Titanium nitride

Skin irritation / corrosion

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

26 July 2013

Deviations:

no

Test guideline 2

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)

Test material

Test material information

Constituent 1

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:

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

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.

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 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

Test system

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:

% 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

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:

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.