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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 (OECD 437). 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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
26 July 2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
06 July 2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Firstly, 5 µl distilled water (aqua dest.) were applied by a pipette to the epidermal surface in order to improve further contact between the powder and the epidermis. The water was gently spread with the pipette. Afterwards, approximately 10 mg (26.3 mg/cm²) of the powder were applied to the epidermis surface. The volume of water was increased to 5 µl and the test item was spread to match size of the tissue.
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EPISKIN-SM(TM) reconstructed human epidermis model (Skin Ethic) which consists of human 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 skin, i.e. the epidermis.
Vehicle:
water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM(TM)
- Tissue batch number(s): 14-EKIN-043
- Production date: November 12, 2014
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 11 November 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: not specified
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: final concentration 0.3 mg/ml
- Incubation time: 3h ± 5 min
- Wavelength: 550 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: exceeded specifications
- Morphology: Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum. Exceeded specifications.

NUMBER OF REPLICATE TISSUES: The test was performed on a total of 3 tissues per dose group.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- - The test item showed no reduction of MTT compared to the solvent and showed no colouring detectable by unaided eye-assessment

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

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 hours of post-incubation is less than or equal to 50%. The test substance may be considered non-irritant to skin if the tissue viability after exposure and post-treatment incubation is higher than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 10 mg (26.3 mg/cm²)

VEHICLE
- Amount(s) applied: 10 µl
- Purity: distilled water

NEGATIVE CONTROL
- Amount(s) applied: 10 µl

POSITIVE CONTROL
- Amount(s) applied: 10 µl
- Concentration: 5% SDS
Duration of treatment / exposure:
15 ± 0.5 min
Duration of post-treatment incubation (if applicable):
42 ± 1 hours
Number of replicates:
3
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:
% tissue viability
Run / experiment:
Test item (Mean of three replicates)
Value:
98.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

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:
GHS criteria not met
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-SM(TM) was topically exposed to 10 mg of titanium nitride (>99% purity) in water for 15 minutes. After exposure the tissue was washed 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, other
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:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Version / remarks:
26 July 2013
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- The test item was suspended with physiological saline (0.9% NaCl) to gain a 20% concentration
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): On the test day, fresh eyes were collected from the slautherhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
- Time interval prior to initiating testing: Immediately after arrival
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were carefully examined for defects and any defective eyes were discarded.
- Indication of any antibiotics used: penicillin/ streptomycin (Gibco, lot no. 1546516, expiry date: 02/2015)
- Selection and preparation of corneas: The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS.
- Quality check of the isolated corneas: Before the corneas were mounted in corneal holders (MC2, Clermont, France) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded.
Vehicle:
physiological saline
Remarks:
0.9% NaCl
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- 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
- Concentration (if solution): 0.9 %
- Lot/batch no. (if required): 1307034

Duration of treatment / exposure:
4h ± 5 minutes at 32 ± 1 °C
Observation period (in vivo):
Not applicable
Duration of post- treatment incubation (in vitro):
90 minutes at 32 ± 1 °C.
Number of animals or in vitro replicates:
- 3 corneas for the test item
- 3 corneas as negative controls treated with physiological saline 0.9% NaCl
- 3 corneas as positive control treated with imidazole 20% in physiological saline 0.9% NaCl
Details on study design:
NUMBER OF REPLICATES: Three

NEGATIVE CONTROL USED: physiological saline (0.9% NaCl)

POSITIVE CONTROL USED: 20% imidazole in physiological saline (0.9% NaCl)

APPLICATION DOSE AND EXPOSURE TIME: 750 µl for 4h ± 5 minutes at 32 ± 1 °C

TREATMENT METHOD: closed chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times with minimum essential medium (MEM)
- POST-EXPOSURE INCUBATION: yes. 90 minutes at 32 ± 1 °C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacitometer (MC2, Clermont, France) had been switched on 15 min before the calibration procedure was started. Empty cornea holders were placed into the opacitometer and the readout was adjusted to zero using the “BAL”-turning knob. For calibration the polyester foil no. 1 was introduced into the test chamber and the readout was adjusted to 75 using the “CAL”-turning knob. To test the linearity of the measurement, two additional calibration foils, polyester foil no. 2 and polyester foil no. 3, were measured. For these, the opacitometer was supposed to display 150 and 225, respectively (± 3%). If this had not been the case, the calibration procedure would have had to be repeated. The calibration procedure was performed before each test and was documented in the raw data.
An initial opacity measurement was performed after the equilibration period when the medium was removed from both chambers and replaced with fresh complete RPMI. Following the incubation period, the epithelium was washed at least three times with MEM (containing phenol red). Once the medium was free of test substance, the cornea was finally rinsed with complete RPMI (without phenol red). The anterior chamber was refilled with complete RPMI and an opacity measurement was performed.
The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. These values were corrected by subtracting from each the average change in opacity observed for the negative-control corneas. The mean opacity value for each treatment was calculated by averaging the corrected opacity values of each cornea for a given treatment. The mean OD490 for the blank wells were calculated.
- Corneal permeability: After the opacity measurement the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 ml of a 5 mg/ml sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer.
The mean blank OD490 was subtracted from the OD490 of each well (corrected OD490). Any dilutions that were made to bring the OD490 values into the linear range of the spectrophotometer (OD490 should be less than 1.500), were taken into account by multiplying the OD490 value of the dilution by the dilution factor. The final-corrected OD490 of the test article and the positive control were calculated by subtracting the average corrected OD490 of the negative control corneas from the corrected OD490 value of each treated cornea:

Final-corrected OD490 = (OD490 – mean blank OD490) – average-corrected negative control OD490

The mean OD490 value of each treatment group was calculated by averaging the final corrected OD490 values of the treated corneas for that treatment condition.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15x mean permeability OD490 value)

DECISION CRITERIA:
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

The assay is considered to be valid if the in vitro irritation score obtained with the positive control falls within the two standard deviations of the current historical mean.
Irritation parameter:
in vitro irritation score
Run / experiment:
Test item (mean from three replicates)
Value:
0.03
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

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:
GHS criteria not met
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 to be 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 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 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.