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EC number: 257-467-0 | CAS number: 51839-25-9
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
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- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
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- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- Feb - June 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 022
- Report date:
- 2022
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:
- 2018
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Zinc carbonate
- EC Number:
- 222-477-6
- EC Name:
- Zinc carbonate
- Cas Number:
- 3486-35-9
- Molecular formula:
- CH2O3.Zn
- IUPAC Name:
- zinc carbonate
- Test material form:
- solid: particulate/powder
- Details on test material:
- Chemical name: Zinc carbonate
CAS number: 51839-25-9
Batch/Lot number: 210205A
Description: White powder
Purity: 72 M-% (ZnO)
Manufacturing date: February 2021
Expiry date: 28 February 2031
Stability of Bulk Test Item: The test items are considered stable when stored under appropriate storage conditions: room temperature (15-25°C, ≤70% relative humidity), protected from humidity (tightly closed container). The test items are stored in the Pharmacy of the Test Facility.
Safety: All precautions required in the handling and disposal of the test item were outlined by the Sponsor. Enhanced safety precautions (nitrile gloves, goggles, face mask (ABEK-P3-filter), lab coat) for unknown materials will be applied to assure personnel health and safety.
The test item will be applied in its original form; no formulation is required.
Constituent 1
- Specific details on test material used for the study:
- Chemical name: Zinc carbonate
CAS number: 51839-25-9
Batch/Lot number: 210205A
Description: White powder
Purity: 72 M-% (ZnO)
Manufacturing date: February 2021
Expiry date: 28 February 2031
Stability of Bulk Test Item: The test items are considered stable when stored under appropriate storage conditions: room temperature (15-25°C, ≤70% relative humidity), protected from humidity (tightly closed container). The test items are stored in the Pharmacy of the Test Facility.
Safety: All precautions required in the handling and disposal of the test item were outlined by the Sponsor. Enhanced safety precautions (nitrile gloves, goggles, face mask (ABEK-P3-filter), lab coat) for unknown materials will be applied to assure personnel health and safety.
The test item will be applied in its original form; no formulation is required.
Test animals / tissue source
- Species:
- chicken
- Strain:
- other: ROSS 308 or COBB 500
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Eyes will be collected from healthy young chickens (approximately 7 weeks old, mean weight: 2.5 kg)
obtained from a slaughterhouse where they will be killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old, mean weight: 2.5 kg
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Chicken heads were collected by a slaughterhouse 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. The heads were received at Charles River Laboratories Hungary Kft. and processed within 2 hours of collection in experiment.
- Eye Selection: After removing the head from the plastic box, it will be put on a paper. The eyelids will be carefully cut away with scissors, avoiding damaging the cornea. Corneal integrity will be checked by
applying one small drop of 2% (w/v) fluorescein solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline solution. Then the head with the fluorescein treated cornea will be examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea is not damaged (i.e. fluorescein retention and corneal opacity scores ≤ 0.5). If the cornea is not damaged, the eyeball will be removed from the orbit.
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount(s) applied: 30 mg of zinc carbonate was applied 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.
The positive control eyes were treated in a similar way with 30 mg of powdered imidazole.
The negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution). - Duration of treatment / exposure:
- 10 seconds
- Observation period (in vivo):
- not applicable
- Duration of post- treatment incubation (in vitro):
- None intended
- Number of animals or in vitro replicates:
- Three test item treated eyes, one negative control eye and three positive control eyes were examined during the study.
- Details on study design:
- SELECTION AND PREPARATION OF ISOLATED EYES
-selection: 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.
-preparation: 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.
EQUILIBRATION AND BASELINE RECORDINGS
- At the end of the acclimatization period, a zero reference measurement will be recorded for cornea thickness and opacity to serve as a baseline (t=0). The corneal thickness of the eyes should not change by more than 5% within approximately 45 minutes before the start of application. Any minor corneal opacity change will be noted. Minor observations or deviations from the above will be considered acceptable provided they are not observed in all of the eyes in a test item group, otherwise the eyes will be replaced.
Following the equilibration period, the fluorescein retention will be measured. Baseline values will be required to evaluate any potential test item related effect after treatment; the location of any minor findings will be marked on the record sheet as a drawing, if applicable. If any eye is considered to be unsuitable following baseline assessment, it will be discarded (not used in the test).
NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED
- yes, Physiological saline (0.9% (w/v) NaCl solution)
POSITIVE CONTROL USED
- yes, Imidazole
APPLICATION DOSE AND EXPOSURE TIME
- exposure time of 10s with the following:
- test substance treated chicken eye: treated with 30 mg zinc carbonate
- positive control chicken eye: treated with 30 mg of powdered imidazole
- negative control eye: treated with 30µL physiological saline (0.9% (w/v) NaCl solution
OBSERVATION PERIOD
- The negative and positive control eyes and all test item treated 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. Haag-Streit BP 900® slit lamp microscope will be used for the measurements.
REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
After an exposure period of 10 seconds from the end of the application, the corneal surface will be rinsed thoroughly with ~20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual material if possible. If test item or positive control material remains on the eye, additional rinsing may be used after the 10-second application and at any subsequent time points (to be documented in the raw data and reported). The eye in the retainer will then be returned to its chamber. The length of time that the eye is out of the chamber will be kept to the minimum. Where test item or positive control material remains on the cornea, the rate of saline solution-drops may be increased at the time of each observation, the amount of saline solution additionally used
for rinsing will be recorded, and the observation of adherence of chemical to the cornea will be recorded.
METHODS FOR MEASURED ENDPOINTS: Haag-Streit BP 900® slit lamp microscope will be used for the
measurements.
- 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.
For corneal thickness measurements, the slit lamp microscope is focused such that the physiological saline solution should appear as a visible, clear (sharp) image as it moves 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.
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the investigator. Morphological findings at any observation point will be recorded, their frequency and degree were taken into evaluation during classification.
- Macroscopic morphological damage to the surface: not observed
- Others (histopathology): At the end of the procedure, the corneas were carefully removed from the eyes and placed individually into labelled containers of preservative fluid (10% neutral buffered formalin, Manufacturer: Lach:ner, Batch number: PP/2021/07941, Expiry date: 09 August 2022).
Histopathology evaluation was performed on all eyes. Slides of each cornea were produced and examined (two slides were prepared for test item treated eyes; one slide was prepared for negative and positive control eyes). The retained corneas were embedded in paraffin wax; sections were cut at 4-6 µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine. Histopathology evaluation was performed by light microscopy. Additional staining and/or photographical records may be performed if needed.
Semi-quantitative microscopic evaluation was performed on the cornea in the ICET. The classification of histopathology findings was performed based on the publications: M.K. Prinsen et al./Toxicology in Vitro 25 (2011), 1475-1479), Elodie Cazelle et al./ Toxicology in Vitro 28 (2014), 657-666 and Atlas of histopathological lesions of Isolated Chicken Eyes, M.V.W. Wijnands and M.K. Prinsen, June 2015 and OECD No. 438 guideline (adopted 25 June, 2018).
Pathology peer review was conducted.
SCORING SYSTEM:
- Mean corneal swelling (%): according to ICE classification criteria for corneal thickness
- Mean maximum opacity score: according to ICE classification criteria. The cornea opacity score for an individual eye will be given based on the following description:
• No opacity: 0
• Very faint opacity: 0.5
• Scattered or diffuse areas, details of iris clearly visible: 1
• Easily discernible translucent area, details of iris slightly obscured: 2
• Severe cornea opacity, no specific details of iris visible, size of pupil barely discernible: 3
• Complete cornea opacity, iris invisible: 4
- Mean fluorescein retention score at 30 minutes post-treatment: according to ICE classification criteria. The fluorescein retention score for an individual eye will be given based on the following description:
• No fluorescein retention: 0
• Very minor single cell staining: 0.5
• Single cell staining scattered throughout the treated area of the cornea: 1
• Focal or confluent dense single cell staining: 2
• Confluent large areas of the cornea retaining fluorescein: 3
DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. - yes
Results and discussion
In vitro
Resultsopen allclose all
- Irritation parameter:
- corneal swelling
- Run / experiment:
- 240 min
- Value:
- 17.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- positive indication of irritation
- Remarks:
- Value of max change up to 240min was 17.1% ; ICE Class II
- Irritation parameter:
- cornea opacity score
- Run / experiment:
- 1
- Value:
- 0
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- not determinable because of methodological limitations
- Remarks:
- Not detected. The Test item was severely stuck to the cornea surface, therefore the corneal opacity could not be measured in two eyes. In case of the third eye particles of the test item could be washed off at 180 minutes after the post-treatment rinse, so the corneal opacity could be measured but one result of the opacity measurement is not compatible with this test. This value will not be included in the evaluation.
- Irritation parameter:
- fluorescein retention score
- Run / experiment:
- 1
- Value:
- 0.067
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- no indication of irritation
- Remarks:
- The mean fluorescein retention score was 0.067%. ICE Class II.
- Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: not observable
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, within the historical control data range
- Acceptance criteria met for positive control: yes, within the historical control data range
Any other information on results incl. tables
Criteria for “No category” (all true) |
|
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II or 1 endpoint classed as I and 2 endpoints classed as II: | N/A |
No severe corneal morphological changes: | True |
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse: | False |
Criteria for “Category 1” (one or more true) |
|
2 or more endpoints classed as IV: | N/A |
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes): | N/A |
Corneal opacity = 4 at any time point (in at least 2 eyes): | N/A |
Severe loosening of epithelium (in at least 1 eye): | N/A |
Criteria for “No prediction can be made” (one or two true) |
|
Based on the endpoints not classifiable for No Category, or for Category 1: | True |
Particles of test item were stuck to the cornea and could not be washed off during the study: | True
|
Test item
Observation | Value | ICE Class |
Mean maximum corneal swelling at up to 75 min | 16.5% | III |
Mean maximum corneal swelling at up to 240 min | 17.1% | II |
Mean maximum corneal opacity change | not detected | no data |
Mean fluorescein retention change | 0.067 | I |
Other Observations | Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces were not cleared in two eyes at 240 minutes after the post-treatment rinse. In case of the third eye particles of it could be washed off at 180 minutes after the post-treatment rinse. | |
Overall ICE Class | no classification |
Slight fluorescein retention change (severity 0.5 on two eyes and severity 1 on one eye) was observed. The opacity change was not detected in two eyes, because the test item fully stuck on the cornea surfaces. In case of the third eye, particles of the test item could be washed off at 180 minutes after the post-treatment rinse, so slight corneal opacity change (severity 1) was measured at 240 minutes. As one result of the opacity measurement is not compatible with this test, this value will not be included in the evaluation. Slight cornea swelling change (mean 17.1%) was detected. Based on this in vitro eye irritation study in isolated chicken eyes with zinc carbonate, the test item was not compatible with the test system. It is concluded that further information is required for classification.
Positive control
Observation | Value | ICE Class |
Mean maximum corneal swelling at up to 75 min | 18.0% | III |
Mean maximum corneal swelling at up to 240 min | 35.3% | IV |
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 | 3xIV
|
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 |
Maximum corneal swelling at up to 75 min | 0.0% | I |
Maximum corneal swelling at up to 240 min | 0.0% | I |
Maximum corneal opacity change | 0.00 | I |
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.
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.
Histopathology
The negative control, 30 µL of 0.9 % sodium chloride (Salsol solution) cornea showed no epithelial erosion and no vacuolation of top part of the corneal epithelium. The positive control, 30 mg of Imidazole caused severe epithelial erosion of corneal epithelium in 3/3 cases. Vacuolation was seen in 1/3 cases. Mid epithelium was affected with moderate vacuolation in 1/3 cases. Low epithelium was altered with moderate vacuolation in 1/3 cases. In the top region of the stroma, slight presence of pyknotic nuclei was seen in 1/3 cases, and moderate presence of pyknotic nuclei was observed in 2/3 cases. The detachment of the epithelium from the stroma was observed in 2/3 cases. The basement membrane was compromised in 2/3 cases. No endothelial changes were recorded.
Zinc carbonate produced very slight erosion of the corneal epithelium in 6/6 cases, accompanied with very slight necrosis of top part of the epithelium in 3/6 cases. The top epithelium was altered with very slight vacuolation in 1/6 cases, and with slight severity in 5/6 cases. Mid part of epithelium was affected with very slight vacuolation in 1/6 cases. No prediction can be made for this test item.
Test material | Eye number | epithelium | stroma | |||||||
erosion | necrosis | vacuolation | disorder of fibers | pyknotic nuclei | ||||||
top | mid | low | notes | outer region (adj. epithelium) | inner region (adj. epithelium) | |||||
Zn carbonate | 4A | 1/2 | - | 1 | - | - | - | - | - | - |
Zn carbonate | 4B | 1/2 | - | 1 | - | - | - | - | - | - |
Zn carbonate | 5A | 1/2 | - | 1/2 | - | - | - | - | - | - |
Zn carbonate | 5B | 1/2 | 1/2 t | 1 | - | - | - | - | - | - |
Zn carbonate | 6A | 1/2 | 1/2 t | 1 | 1/2 | - | - | - | - | - |
Zn carbonate | 6B | 1/2 | 1/2 t | 1 | - | - | - | - | - | - |
Imidazole | 7 | 3 | 3 | - | 2 | 2 | - | - | 2 | - |
Imidazole | 8 | 3 | 3 | - | - | - | x, y | - | 2 | - |
Imidazole | 9 | 3 | - | - | - | - | x, y | - | 1 | - |
Salsol solution, 0.9% (w/v) NaCl solution | 10 | - | - | - | - | - | - | - | - | - |
- = not observed; ½ = very slight; 1 = slight; 2 = moderate; 3 = severe, x= basement membrane compromised, y=detachment of the epithelium from the stroma, t = top part of the epithelium
Necrosis of the epithelium was not observed for any of the eyes.
Applicant's summary and conclusion
- Interpretation of results:
- Category 2 (irritating to eyes) based on GHS criteria
- Conclusions:
- Based on the OECD No. 438 Guideline for histopathological changes, zinc carbonate was not classified as severe irritant (no prediction can be made). Nonetheless, since the test item could not be 'not classified', a category 2 classification is derived for the test item.
- 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 (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 30 mg test item. The three positive control eyes were treated in a similar way with 30 mg of 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 experiment. Thus, the study was considered to be valid.
Slight fluorescein retention change (severity 0.5 on two eyes and severity 1 on one eye) was observed. The opacity change was not detected in two eyes because the test item fully stuck on the cornea surfaces. In case of the third eye, particles of the test item could be washed off at 180 minutes after the post-treatment rinse, so slight corneal opacity change (severity 1) was measured at 240 minutes. Slight cornea swelling change (mean 17.1%) was detected. Based on this in vitro eye irritation study in isolated chicken eyes with zinc carbonate, the test item was not compatible with the test system. It is concluded that further information is required for classification.
No other corneal effect was observed.
Based on this in vitro eye irritation assay in isolated chicken eyes with zinc carbonate, the test item was not compatible with the test system. It is concluded that further information is required for classification.
Histopathology evaluations were performed at the Sponsor request. One slide per each control cornea and 2 slides per each test item treated cornea were prepared. Histopathological observations were made on two sections of each of the test item treated corneas (a total of 6 sections per test item) by the Pathologist. The test item zinc carbonate produced very slight erosion of the corneal epithelium in 6/6 cases, accompanied with very slight necrosis of top part of the epithelium in 3/6 cases. The top epithelium was altered with very slight vacuolation in 1/6 cases, and with slight severity in 5/6 cases. Mid part of epithelium was affected with very slight vacuolation in 1/6 cases. No prediction can be made for this test item.
When applied to the cornea, the test item adhered to the surface and could not readily be washed off, however the histological corneal damage was limited to the upper epithelial layers, indicating a lack of severe effect.
Based on the OECD No. 438 Guideline for histopathological changes, zinc carbonate was not classified as severe irritant (no prediction can be made).
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