Titanium nitride

Eye irritation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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)

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
- Analytical purity: > 99%
- Physical state: solid
- Colour: yellow
- 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
- The test item was suspended with physiological saline (0.9% NaCl) to gain a 20% concentration

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results

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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).