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Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Remarks:
Isolated Chicken Eye Test (ICET)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
8 June 2020 - 23 December 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

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)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

1
Reference substance name:
Nickel Cobalt Manganese Hydroxide
Cas Number:
189139-63-7
Molecular formula:
Molecular formula: Ni1-x-yMnxCoy(OH)2, Stoechiometry of (Me = Ni + Co + Mn) equals 1, ranges of the elements approx. as: Ni/Me = 0.25 – 0.95 Mn/Me = 0.02 – 0.40 Co/Me = 0.02 – 0.40 OH = 2.00
IUPAC Name:
Nickel Cobalt Manganese Hydroxide
Constituent 1
Chemical structure
Reference substance name:
Manganese dihydroxide
Cas Number:
18933-05-6
Molecular formula:
Mn(OH)2
IUPAC Name:
Manganese dihydroxide
Constituent 2
Chemical structure
Reference substance name:
Cobalt dihydroxide
EC Number:
244-166-4
EC Name:
Cobalt dihydroxide
Cas Number:
21041-93-0
Molecular formula:
CoH2O2
IUPAC Name:
cobalt(2+) dihydroxide
Constituent 3
Chemical structure
Reference substance name:
Nickel dihydroxide
EC Number:
235-008-5
EC Name:
Nickel dihydroxide
Cas Number:
12054-48-7
Molecular formula:
H2NiO2
IUPAC Name:
nickel(2+) dihydroxide
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch/Lot number: PVX-089 PVX 14
Description: Dark Grey Powder
Purity: 100% (6.31% Co ; 5.96% Mn ; 50.87% Ni)
Manufacturer: Umicore
Expiry date: 30 April 2021
Storage conditions: Controlled room temperature (15-25 °C, ≤70% relative humidity). Protected from light and humidity (store in a tightly closed container).
The Certificate of Analysis is attached in Appendix 1 of the study report.

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. (Address: H-9600 Sárvár, Rábasömjéni utca 129., Hungary)
- Number of animals: 7 (3 test item, 3 positive control, 1 negative control)
- Characteristics of donor animals (e.g. age, sex, weight): Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue: Heads were collected by a slaughter house technician and heads transported to Charles River Laboratories Hungary Kft. 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). The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection.
- Time interval prior to initiating testing: 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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit. The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition.
- Indication of any antibiotics used: no data

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 105 mg


Duration of treatment / exposure:
exposure: 10 seconds
Duration of post- treatment incubation (in vitro):
ex vivo: 4 hours observation period
Number of animals or in vitro replicates:
test item: 3 eyes
positive control: 3 eyes
negative control: 1 eye
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
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 (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eyeball 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.
Each eye was located in chamber identified by a unique number within the Test Facility.
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 avoid too much pressure on the eye by the clamp. 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 after being 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 the physiological saline which 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, acclimatization 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 acclimatization and treatment periods

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization 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 significant corneal thickness changes (1.6% was observed in one eye and 1.7 was observed in one eye) and no corneal thickness changes were observed in the other eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
test item: 3 eyes
positive control: 3 eyes
negative control: 1 eye

NEGATIVE CONTROL USED : physiological saline (0.9% (w/v) NaCl solution

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED : Imidazole

APPLICATION DOSE
onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea
test item: 105 mg
positive control: 30 mg powdered Imidazole
negative control: 30 µL of physiological saline (0.9% (w/v) NaCl solution)

EXPOSURE TIME
10 seconds

OBSERVATION PERIOD
4 hours

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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 3x20 mL saline was performed (with two different rinsing method) at each time point when the test material or the positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
The negative and positive control eyes and all test item treated eyes were evaluated pre-treatment (as described in Section 11.2. Baseline assessments) and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable. Haag-Streit BP 900® slit lamp microscope was used for the measurements.
Corneal thickness and corneal opacity were measured at each time point indicated above. Fluorescein retention was measured on two occasions, at base line (t=0) and approximately 30 minutes after the post-treatment rinse.

- Corneal opacity: For opacity determination, the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface.

- Damage to epithelium based on fluorescein retention: The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used.

- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: For thickness measurements, the slit lamp microscope was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface.

- Macroscopic morphological damage to the surface: In the test item treated eyes: the cornea opacity was not observed until 30 minutes observation because the test item fully stuck the all cornea surfaces. Test item 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.
In the positive control group: Imidazole 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.

SCORING SYSTEM:
- Mean corneal swelling (%): Corneal swelling is determined from corneal thickness measurements made with an optical pachymeter on a slit lamp microscope. For the calculation of maximum corneal swelling, small negative numbers for swelling following application (0 to -5%) are counted as zero (scored 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).
- Mean maximum opacity score : Corneal opacity is scored using the area of the cornea that is most densely opacified. at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: decision criteria as indicated in the TG

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
at up to 75 min
Value:
-15.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.6%, ICE Class I
Positive controls validity:
valid
Remarks:
9.8%, ICE Class II
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
percent corneal swelling
Remarks:
at up to 240 min
Value:
-15.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.6%, ICE Class I
Positive controls validity:
valid
Remarks:
20.7%, ICE Class III
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
cornea opacity score
Remarks:
Mean maximum cornal opacity change
Value:
4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.00, ICE Class I
Positive controls validity:
valid
Remarks:
4.00, ICE Class IV
Remarks on result:
other: ICE Class IV
Irritation parameter:
fluorescein retention score
Remarks:
Mean fluorescein retention change
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.00, ICE Class I
Positive controls validity:
valid
Remarks:
3.00, ICE Class IV
Remarks on result:
other: ICE Class IV
Irritation parameter:
other: Observations
Positive controls validity:
other: Imidazole 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.
Remarks on result:
other: The cornea opacity was not observed until 30 minutes observation because the test item fully stuck the all cornea surfaces. Test item was stuck on all cornea surfaces after the post-treatment rinse.
Remarks:
Test item 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.
Other effects / acceptance of results:
OTHER EFFECTS:
Test item
Other observations: The cornea opacity was not observed until 30 minutes observation because the test item fully stuck the all cornea surfaces. Test item 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.
Overall ICE Class: 3xIV
Based on this in vitro eye irritation study in isolated chicken eyes with Nickel Cobalt Manganese Hydroxide (8:1:1), for OECD/GHS classification the test item was severely irritant. UN GHS Classification: Category 1.

Positive control
Other observations: Imidazole 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.
Overall ICE Class: 1xIII 2xIV
Based on these observations, the positive control substance Imidazole was classified as severe irritant according to the EU regulations. UN GHS Classification: Category 1.

Negative control
Other observations: None
Overall ICE Class: 3xI
The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.





Any other information on results incl. tables

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The conclusion on eye irritancy was based on the relevant OECD guideline quantitative assessments.

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention change ICE classes are used for EC and GHS classification.

Test item: pNMC-hydroxide (8:1:1)

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

-15.4 %

IV

Mean maximum corneal swelling at up to 240 min

-15.4 %

IV

Mean maximum corneal opacity change

4.00

IV

Mean fluorescein retention change

3.00

IV

Other Observations

The cornea opacity was not observed until 30 minutes observation because the test item fully stuck the all cornea surfaces. Test item 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.

Overall ICE Class

3xIV

Based on thisin vitroeye irritation study in isolated chicken eyes withNickel Cobalt Manganese Hydroxide (8:1:1), for OECD/GHS classificationthe test item was severely irritant.UN GHS Classification: Category 1.

Positive control

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

9.8%

II

Mean maximum corneal swelling at up to 240 min

20.7%

III

Mean maximum corneal opacity change

4.00

IV

Mean fluorescein retention change

3.00

IV

Other Observations

Imidazole 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.

Overall ICE Class

1xIII 2xIV

Based on these observations, the positive control substance Imidazole was classified as severe irritant according to the EU regulations. UN GHS Classification: Category 1.

Negative control

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.6%

I

Mean maximum corneal swelling at up to 240 min

1.6%

I

Mean maximum corneal opacity change

0.00

I

Mean fluorescein retention change

0.00

I

Other Observations

None

Overall ICE Class

3xI

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: No Category.

Validity

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

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
Observed effects in the in vitro OECD438 test indicate that pNMC hydroxide (8:1:1) should be classified as Eye Dam. Cat.1, but the test conditions did not meet those that are specified in the testing procedure: it was not possible to remove pNMC hydroxide (by rinsing) after the initial exposure. Based on the test data, it is therefore not possible to draw a conclusive decision on the potential hazard of pNMC hydroxide (8:1:1) for the eye irritation endpoint.
Executive summary:

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline
(25 June 2018).

After the zero reference measurements, the eyes were held in a horizontal position and the test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered in all cases. After 10 seconds exposure time, the surface of the eyes was rinsed with physiological saline solution. Three eyes were treated with approximately105 mg test item. Three positive control eyes were treated in a similar way with 30 mg powdered Imidazole and the negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

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 experiments. Thus, the study was considered to be valid.Severe corneal swelling change (mean = -15.4%) was observed during the four-hour observation period on test item treated eyes. Severe cornea opacity change (severity 4) was observed. Severe fluorescein retention change (severity 3) was noted.The cornea opacity was not observed until 30 minutes observation because the test item fully stuck the all cornea surfaces. Test item 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.