Registration Dossier

Administrative data

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

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 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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Batch No: 81704059
- Physical state: solid, powder
- Colour: grey
- Purity: 99.47%
- Expiry date: 2019-04-28
- Storage conditions: room temperature

Test animals / tissue source

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.

Test system

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%.

Results and discussion

In vitro

Results
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"

In vivo

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".

Any other information on results incl. tables

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
the duplicates
(blank-corrected)

1.401

1.396

0.450

0.465

1.582

1.637

Total mean OD570of
2 replicate tissues
(blank-corrected)

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%

Applicant's summary and conclusion

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”.