Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

EC Number:

945-733-5

Molecular formula:

See remarks

IUPAC Name:

Reaction mass of dieuropium tricarbonate and digadolinium tricarbonate and disamarium tricarbonate

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Natural white powder
- Storage conditions: Room temperature (15-25 °C), protected from light and humidity

Specific details on test material used for the study:

The solubility of the test material in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test material did not dissolve in physiological saline.

Test animals / tissue source

Species:

chicken

Strain:

other: Ross 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to Citoxlab Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box).
- Time interval prior to initiating testing: The heads were received at Citoxlab Hungary Ltd. and processed within 2 hours of collection.
- Indication of any existing defects or lesions in ocular tissue samples: After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test material was applied onto the entire surface of the cornea.

Duration of treatment / exposure:

10 seconds.

Duration of post- treatment incubation (in vitro):

Observations were made at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

Three eyes were exposed to the test material.

Details on study design:

PREPARATION OF ISOLATED EYES
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
- Eyes examination and acclimatisation time:
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head).
Again too much pressure on the eye by the clamp was avoided. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and then placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly that the physiological saline was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10% from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32 ± 1.5°C) during the acclimatisation and treatment periods.

- The baseline assessments
At the end of the acclimatisation period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes in the experiment. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test material related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
One negative control, three posititve control and three test material-treated.

NEGATIVE CONTROL USED
Negative control eye was treated with 30 μL of physiological saline.

POSITIVE CONTROL USED
Positive control eyes were treated with 30 mg powdered imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. 30 mg of the test material was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test material, taking care not to damage or touch the cornea. An exposure period of 10 seconds from the end of the application was used.

OBSERVATION PERIOD
- Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ± 5 minutes were considered acceptable.

REMOVAL OF TEST MATERIAL
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with at least 20 mL saline was performed at each time point when the test material or positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Haag-Streit Bern 900 slit-lamp microscope was used for the measurements

EVALUATION
Corneal swelling was calculated according to the following formulae:

CS at time t = ((CT at time t –CT at t=0) / (CT at t=0)) x 100

Mean CS at time t = (FECS(at time t)+ SECS(at time t) + TECS(at time t)) / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point

Small negative numbers for swelling (0 to -5%) following application are evaluated as class I. Large negative numbers (>12% below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12% are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t=0

Mean ΔCOmax = (FECOmax(30min to 240min)+ SECOmax(30min to 240min) + TECOmax(30min to 240min)) / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t – FR at t=0

Mean ΔFR = (FEFR (30min) + SEFR(30min) + TEFR(30min)) / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention


DECISION CRITERIA:
ICE classification criteria for corneal thickness:
Mean Corneal Swelling (%) ICE Class
0 to 5 I
>5 to 12 II
>12 to 18 ( >75 min after treatment ) II
>12 to 18 ( <75 min after treatment ) III
>18 to 26 III
>26 to 32 ( >75 min after treatment ) III
>26 to 32 ( <75 min after treatment ) IV
>32 IV

ICE classification criteria for corneal opacity:
Mean Maximum Opacity Score ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 4.0 IV

ICE classification criteria for mean fluorescein retention:
Mean Fluorescein Retention Score at 30 minutes post - treatment ICE Class
0.0 – 0.5 I
0.6 – 1.5 II
1.6 – 2.5 III
2.6 – 3.0 IV

Assessment of the general in vitro eye irritancy and regulatory UN GHS classification:
The following table is used to identify the probably eye irritancy potential of test materials. In the case where the result indicates Non-irritant or Corrosive/Severely Irritating, then the test material can be classified. In all other cases the probable level of irritancy can be reported, but a regulatory in vivo rabbit eye irritation test is required for regulatory classification and labelling purposes.

UN GHS Classification Combinations of the three ICE Classes
No Category 3×I
2×I, 1×II
No prediction can be made Other combinations
Category 1 3×IV
2×IV, 1×III
2×IV, 1×II*
2×IV, 1×I*
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes)
Corneal opacity = 4 at any time point (in at least 2 eyes)
Severe loosening of epithelium (in at least 1 eye)
* Combinations of categories less likely to occur.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

Up to 75 min

Run / experiment:

Mean

Value:

2.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

10.9 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

Up to 240 min

Run / experiment:

Mean

Value:

5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

25.1 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Results 3

Irritation parameter:

cornea opacity score

Remarks:

Mean maximum

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

4.0 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Results 4

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.0 %

Positive controls validity:

valid

Remarks:

3.0 %

Remarks on result:

no indication of irritation

Remarks:

Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: Test material was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
- The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse.
No other morphological effect was observed in the study.

RESULTS:
Based on this in vitro eye irritation in the isolated chicken eyes test with the test material, the test maerial is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The physological saline gave no indication of irritation.
- Acceptance criteria met for positive control: The imdazole positive control was severly irritating.
- Range of historical values if different from the ones specified in the test guideline: The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the test, the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

Executive summary:

An in vitro eye irritation study of the test material was performed with isolated chicken’s eyes. The irritation effects of the test material were evaluated according to the standardised guideline OECD 438 (26 July 2013).

After the zero reference measurements, the eye was held in horizontal position and 30 mg test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9% (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined.

The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in the experiment. Thus, the experiment was considered to be valid.

No significant corneal swelling (mean ≤ 5%) was observed during the four-hour observation period on test material treated eyes. No significant cornea opacity change (severity 0.5) was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test material was stuck on all cornea surfaces after the post-treatment rinse. All three cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. Although there were no adverse corneal effects observed, no conclusion of in vivo significance could be established due to the adherence of the test material to the cornea, since in vivo eye lids may clear the surface, but abrasion may occur.

Under the conditions of the test the test material is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.