± 10 min at 37 ± 1 °C, 5.0% CO2 / 95% air.
- After the 3 h MTT incubation period the inserts were removed, the bottom of the inserts blotted on blotting paper, and then transferred into new 6-well “extraction plates“, containing 2 mL of isopropanol to extract the formazan only from the bottom of the tissues to avoid possible contamination of test material. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out immediately by shaking on an orbital plate shaker for 2 - 3 h at room temperature. At the end of the extraction period the tissues were not pierced to avoid contamination of the extract with remaining test item.
Then the inserts were discarded and the extracts were mixed three times using a pipette. If any visible cell/tissue fragments were in suspension, extracts were centrifuged to eliminate the fragments and avoid further possible interference with the absorbance readings.
- For each tissue 2 x 200 µL aliquots of the extract were transferred into a 96-well plate and OD was measured at 570 nm using a filter band pass of maximum ± 30 nm in a plate spectrophotometer using isopropanol as a blank.

Irritation parameter:

other: mean relative tissue viability

Value:

> 0.6

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:

OTHER EFFECTS:
- Visible damage on test system: None

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Interpretation of results:

GHS criteria not met

Conclusions:

In this study under the given conditions the test item showed no irritant effects. The test item is classified as “non-irritant“ in accordance with UN GHS “No Category”

Executive summary:

In a primary eye irritation study conducted according to OECD guideline 492, 50 mg of zirconium oxide, hafnium and ytterbium doped (purity >99.5%) was applied topically on EpiOcular tissue, a reconstituted three-dimensional human corneal epithelium model for 6 h and a post-treatment period of 18 h compared to that of the concurrent negative control. Irritation was scored by the method of mean relative tissue viability.

 The mean relative tissue viability (% negative control) was > 60% (60.2%). As the results were within the borderline values (60 ± 5%), a second experiment was performed to confirm the first results. The mean relative tissue viability (% negative control) of the second experiment was > 60% (99.2%) and the test item did not show irritant effects. Based on these results, the test item can be classified as a non-irritant according to the UN GHS Criteria.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The potential of zirconium oxide, hafnium and ytterbium doped (target substance) to induce skin irritation (OECD 439, GLP) and eye irritation (OECD 492, GLP) was tested in suitable in vitro test methods. Based on the results, the target substance can be considered non-irritant to the skin and eye.

Justification for classification or non-classification

Based on available data, no classification for skin and/or eye irritation is warranted based on the results from suitable in vitro tests (OECD 439, 492).