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Description of key information

In an in vitro skin irritation assay in accordance with OECD 439, titanium nitride was applied topically to reconstituted three-dimensional human epidermis. Based on the results, titanium nitride was considered to be "non-irritant" to the skin.
The eye irritancy potential of titanium nitride was assessed by using the bovine corneal opacity and permeability assay: Based on the results, titanium nitride was considered to be "non-irritant" to the 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:
2014-11-04 to 2014-12-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP 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)
Species:
other: human epidermis model: Episkin
Strain:
not specified
Details on test animals or test system and environmental conditions:
n.a.
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%.

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)

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
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.

 

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:
2014-11-03 to 2014-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
other: BCOP
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany
Vehicle:
physiological saline
Controls:
yes
Amount / concentration applied:
TEST MATERIAL
- Concentration (if solution): test item was suspended with 0.9% NaCl to gain a 20% concentration.

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µl
Duration of treatment / exposure:
4h ± 5 minutes at 32 ± 1 °C
Observation period (in vivo):
Opacity measurements were performed before and directly after exposure.
The optical density at 490 nm was measured upon 90 minutes of incubation with fluorescein after exposure to the test item by using a spectrophotometer.
Number of animals or in vitro replicates:
Three corneas each for titanium nitride, negative control (physiological saline 0.9% NaCl) and positive control (20 % imidazole in physiological saline 0.9 NaCl%).
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): at least 3 times with minimum essential medium (MEM)
- Time after start of exposure: 4h ± 5 minutes

SCORING SYSTEM:
In vitro irritation score (IVIS) = mean opacity value + (15x mean permeability OD490 value)
The IVIS cut-off values for identifiying test substances as inducing serious eye damage (UN GHS Category 1) and test substances not requiring classification for eye irritation of serious eye damage (UN GHS No Category):
IVIS: ≤ 3 - UN GHS: No Category
IVIS: > 3 - ≤ 55 - UN GHS: No prediction can be made
IVIS: > 55 - UN GHS: Category 1

TOOL USED TO ASSESS SCORE:
Opacity measurement was performed with an opacitometer
Irritation parameter:
other: In vitro irritation score (IVIS)
Basis:
mean
Remarks:
(triplicates)
Time point:
other: 90 minutes after 4h exposure
Score:
0.03
Max. score:
202
Remarks on result:
other: Max score= mean in vitro irritation score of positive control

Table 1: In Vitro Irritation Score

Cornea No.

Test Item

Corrected Opacity

Corrected OD490 Value

IVIS

1

Negative Control

0.00

0.005

 

2

1.00

0.009

 

3

0.00

0.023

 

MV

0.33

0.012

0.52

4

Positive
Control

187.67

1.791

 

5

174.67

2.019

 

6

155.67

2.026

 

MV

172.67

1.945

201.84

7

Test Item

-0.33

-0.001

 

8

-0.33

0.008

 

9

0.67

-0.001

 

MV

0.00

0.002

0.03

MV = mean value

Table 2: Historical mean in vitro irritation score of the positive control
  IVIS (positive control)
Mean Value (MV) 206.77
Standard deviation (SD) 24.53
MV - 2 x SD 157.71
MV + 2 x SD 255.84

IVIS: in vitro irritation score

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: UN GHS
Conclusions:
In conclusion, based on the mean in vitro irritation score of 0.03 obtained in the bovine cornea opacity and permeability assay (OECD 437), titanium nitride is considered to be not irritating under the test conditions reported.
Executive summary:

The eye irritation potential of titanium nitride (> 99% purity) was investigated in the bovine cornea opacity and permeability assay in accordance to OECD 437. The test item was suspended with 0.9% NaCl to gain a 20% concentration. The corneal opacity was measured before and after treatment (4 hours). The mean in vitro irritation score was determined with 0.03. The positive control induced the appropriate responses, indicating the validity of the assay. Based on the results obtained, titanium nitride can be considered as not irritating to the eye ( UN GHS No Category).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The irritancy potential of titanium nitride towards skin and eye was addressed by using two in vitro assays, the EPISKIN-SM, a reconstituted three-dimensional human epidermis model (OECD 439), and the bovine corneal opacity and permeability (BCOP) test (OECD 437).

For the in vitro skin irritancy test, 10 mg of titanium nitride (>99% purity) was topically applied with to the reconstituted three-dimensional human epidermis tissue for a period of 15 minutes. Following washing with phosphate buffered saline and a 42 h post-incubation time, the tissue viability was measured using the vital dye MTT. Titanium nitride resulted in a mean tissue viability score of 98.8 %, classifying titanium nitride as non-irritant to the skin.

The BCOP assay was performed in triplicates, exposing fresh corneas for 4 h to a 20% solution of titanium nitride (>99% purity) solved in physiological saline. Negative and positive controls were run in parallel. For assessment of the irritancy potential the in vitro irritation score (IVIS) was calculated. Titanium nitride resulted in a mean IVIS of 0.03, clearly classifying titanium nitride as non-irritant to the eye.


Justification for selection of skin irritation / corrosion endpoint:
GLP guideline study

Justification for selection of eye irritation endpoint:
GLP guideline study

Justification for classification or non-classification

The in vitro methods used are suitable methods for testing the irritancy potential of titanium nitride to the skin and eye (OECD 439 and OECD 437). For both target organs, addressed by the methods used, titanium nitride can be considered as non-irritating. Therefore, based on the available data, titanium nitride does not warrant classification for skin and/or eye irritation.

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