Gadolinium trinitrate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 May 2016 - 23 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

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:

26 July 2013

Deviations:

yes

Remarks:

deviation from study plan, see "Any other information on results"

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Gadolinium trinitrate

EC Number:

233-437-2

EC Name:

Gadolinium trinitrate

Cas Number:

10168-81-7

Molecular formula:

Gd(NO3)3

IUPAC Name:

gadolinium trinitrate

Test material form:

solid: crystalline

Details on test material:

- Name of test material (as cited in the report): gadolinium trinitrate
- CAS = 19598-90-4 (i.e. the hexahydrate form Gd(NO3)3.6H2O). According to Annex V, point 6 of the REACH regulation (CE) No. 1907/2006, this form will be covered by the registration of the anhydrous form (CAS number: 10168-81-7).
- Physical state: crystalline solid
- Further information on test item is confidential

Specific details on test material used for the study:

Correction factor: No correction factor was applied for this type of study.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Abattoir EVA, Saint-Pierre-sur-Dives, France
- Number of animals: At least 5 (9 corneas used in total during the study). Not specified if the eyes came from different animals.
- Characteristics of donor animals (e.g. age, sex, weight): Freshly slaughtered calves < 12 months old.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were transported at ambient temperature in a cool box, immerged in cooled buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)]. A container with smooth internal surfaces was used for transport to avoid damage to the corneas.
- Time interval prior to initiating testing: Maximum 24 hours, stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C.
- Indication of any existing defects or lesions in ocular tissue samples: no
- Indication of any antibiotics used: Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final)

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

- Amount(s) applied (volume or weight with unit): 750 mg (± 75 mg)

Duration of treatment / exposure:

4 hours

Duration of post- treatment incubation (in vitro):

90 minutes ± 5 minutes

Number of animals or in vitro replicates:

3 of each per group (test item, positive control, negative control)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
- Macroscopic examination was performed on all eyes to detect the presence of any defects. Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swiveled in order to observe the fringe areas and any scratches directly under the light. Tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out.
- The corneas were washed 3 times for 15 min in HBSS plus penicillin/streptomycin (100 units/100 μg/mL final) at room temperature. The corneas were used within a maximum of 24 hours. Each cornea was store individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.
- The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders (one cornea per holder) with the endothelial side against the O-ring of the posterior chamber. The anterior half of the holder was then positioned on top of the cornea and tightened with screws.
- For pre-incubation, both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea. After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C).
- At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM (previously heated to +32°C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0.

QUALITY CHECK OF THE ISOLATED CORNEAS: Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride

POSITIVE CONTROL USED: 20% imidazole solution in 0.9% NaCl (w/v)

APPLICATION DOSE AND EXPOSURE TIME: 750 mg (± 75 mg) of test item, and 750 μL (± 8 μL) of both positive and negative controls. Total treatment time was 4 h.

TREATMENT METHOD
Closed chamber method for negative and positive controls.
- Open chamber method for test item treatment.
- Application of test item: The window-locking ring and glass window from the anterior chamber were removed. The test item was gently applied onto the epithelium of the cornea, as uniformly as possible in order to ensure that it covers the whole epithelial surface. The glass window of the anterior chamber was then replaced (without the window-locking ring).

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 6 times with pre-warmed cMEM containing phenol red, then the corneas were finally rinsed with pre-warmed cMEM without phenol red.

POST-EXPOSURE INCUBATION: Yes, 90 min in 5 mg/mL fluorescein stain at +32°C.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
- Corneal permeability: Passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry (OD490).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS).

ACCEPTANCE CRITERIA:
For the validation of an experiment, the following criteria had to be fulfilled:
- the mean In Vitro Irritancy Score (IVIS) of the positive control corneas should fall within two standard deviations of the historical mean;
- the mean opacity of the negative control corneas should be < 4.4;
- the mean OD490 nm of the negative control corneas should be < 0.0296.

DECISION CRITERA:
- If the test item induces an IVIS <= 3, the test item is not assigned to any category for eye irritation under UN GHS.
- If the test item induces an IVIS < 3 but <= 55, no prediction can be made.
- If the test item induces an IVIS > 55, the test item is assigned to Category 1 for eye irritation (Eye Damage 1) under UN GHS.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3

Value:

130

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Opacity, fluorescein fixation and thickening of the corneas were observed on the corneas treated with the test item and the positive control.
- No notable opaque spots or irregularities were observed on those treated with the negative control.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Any other information on results incl. tables

Deviation from the study plan

The mean OD490nm of the negative control corneas was found at 0.040 instead of below 0.0296, as specified in the study plan. This deviation would lead to an over-correction of the cOD490nm. The mean cOPT obtained for test item-treated corneas was higher than the one obtained with the positive control-treated corneas and above the eye corrosion positivity threshold of 55. Therefore and based on the obtained results, this deviation is considered not to have any impact on the conclusion of the study. Indeed, even with this over-correction, acceptance criteria of positive control are met and results of test item-treated corneas allowed unequivocal test item classification.

Results table

Group		Opacity				Permeability		Score
	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490nm	cOD490nm
Negative control	38	1	3	2		0.057
	29	1	0	-1		0.011
	13	1	7	6		0.051
	Mean			2		0.040
	SD			4		0.025
	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490nm	cOD490nm
Positive control	23	0	139	139	137	0.340	0.300	141
	42	2	103	101	99	0.983	0.943	113
	34	2	131	129	127	0.576	0.536	135
	Mean				120.7		0.593	130
	SD				19.7		0.3	14.9
	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490nm	cOD490nm
Test item	46	0	120	120	118	2.404	2.364	153
	5	1	120	119	117	2.772	2.732	158
	36	2	121	119	117	3.576	3.536	170
	Mean				117.0		2.878	160
	SD				0.6		0.599	8.6

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

As the test item induced a mean IVIS > 55, it was considered as a test chemical inducing serious eye damage. Under the experimental conditions of this study, the test item was identified as a test item inducing serious eye damage (UN GHS Category 1).