Carbonic acid, zinc salt, basic

Endpoint summary

Description of key information

Skin irritation/corrosion:

An in vitro skin irritation study was performed with an EpiDerm model with test item zinc carbonate (Gorbay-Nagy, 2022). In this in vitro Epiderm model test with zinc carbonate, the results indicate that the test item is non-irritant to the skin.

Eye irritation:

An in vitro eye irritation study was performed in isolated chicken’s eyes with zinc carbonate (Gorbay-Nagy, 2022). The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (2018). The test item was not compatible with the test system as it remained stuck to the cornea surface so further histopathology evaluations were performed.

Histopathological observations were made on two sections of each of the test item treated corneas (6 sections per test item). The test item 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. Based on the published criteria for histopathological changes, the criteria triggering serious eye damage were not met for Zinc.

The histopathology evaluations showed slightly irritating effects. Taking into account the OECD No. 438 guideline data with the supplemental histopathology data for zinc carbonate, the weight of evidence approach indicates an overall conclusion classification as eye irritant category 2.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

February - April 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

(2021)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: The test item of a suitable chemical purity was provided by the Sponsor. Batch/Lot number: 210205A
- Purity, including information on contaminants, isomers, etc.: 72 M-% (ZnO)
- Manufacturing date: February 2021
- Expiry date: 28 February 2031

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test item is considered stable when stored under appropriate storage conditions: controlled room temperature (15-25°C, ≤70% relative humidity). The test item is stored in the Pharmacy of the Test Facility.

The test item will be applied in its original form; no formulation is required.

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 were applied to ensure personnel health and safety.

OTHER SPECIFICS
- Other relevant information needed for characterising the tested material, e.g. if radiolabelled, adjustment of pH, osmolality and precipitate in the culture medium to which the test chemical is added: test material is a white powder

Test system:

human skin model

Remarks:

EpiDerm™ (Source: MatTek, Bratislava, Slovakia) units consist of normal, human-derived epidermal keratinocytes (NHEK) which have been cultured to form a multilayered, highly differentiated model of the human epidermis (0.6 cm2).

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: as provided by MatTek Corporation, Bratislava, Slovakia

Source strain:

other: human-derived epidermal keratinocytes (NHEK)

Details on animal used as source of test system:

Not applicable - EpiDerm™ (Source: MatTek, Bratislava, Slovakia) units consist of normal, human-derived
epidermal keratinocytes (NHEK).

Justification for test system used:

The EpiDerm™ model has been validated for in vitro irritation testing in multiple laboratories
and accepted by OECD No 439, thus it is considered to be suitable for this study.
This test is designed to predict and classify the skin irritant potential of chemicals/formulations/products/mixtures according to chemical safety regulations, using the
reconstructed human epidermis model EpiDerm™ Model and parameters related to skin
irritation. This method is approved by international regulatory agencies as a replacement for
the identification of irritants / corrosives in the in vivo rabbit skin assay (OECD No. 404) and
is specifically approved as a replacement for the in vivo skin irritation test within OECD No.
439.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ (Source: MatTek, Bratislava, Slovakia)
- Tissue batch number(s): EPI-200-SIT-MD lot number 36125
- Shipping date: 28 February 2022
Identification data for the components of the kits:
Name: Assay Medium ; Batch numbers: 022422LHC ; Expiry date: 10 March 2022
Name: MTT-100-CON ; Batch numbers: 011822MSA ; Expiry date: 22 March 2022
Name: MTT-100-DIL ; Batch numbers: 2293733 ; Expiry date: 31 March 2022
Name: MTT-100-EXT ; Batch numbers: 121421LHA ; Expiry date: 14 December 2022
Name: 5% SDS Solution (TC-SDS-5%) ; Batch numbers: 081221NMA ; Expiry date: 12 August 2022
Name: DPBS ; Batch numbers: 120721MSA ; Expiry date: 07 December 2022
- Date of initiation of testing: not specified

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time the EpiDerm™ units will be removed and rinsed into clean beakers containing 100 mL of DPBS each (20 times). Any remaining liquid will be removed onto an
absorbent paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: final concentration: 300 µL MTT medium (1 mg MTT / ml medium)
- Incubation time: 3 hours (±5 minutes)
- Wavelength: 570 ± 30 nm

Quality Control
EpiDerm™ Model (EPI-200-SIT) kits are manufactured according to defined quality assurance procedures. All biological components of the epidermis and the kit culture medium have been tested for the presence of viruses, bacteria and mycoplasma.
The RhE tissue construct was only used if the developer/supplier demonstrated that each batch of the RhE tissue construct met the defined production release criteria, among which those for viability and barrier function were the most relevant. An acceptability range (upper and lower limits) for the barrier functions as measured by the ET50 was established by the RhE tissue construct developer/supplier. The ET50 acceptability range used as QC (Quality Control) batch release criterion by the developer/supplier of the RhE tissue constructs was documented in OECD No. 439.
Tissue viability and the barrier function test are within the acceptable ranges and indicate appropriate formation of the epidermal barrier, the presence of a functional stratum corneum, a viable basal cell layer and intermediate spinous and granular layers.

NUMBER OF REPLICATE TISSUES: At least three replicates will be used for test item; three replicates will be used for both the negative control and positive control. For additional controls, at least two replicates will be used for NSMTT controls, NSCliving and NSCkilled controls (if nessesary).

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
9.9.1 Check-method for possible direct MTT reduction with test item
25 mg test item was added to 1 mL MTT working solution and mixed. The mixture was incubated at 37°C for 1 hours protected from light, and then any colour change was recorded:
• Test item which does not react with MTT: other colours
• Test item which directly reacts with MTT: blue or purple
After one hour of incubation, red colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.

PREDICTION MODEL / DECISION CRITERIA
The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean relative viability of three individual tissues after 60 minutes exposure to the test item and 42 hours post incubation is less or equal (≤) to 50% of the mean viability of the negative controls. In case the test chemical is found to be non-corrosive, and shows tissue viability after exposure and post-treatment incubation is less than or equal (≤) to 50%, the test chemical is considered to be irritant to skin in accordance with UN GHS Category 2. The test item may be considered to be non-irritant to skin in accordance with UN GHS (No Category), if the mean relative viability of three individual tissues after 60 minutes exposure to the test item and 42 hours post incubation is more than (>) 50% of the mean viability of the negative controls.
In case of 45-55% relative viability values, a confirmatory run is suggested according to the OECD No. 439 guideline.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at least 25 mg of the test item will be applied evenly to the epidermal surface

VEHICLE
- Amount(s) applied (volume or weight with unit): na; no formulation was required

30 µL of positive (5% (w/v) SDS solution) or negative (DPBS) control will be added to each skin unit by using a suitable pipette. Chemicals may be spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Duration of treatment / exposure:

The plates with the test item, negative and positive control treated epidermis units will be incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere. After 35 minutes, all plates will be removed from the incubator, the plates will be placed them into the sterile hood and will be waited until the period of 60 minutes is completed for the first dosed tissue (35 minutes in incubator and 25 minutes at room temperature).

Duration of post-treatment incubation (if applicable):

42h

Number of replicates:

In this assay, three replicates were used for the test item. Three negative controls and three positive controls were also run in the assay. As the test item was coloured, two additional test item-treated living tissues were used for the non-specific optical density (OD) evaluation.

Details on study design:

Pre-incubation (Day [-1])
The appropriate number of wells in an assay plate will be filled with the EpiDerm Assay medium (0.9 mL per well). The epidermis units will be placed with the media below them, in contact with the epidermis into each prepared well and then incubated 1 hour at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere. Afterwards the medium will be replaced and continued with overnight pre-incubation.

Application (Day 0)
At least three skin units will be used for each test or control material.
Test Item:
As the test item is solid, first an appropriate amount 25 μL of sterile DPBS will be applied to
the epidermal surface (in order to improve further contact between powder and epidermis) and
then at least 25 mg of the test item will be applied evenly to the epidermal surface. Higher amount of test item may be used in order to properly cover the surface of the EpiDerm™ units (the applied volume will be documented in the raw data and reported). If necessary, the test item may be spread gently on the skin surface with a curved flat spatula or pipette tip (or other appropriate tool) taking care not to damage the epidermis. For sticky materials, viscous pastes or other difficult materials, alternative application methods may be appropriate (and will be documented in the raw data and reported).
Positive and negative controls:
30 µL of positive (5% (w/v) SDS solution) or negative (DPBS) control will be added to each skin unit by using a suitable pipette. Chemicals may be spread gently with the pipette tip in order to cover evenly all the epidermal surface if necessary.

Incubation
The plates with the test item, negative and positive control treated epidermis units will be incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere. After 35 minutes, all plates will be removed from the incubator, the plates will be placed them into the sterile hood and will be waited until the period of 60 minutes is completed for the first dosed tissue (35 minutes in incubator and 25 minutes at room temperature). The plate lids and the epidermis units will be identified with the exposure time, the replicate number and the code of the chemicals to be tested.

Rinsing (Day 0)
After the incubation time the EpiDerm™ units will be removed and rinsed into clean beakers containing 100 mL of DPBS each (20 times). Any remaining liquid will be removed onto an absorbent paper.

Incubation (Day 0)
After rinsing, the units will be placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 24 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.

Incubation (Day 1)
At the end of the 24 hours incubation period the units will be placed into the plate wells with fresh Assay Medium (0.9 mL/well) below them and then incubated for 18 hours (± 2hours) at 37°C in an incubator with 5 % CO2, in a >95 RH % humidified atmosphere.

MTT test after 42 hours incubation (Day 2)
After the 42 hours incubation, 300 µL MTT medium (1 mg MTT / ml medium) will be added to each well of plate and the skin units will be transferred to the MTT medium (except colour control units which will be incubated in Assay Medium). The lid will be replaced and the plate incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours (±5 minutes).

Formazan extraction (Day 2)
MTT will be gently aspirated from all wells (e.g. using a suction pump), then wells will be refilled with DPBS and aspirated. The rinsing will be repeated twice and made sure that tissues are dry after the last aspiration. Inserts will be transferred to new 24-well plates. The inserts will be immersed by gently pipetting 2 mL extractant (solution extractant - MTT-100-EXT) into each insert. The level will rise above the upper edge of the insert, thus completely covering the tissue from both sides. The 24 well plate will be sealed (e.g. with a zip bag) to inhibit the
extractant solution evaporation. Extraction will be performed 2 hours with shaking (~120 rpm) at room temperature. After the extraction period is complete, the inserts will be pierced with an injection needle and allowed the extract to run into the well from which the insert was taken. Afterwards the insert can be discarded. The 24-well plates will be placed on a shaker for 15 minutes until solution is homogeneous in colour. A blank sample containing 2 mL of extracting solution (MTT-100-EXT) will be processed in parallel.

Cell viability measurements (Day 2)
Following the formazan extraction, per each tissue will be transferred 2×200 µL of the blue formazan solution into a 96-well flat bottom microtiter plate. The OD (Absorbance / Optical Density) of the samples will be read in a 96-well plate spectrophotometer, at a wavelength of 570 ± 30 nm, using isopropanol solution (MTT-100-EXT) as blank (at least 5×200 µL).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

80.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Following exposure to Zinc carbonate, the mean cell viability was 80.4% compared to the negative control. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered as being non-irritant to skin.

Other effects / acceptance of results:

- Direct-MTT reduction: 25 mg test item was added to 1 mL MTT working solution and mixed. The mixture was incubated at 37°C for 1 hours protected from light, and then any colour change was recorded:
• Test item which does not react with MTT: other colours
• Test item which directly reacts with MTT: blue or purple
After one hour of incubation, red colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT and therefore the use of additional controls was not necessary.
- Colour interference with MTT: Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water and/or isopropanol* (simulating a tissue humid environment). As the test item had an intrinsic colour, thus further evaluation to detect colouring potential was necessary. Non-specific Colour % (NSCliving %) was determined in order to evaluate the ability of test items to stain the epidermis by using additional control tissues.
*Note: Water is the environment during exposure, isopropanol is the extracting solution.
In addition to the normal procedure, two additional test item-treated living tissues were used for the non-specific OD evaluation. These tissues followed the same test item application and all steps as the other tissues, except for the MTT step: MTT incubation was replaced by incubation with fresh Assay Medium to mimic the amount of colour from the test item that may be present in the test disks. OD reading was conducted following the same conditions as for the other tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Results

Additional Controls:
No colour change was observed after three hours of incubation of the test item in MTT working solution, thus the test material did not interact with MTT. Therefore, additional controls and data calculations were not necessary. The false estimation of viability can be excluded.
As the test item was coloured, two additional test item-treated living tissues were used to determine the non-specific OD value and a non-specific colour percentage (NSC_living%). Results of this non-specific colour determination are shown in Table 1. The mean value for optical density (measured at 570 nm) was 0.006, and the NSC_living % value for the test item was calculated as 0.3%. This value was below 5%, therefore additional data calculation was not necessary.

Optical Density (OD) and the Calculated Non-Specific Colour % (NSC_living%) of the Additional Control Tissues: 

Additional control	Optical Density (OD)			NSC % living
		Measured	Blank corrected
Treated with test item	1	0.054	0.009	0.3
	2	0.047	0.002
	Mean		0.006

Notes:

1. Mean blank value was 0.045.


2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Viability results:

The results of the OD measured at 570 nm for the test item, positive and negative controls as well as the calculated relative viability percent values (% RV) are presented in the table below. The OD values for the test item treated skin samples showed 80.4% relative viability compared to the negative control.

Optical Density (OD) and the Calculated Relative Viability % of the Samples:

Substance	Optical Density (OD)			Viability (% RV)	Standard deviation (SD)
		Measured	Blank corrected
Negative control	1	2.002	1.957	98.6
	2	2.103	2.058	103.7
	3	1.985	1.940	97.7
	mean	--	1.985	100.0	3.2
Positive control	1	0.091	0.046	2.3
	2	0.091	0.046	2.3
	3	0.102	0.057	2.9
	mean	--	0.050	2.5	0.3
Test item	1	1.649	1.604	80.8
	2	1.592	1.547	77.9
	3	1.683	1.638	82.5
	mean	--	1.596	80.4	2.3

1. Mean blank value was 0.045.


2. Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).

Negative control is dulbecco's phosphate buffer saline
Positive control is 5% (w/v) SDS solution
Test item is Zinc carbonate

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro Epiderm model test with zinc carbonate, the results indicate that the test item is non-irritant to the skin.

Executive summary:

An in vitro skin irritation test was conducted on zinc hydroxide in a reconstructed human epidermis model. The EpiDerm^TM Model is designed to predict and classify the irritation potential of chemicals by measuring its cytotoxic effect as reflected in the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; CAS number: 298-93-1) assay. The irritation potential of the test item was evaluated according to the OECD guideline No. 439.

Disks of EpiDerm™ Model (three units) were treated with the test item and incubated for 35 minutes 37°C, 5 % CO2 >95 RH% humidified atmosphere and 25 minutes at room temperature. Exposure of the test item was terminated by rinsing with Dulbecco’s Phosphate Buffered Saline (DPBS). The epidermis units were then incubated at 37°C for 24 hours in an incubator with 5% CO2, in a >95% humidified atmosphere and 18 hours at 37°C in an incubator with 5% CO2, in a >95% humidified atmosphere (after medium change). The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light, in a >95% humidified atmosphere. The precipitated formazan crystals were then extracted using extracting solution (Isopropanol) for 2 hours with shaking at room temperature and quantified spectrophotometrically at 570 nm.

The negative control epidermis units were treated with DPBS, whilst the positive control epidermis units were treated with 5% (w/v) sodium dodecyl sulphate (SDS) (three units / control). Two additional disks were used to provide an estimate of colour contribution (NSC_living) from the test item. For each treated tissue, the viability was expressed as a percentage relative to the negative control. If the mean relative viability is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin.

Following exposure with zinc carbonate, the mean cell viability was 80.4% compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiDerm^TM model test with zinc carbonate, the results indicate that the test item is non-irritant to skin.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

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)

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.

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.

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

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

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.

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Additional information

Skin irritation/corrosion:

An in vitro skin irritation study was performed with an EpiDerm model with test item zinc carbonate (Gorbay-Nagy, 2022). In this in vitro Epiderm model test with zinc carbonate, the results indicate that the test item is non-irritant to the skin.

Following exposure to zinc carbonate, the mean cell viability was 80.4% compared to the negative control. This is above the threshold of 50%, therefore under the condition of this assay the test item was considered as being non-irritant to skin.

Eye irritation:

An in vitro eye irritation study was performed in isolated chicken’s eyes with zinc carbonate (Gorbay-Nagy, 2022). The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (2018). Because the test item was severely stuck to the cornea surface, histopathology evaluations were performed.

Histopathological observations were made on two sections of each of the test item treated corneas (a total of 6 sections per test item). 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. Based on the published criteria for histopathological changes, the criteria triggering serious eye damage were not met for Zinc carbonate.

These evaluations showed slightly irritating effects of the test item.

Based on this in vitro eye irritation study in isolated chicken eyes with zinc carbonate, the test item did not have severe irritating effects and was not a non irritant. A category 2 classification is therefore derived.

Justification for classification or non-classification

Skin irritation:

Based upon OECD 439 in vitro skin irritation data, zinc carbonate is not irritating to the skin and therefore does not require classification

 

 

Eye irritation:

 

Based upon the ICE test OECD 438 and additional histopathology evaluations, zinc carbonate is classified as category 2 for eye irritation.