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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-11-04 to 2014-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report date:
2014

Materials and methods

Test guidelineopen allclose all
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 material

Constituent 1
Chemical structure
Reference substance name:
Titanium nitride
EC Number:
247-117-5
EC Name:
Titanium nitride
Cas Number:
25583-20-4
Molecular formula:
NTi
IUPAC Name:
titanium nitride
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Titanium nitride
- Physical state: solid
- Colour: yellow
- Analytical purity: > 99%
- Lot/batch No.: 84958
- Expiration date of the lot/batch: 2016-09-16
- Storage condition of test material: at room temperature

Test animals

Species:
other: human epidermis model: Episkin
Strain:
not specified
Details on test animals or test system and environmental conditions:
n.a.

Test system

Vehicle:
water
Controls:
not required
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (= 26.3 mg/cm²) + 10 µL Aqua dest.

Duration of treatment / exposure:
15 ± 0.5 min
Observation period:
42 h post-incubation period
Number of animals:
N.A.
Details on study design:
TEST SITE
- Area of exposure: 0.38 cm²
- % coverage: 100 %

REMOVAL OF TEST SUBSTANCE
- Washing (if done): phosphate buffered saline
- Time after start of exposure: 15 ± 0.5 min

PRE-EXPERIMENT
To check the non specific MTT-reducing capability of the test item 10 mg of the test item were mixed per 2 mL MTT medium and incubated for 3 h at 37 ± 1 °C in the dark. If the mixture turns blue/purple, the test item is presumed to have reduced MTT. To check the colouring potential of the test item 10 mg of the test item were mixed per 90 μL Aqua dest. in a transparent recipient for 15 min.

EXPERIMENTAL PROCEDURE
Upon receipt of the EPISKIN-SM, the tissues were transferred into 12-well plates containing 2 mL prewarmed maintenance medium per well. The 12-well plates were incubated in a humidified incubator at 37 ± 1 °C, 5.0% CO2 for at least 24 h.
After this pre-incubation the tissues were treated with each dose group in triplicate, starting with the negative control. Start time was recorded with dosing of the first tissue. Then the tissues were incubated at room temperature for 15 ± 0.5 min. Afterwards, the tissues were washed with PBS to remove any residual test item. Excess PBS was removed by blotting bottom with blotting paper. The inserts were placed in a prepared 12-well plate containing 2 mL prewarmed fresh maintenance medium and post-incubated at 37 ± 1 °C, 5.0% CO2 for 42 ± 1 h.
After this incubation period the plates were placed for 15 ± 2 min. on a plate shaker. Then the inserts were transferred in a prepared 12-well plate containing 2 mL prewarmed MTT medium and further incubated for 3 h ± 5 min. at 37 ± 1 °C, 5.0% CO2.
After the 3 h MTT incubation period the tissues were placed on blotting paper to dry the tissues. Afterwards a total biopsy of the epidermis by using the special biopsy punch was performed and the epidermis was separated from the collagen matrix with the aid of forceps. Both parts (epidermis and collagen matrix) were transferred into suitable tubes and 500 μL of acidic isopropanol were added. Extraction was carried out protected from light over the weekend at 2 - 8°C.
At the end of the formazan extraction period the tubes were mixed by vortexing until solution colour became homogeneous. If any visible cell/tissue fragments were in suspension, the tubes were centrifuged at 500 rpm to eliminate the fragments and avoid further possible interference with the absorbance readings. Per tissue 2 x 200 μL aliquots of the extract were transferred into a 96-well plate and OD was measured at 550 nm without reference wavelength in a plate spectrophotometer.

SCORING SYSTEM:
Irritant potential of the test item was predicted from the relative mean tissue viabilities compared to the negative control tissues concurrently treated with PBS. The test item is considered to be irritant to skin in accordance with regulation EC1272/2008 and UN GHS "Category 2", if the tissue viability after 15 min of exposure and 42 h of post-incubation is less or equal to 50%. The test substance may be considered as non-irritant to skin in accordance with UN GHS "No category" if the tissue viability after exposure and post-treatment incubation is higher than 50%.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
other: other: mean relative tissue viability
Value:
98.8
Remarks on result:
other:
Remarks:
Basis: mean (triplicates). Time point: 42 h post-incubation . Max. score: 100.0. Remarks: max. score: mean relative viability obtained by the negative control. (migrated information)

Any other information on results incl. tables

Table 1: Result of the Test Item Titanium nitride
Name Negative Control Positive Control Test Item  
Tissue 1 2 3 1 2 3 1 2 3  
absolute OD550 0.902
0.886
0.806
0.791
0.905
0.872
0.115
0.098
0.093
0.089
0.082
0.077
0.771
0.743
0.999
0.958
0.889
0.745
 
OD550(blank-corrected) 0.859
0.844
0.763
0.748
0.862
0.830
0.073
0.056
0.050
0.046
0.039
0.034
0.728
0.700
0.956
0.915
0.847
0.702
 
mean OD550of the duplicates (blank-corrected) 0.851 0.756 0.846 0.064 0.048 0.037 0.714 0.936 0.774  
total mean OD550of 3 replicate tissues (blank-corrected) 0.818* 0.05 0.808  
SD OD550 0.054 0.014 0.114  
relative tissue viabilities [%] 104.1 92.4 103.4 7.9 5.9 4.5 87.3 114.4 94.7  
mean relative tissue viability [%] 100.0 6.1** 98.8  
SD tissue viability [%]*** 6.6 1.7 14.0  
CV [% viability] 6.6 28.0 14.2  
* Corrected mean OD550 of the negative control corresponds to 100% absolute tissue viability.  
** mean relative tissue viability of the three positive control tissues is ≤ 40%  
*** The standard deviation (SD) obtained from the three concurrently tested tissues is < 18%.  

Table 2: Quality criteria
 
  Value Cut Off pass/fail
Mean OD550 nmBlank 0.043 < 0.1 pass

Mean Absolute OD550 nm NC

0.86 0.6≤ NC ≤ 1.5 pass
Mean Relative viability [%] PC 6.1 ≤ 40% pass
SD of % Viability [%] 1.7 - 14.0 ≤ 18% pass

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In conclusion, titanium nitride showed no irritant effects in an in vitro skin irritation study according to OECD 439.
Executive summary:

In an in vitro skin irritation study (OECD 439), the reconstituted three-dimensional human skin model EPISKIN-SMTM was topically exposed to 10 mg of titanium nitride (>99% purity) in water for 15 minutes. After exposure the tissue was washed off with phosphate buffered saline and incubated for additional 42 h. Tissue viability was assessed measuring the enzymatic reduction of the vital dye MTT.

PBS served as negative control, corresponding to 100% tissue viability. As positive control SDS (5%) was used. Measurements were performed in triplicates and mean values were calculated.

In summary, titanium nitride showed no irritant effects, based on a mean tissue viability of 98.8% after treatment with the test substance in comparison to the negative control.