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EC number: 234-911-1 | CAS number: 12039-90-6
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Endpoint summary
Administrative data
Description of key information
In an in vitro skin irritation assay conducted in accordance with OECD test guideline 439, Zirconium disilicide (> 99.47% purity) was applied topically to a reconstituted three-dimensional human epidermis. Based on the results, the target substance was considered to be non-irritating to the skin.
In an in vitro eye irritation assay conducted in accordance with OECD test guideline 492, Zirconium disilicide (> 99.47% purity) was applied atop the EpiOcular™ tissue. Based on the results, the target substance was considered to be non-irritating to the eye.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-06-20 to 2017-07-20
- 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)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- 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, i.e. the epidermis.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model (MatTek).
- Tissue batch number(s): Lot 25822
EpiDerm Kit:
The EpiDerm™ tissues were provided as kits (e.g. EPI-200-SIT, MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing e.g. 24 reconstructed epidermis units (area: 0.63 cm2); each reconstructed epidermis is attached to a cell culture insert and maintained on nutritive agar for transport (Lot No.: 25822)
2x 24-well plates
8x 6-well plates
1x bottle of assay medium (DMEM-based medium, Lot No.: 061517 THB)
1x bottle of DPBS Rinse Solution
1x 1 vial 5% SDS Solution (TC-SDS-5%)
25 pieces Nylon Mesh circles (8 mm diameter, 200 µm pore)
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for the first 35 +/- 1 min, afterwards the plates were placed under the sterile flow until 60 +/- 1 min incubation time of the first dosed tissue was over.
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C
REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After exposure, the tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, staggered again in e.g. one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.
MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm
NUMBER OF REPLICATE TISSUES: 3
PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 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 + 25 µL DPBS
NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL DPBS (DPBS; Gibco, Cat. No. 14040-091, Lot No.: 1838067)
POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 µL 5% SDS solution (AppliChem, CAS No.: 151-21-3, Lot No.: 4O015277) in H2O (Sigma, Lot No.: RNBF3331). - Duration of treatment / exposure:
- 60 min +/ 1 min
- Duration of post-treatment incubation (if applicable):
- 42 h post-incubation
- Number of replicates:
- 3 tissues per dose group
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- mean of three tissues
- Value:
- 104.7
- 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:
- For detailed results see Table 1 in box "Any other information on results" .
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, in this in vitro skin irritation study (OECD 439), Zirconium disilicide is considered to be non-irritating to the skin.
- Executive summary:
In the present study the skin irritant potential of Zirconium disilicide (99.47% purity) was analysed according to OECD 439 using the EpiDermTM standard model (EPI-200TM), a reconstructed human epidermis model to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, 25 mg of the test item was applied directly atop the EpiDermTM tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 minutes exposure and 42 hours post-incubation period and compared to those of the concurrent negative controls. The test item showed no non-specific reduction of MTT and no colouring potential. Therefore, no additional controls for correction of results were necessary. The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (104.7%) after 60 minutes treatment and 42 hours post-incubation. Therefore, Zirconium disilicide is considered to be non-irritating to the skin in accordance with UN GHS “No Category”.
Reference
Results of the Pre-Experiments:
The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
The mixture of 25 mg of the test item per 300 µL aqua dest. and per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.Therefore, NSClivingequalled 0%.
Results of the main experiment:
Table 1: Result of the Test Item Zirconium disilicide
Name |
Negative Control |
Positive Control |
Test Item |
||||||
Tissue |
1 |
2 |
3 |
1 |
2 |
3 |
1 |
2 |
3 |
absolute OD570 |
1.923 |
2.198 |
2.195 |
0.100 |
0.125 |
0.132 |
2.296 |
2.226 |
2.245 |
1.901 |
2.146 |
2.236 |
0.099 |
0.128 |
0.131 |
2.134 |
2.126 |
2.148 |
|
OD570 |
1.882 |
2.156 |
2.154 |
0.059 |
0.084 |
0.091 |
2.255 |
2.185 |
2.203 |
1.860 |
2.105 |
2.195 |
0.058 |
0.087 |
0.090 |
2.093 |
2.085 |
2.107 |
|
mean OD570of the duplicates |
1.871 |
2.131 |
2.174 |
0.059 |
0.085 |
0.090 |
2.174 |
2.135 |
2.155 |
total mean OD570of 3 replicate tissues (blank-corrected) |
2.059* |
0.078 |
2.155 |
||||||
SD OD570 |
0.164 |
0.017 |
0.019 |
||||||
relative tissue viabilities [%] |
90.9 |
103.5 |
105.6 |
2.8 |
4.1 |
4.4 |
105.6 |
103.7 |
104.7 |
mean relative tissue viability [%] |
100.0 |
3.8** |
104.7 |
||||||
SD tissue |
8.0 |
0.8 |
0.9 |
||||||
CV [% viability] |
8.0 |
21.8 |
0.9 |
*Blank-corrected mean OD570 nmof the negative control corresponds to 100% absolute tissue viability.
*Mean relative tissue viability of the three positive control tissues is ≤ 20%.
***Standard deviation (SD) obtained from the three concurrently tested tissues is ≤ 18%.
Table 2: Quality Criteria
|
Value |
Cut off |
pass/fail |
Mean Absolute OD570 nmNK |
2.100 |
0.8 ≤ NK ≤ 2.8 |
pass |
Relative Viability [%] PC |
3.8 |
≤ 20% |
pass |
SD Viability[%] |
0.8 – 8.0 |
≤ 18% |
pass |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2017-06-22 to 2017-08-03
- 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:
- 28 July 2015
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- human
- Details on test animals or tissues and environmental conditions:
- 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 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 ± 0.25 h
- Observation period (in vivo):
- N.A.
- Duration of post- treatment incubation (in vitro):
- Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C - Number of animals or in vitro replicates:
- 2 tissues per dose group
- Details on study design:
- - Details of the test procedure used:
Upon receipt of the EpiOcular™, the tissues were equilibrated in the 24-well shipment plate to room temperature for about 15 min. Then, the EpiOcular™ tissues were transferred into 6-well plates containing 1 mL pre-warmed assay medium per well and incubated for 1 h. Then the inserts 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-buffer 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. While the test item was applied, the tissue inserts were placed on a sterile surface. After dosing, the inserts were placed back into the culture medium. Then the 6-well plate(s) were incubated for 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air. 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 only the bottom of the tissues. The extraction plates were sealed to inhibit isopropanol evaporation. Extraction was carried out either after storage overnight in the dark at 2 - 8 C° or 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.
Per 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.
- RhCE tissue construct used, including batch number:
EpiOcular™ reconstructed human cornea-line epithelium (RhCE) model (MatTek), consisting of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified, highly differentiated squamous epithelium 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.
The EpiOcular™ tissues were provided as kits (e.g. OCL-200-EIT; MatTek), consisting of the following components relevant for this study:
1x sealed 24-well plate containing 24 inserts with EpiOcular™ tissues on agarose (Lot No.: 23793),
1x bottle EpiOcularTM assay medium (Lot No.: 071017ISA),
1x bottle Ca2+/Mg2+-free DPBS buffer (Lot No.: 022217ZSB),
1x vial Methyl acetate, CAS No. 79-20-9 (positive control; Lot No.: S6943111)
- Doses of test chemical and control substances used:
1. Negative Control 50 µL Aqua dest.
2. Positive Control 50 µL methyl acetate
3. Test Item 50 mg
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
Exposure: 6 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air.
Post exposure post-soak plate: 25 ± 2 min at room temperature
Post exposure post-treatment plate: 18 ± 0.25 h at 37 ± 1 °C, 5.0% CO2 / 95% air
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals:
See section "Pre-experiments" in box "Any other information on materials and methods incl. tables"
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable):
2 tissues per group
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer):
570 nm ± 30 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model:
Mean tissue viability <= 60 %: Irritant (I): UN GHS “Category 1” or “Category 2”
Mean tissue viability > 60%: Non-Irritant (NI): UN GHS “No Category”
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria:
Historical control data were generated from 2012-2016
Absolute Absolute OD570 ± 30 nm NK: Mean: 1.664; SD: 0.311, N:14
Relative Viability PC [%]: Mean: 28.9, SD: 12.0, N: 14
Difference of Viability [%]: Mean: 9.9, SD: 15.9, N: 53
- Acceptable variability between tissue replicates for positive and negative controls
mean absolute OD570 nm of the negative control is > 0.8 and < 2.5
mean relative tissue viability of the positive control is < 50%
- Acceptable variability between tissue replicates for the test chemical
relative tissue viability difference of replicate tissues is < 20%. - Irritation parameter:
- other: Relative tissue viability
- Run / experiment:
- mean of two replicates
- Value:
- 115.1
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Other effects / acceptance of results:
- ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
See also Table 1 in box "Any other information on results incl. tables" - Irritant / corrosive response data:
- The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 60% (115.1%). For detailed information please refer to Table 2 in box "Any other information on results incl. tables".
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, in this in vitro eye irritation study (OECD 492), Zirconium disilicide is not an eye irritant.
- Executive summary:
In the present study the eye irritant potential of Zirconium disilicide (99.47% purity) was analysed according to OECD 492 using the three-dimensional human corneal epithelium model EpiOcular, consisting of normal, human-derived epidermal keratinocytes mimicking characteristics of the corneal epithelium. Hereby, 50 mg of Zirconium disilicide applied directly atop the EpiOcular™ tissue. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 6 hours exposure and 18 hours post-incubation period and compared to those of the concurrent negative controls. Zirconium disilicide showed no non-specific reduction of MTT and no colouring potential. Therefore, no additional controls for correction of results were necessary. Zirconium disilicide showed no irritant effects. The mean relative tissue viability of two replicates (% negative control) was > 60% (115.1%). Therefore, Zirconium disilicide is considered to be non-irritating to the eye in accordance with UN GHS “No Category”.
Reference
Table 1: Test acceptance criteria
|
Value |
Cut off |
pass/fail |
Mean Absolute OD570 nmNK |
1.441 |
0.8 < NK < 2.5 |
pass |
Mean Relative Viability PC [%] |
32.7 |
< 50% |
pass |
Max. Difference of % Viability [%] |
3.9 |
< 20% |
pass |
The test item showed no non-specific reduction of MTT (NSMTT) and no colouring potential. Therefore, no additional controls for correction of results were necessary.
The controls confirmed the validity of the study. The mean absolute OD570 of the two negative control tissues was > 0.8 and < 2.5 (1.441). The mean relative tissue viability (% negative control) of the positive control was < 50% (32.7%). The inter tissue difference of replicate tissues of all dose groups was < 20% (0.4 - 3.9%).
Table 2: Results of the Test Item Zirconium disilicide
Name |
Negative Control |
Positive Control |
Test Item |
|||
Tissue |
1 |
2 |
1 |
2 |
1 |
2 |
Absolute OD570 |
1.438 |
1.432 |
0.495 |
0.510 |
1.659 |
1.684 |
1.449 |
1.444 |
0.489 |
0.504 |
1.590 |
1.674 |
|
OD570(blank-corrected) |
1.396 |
1.390 |
0.453 |
0.468 |
1.617 |
1.642 |
1.407 |
1.402 |
0.447 |
0.462 |
1.548 |
1.632 |
|
Mean OD570of |
1.401 |
1.396 |
0.450 |
0.465 |
1.582 |
1.637 |
Total mean OD570of |
1.399* |
0.457 |
1.610 |
|||
SD OD570 |
0.01 |
0.01 |
0.04 |
|||
Relative tissue viability [%] |
100.2 |
99.8 |
32.2 |
33.2 |
113.1 |
117.0 |
Relative tissue viability difference [%]*** |
0.4 |
1.1 |
3.9 |
|||
CV [% viability] |
0.4 |
3.3 |
3.4 |
|||
Mean relative tissue viability [%] |
100.0 |
32.7** |
115.1 |
*= corrected mean OD570 of the negative control corresponds to 100% absolute tissue viability
**= mean relative tissue viability of the positive control is < 50%
***= relative tissue viability difference of replicate tissues is < 20%
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Zirconium disilicide (> 99.47% purity) was tested negative for acute dermal and eye irritating properties in studies conducted according to OECD test guideline 439 and 492. Based on these results, Zirconium disilicide is considered to be not irritating to the skin and eye.
Justification for classification or non-classification
Zirconium disilicide (> 99.47% purity) was tested negative for acute dermal irritating and eye irritating/corrosive properties in studies conducted according to OECD 439 and OECD 492. Therefore, Zirconium disilicide does not warrant classification for skin and/or eye irritation.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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