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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From April 2017 (Protocol signed) to October 2017 (Final report)
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
Edition adopted 26 Jul. 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
adopted 14 Feb 2017
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Name TiH2
Batch no. 78562
Appearance black to grey powder
Composition Titanium Hydride Powder VM (Ti total min 94 %)
Purity Min 94 %
Homogeneity homogen metal hydride
Expiry date Feb. 2019
Storage Room Temperature (20 ± 5°C)

Test animals / tissue source

Species:
cattle
Strain:
other: Bos primigenius Taurus (fresh bovine corneas)
Details on test animals or tissues and environmental conditions:
Fresh bovine eyes were obtained from the slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany, on the day of the test. The cattle were between 12 and 60 months old. Within 1 hour 5 minutes, the eyes were transported to the test facility in Hanks’ Balanced Salt Solution with 1% Penicillin-Streptomycin solution (Penicillin 100 U/mL, Streptomycin 100 µg/mL) in a suitable cooled container.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
The test item is a solid substance. It was tested directly, without dilution or preparation of a solution. No 20% solution or suspension of the test item was tested, because this procedure is more close to reality.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The following amounts of the test item were tested neat and applied directly on the cornea using a weighing funnel:
- Replicate 1: 499.2 mg
- Replicate 2: 608.5 mg
- Replicate 3: 482.8 mg
Duration of treatment / exposure:
The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item.
Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded. The cMEM without phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration: 5 mg/mL) was added to the front chamber.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability of the cornea was measured as optical density of the liquid with a microtiter plate photometer at 492 nm.
Duration of post- treatment incubation (in vitro):
Exposure time on the corneas was 4 hours at 32 ± 1 °C.
Number of animals or in vitro replicates:
Three replicates - Test item and positive and negative controls.
Details on study design:
* Preparations
After having carefully cleaned and sterilised the cornea holders, they were kept in the incubation chamber at 32 ± 1 °C.
On the day of the assay, the MEM without phenol red was supplemented with sodium bicarbonate, L-glutamine and 1% fetal calf serum (= complete MEM) and stored in a water bath at 32 ± 1 °C. The same was performed with the MEM with phenol red, but without addition of sodium bicarbonate.
After the arrival of the corneas, they were examined and only corneas which were free from damages were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM (32 ± 1 °C) without phenol red was filled. The holders were then incubated for 1 hour in the incubation chamber at 32 ± 1 °C.

* Experimental Parameters
Date of treatment 26. Jun. 2017
Incubation time 4 h
Negative control HBSS
Positive control Imidazole, 20 % solution in HBSS

* Method Description
The experimental procedure was performed under the fume hood. After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL negative and positive control solution and a defined amount of test item were applied to each replicate.

* Evaluation
- Calculation of Opacity
The change of opacity value of each treated cornea with test item, positive control and negative control was calculated by subtracting the initial basal opacity from the post treatment opacity reading for each cornea. The average change in opacity of the negative control cornea was calculated and this value was subtracted from the change in opacity of each treated cornea with test item and positive control to obtain a corrected opacity.

Opacity= ( I0/I)-b/a

a = 0.0251 and b = 0.9894 being Opacitometer-specific empirically determined variables
I0 = the empirically determined illuminance through a cornea holder with windows and
Medium, here: Io= 1055.62
I = the measured illuminance (unit: LUX)

- Calculation of Permeability
The corrected OD492 value of each cornea treated with test item and positive control was calculated by subtracting the mean negative control cornea value from the original permeability value for each cornea.
The mean OD492 value for each treatment group (test item, positive control and negative control) was determined by averaging the final corrected OD492 values of the treated corneas for one treatment group.

- Calculation of IVIS (In Vitro Irritancy Score)
The IVIS of each replicate of the negative control was calculated from the following equation:
IVIS = opacity difference + (15 x corrected OD492 value)

The IVIS of each replicate of the positive control and of the test item were calculated from the following equation:
IVIS = (opacity difference – mean opacity difference of the negative control) + [15 x (OD492 – mean OD492 of the negative control)]

Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Run / experiment:
Mean opacity difference corrected of the test item (4 hours exposure)
Value:
ca. 5.49
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
cornea opacity score
Run / experiment:
Mean Opacity Difference of the negative controls (4 hours exposure)
Value:
ca. 2.45
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Irritation parameter:
cornea opacity score
Run / experiment:
Mean Opacity Difference Corrected of the positive controls ( 4 hours exposure)
Value:
ca. 106.79
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
* In vitro Irritancy Score (IVIS)
- Negative control HBSS:
Mean IVIS: 2.45
Relative Standard Deviation IVIS: 34.35%
- Test item TiH2
Mean IVIS: 5.49
Relative Standard Deviation IVIS: 103.32%
- Positive control 20% imidazole solution:
Mean IVIS: 106.79
Relative Standard Deviation IVIS: 5.48%
* Optical density at 492 nm of Negative Control, Test Item and Positive Control
Corrected mean of the 3 replicates:
Negative Control: /
Test Item: 0.0267
Positive Control: 1.8741

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Following OECD Guideline no. 437 (BCOP), the test item TiH2 showed effects on the cornea of the bovine eye. However, the calculated IVIS (in vitro irritancy score) is 5.49. Therefore, as the substance has an IVIS between > 3 and ≤ 55 induces effects on the cornea, it cannot be classified in an UN GHS Category for eye damage.
Executive summary:

BCOP Study (OECD 437) with Titanium hydride

One valid experiment was performed.

Bovine corneas were used. They were collected from slaughtered cattle which were between 12 and 60 months old. The test item TiH2 was brought onto the cornea of a bovine eye which had been incubated with cMEM without phenol red at 32 ± 1 °C for 1 hour and whose opacity had been measured. The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured. HBSS-solution was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (in vitro irritancy score) is 2.45. 20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS (in vitro irritancy score) is 106.79.

Under the conditions of this study, the test item TiH2 showed effects on the cornea of the bovine eye. The calculated IVIS (in vitro irritancy score) is 5.49.

According to OECD Guideline no. 437 (Jul. 2013), a substance with an IVIS > 3 and ≤ 55 induces effects on the cornea, that cannot be classified in an UN GHS Category for eye damage.