Registration Dossier

Administrative data

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

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
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

In vitro test system

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

Results and discussion

In vitro

Results
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" .

Any other information on results incl. tables

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
(blank-corrected)

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
(blank-corrected)

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
viability [%]***

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

 

Applicant's summary and conclusion

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”.