Zinc hydroxide

Endpoint summary

Description of key information

Skin irritation:

The in vitro skin irritation test in the EpiDerm model (OECD 439) (Gorbay-Nagy, CRL Hungary kft., 2022) with zinc hydroxide indicates that the test item is non-irritant to the skin. Following exposure to zinc hydroxide, the mean cell viability was 89.8% 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.

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
Name: Zinc hydroxide
Chemical name: Zinc hydroxide
CAS number: 20427-58-1
Batch/Lot number: S4374
Description: White powder
Purity: 99.7 %
Manufacturer/supplier: MP Biomedicals
Expiry date: 30 June 2023

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. The test item was not already a fine powder, therefore it was ground finely in a mortar and pestle.

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): not specified - EpiDerm™ Model (EPI-200-SIT) kits
- Identification data for the components of the kits:
Assay Medium: Batch numbers: 022422LHC ; Expiry date: 10 March 2022
MTT-100-CON: Batch numbers: 011822MSA ; Expiry date: 22 March 2022
MTT-100-DIL: Batch numbers: 2293733 ; Expiry date: 31 March 2022
MTT-100-EXT: Batch numbers: 121421LHA ; Expiry date: 14 December 2022
5% SDS Solution (TC-SDS-5%): Batch numbers: 081221NMA ; Expiry date: 12 August 2022
DPBS: Batch numbers: 120721MSA ; Expiry date: 07 December 2022
- Delivery date: The date of dispatch written on the package was checked before opening the EpiDerm™ kit. The contents of the package were checked according to the map and instructions provided by the supplier.
- Date of initiation of testing: start of experiment on 1 March 2022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): The plates with the test item, negative and positive control treated epidermis units were incubated for 35 minutes (± 1 minute) to the humidified incubator at 37°C with 5 % CO2, in a >95% humidified atmosphere and for the exposure time of 25 minutes (± 0.5 minute) at room temperature (21.5-23.2°C).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the incubation time, the EpiDermTM units were removed and rinsed into clean beakers containing 100 mL of DPBS each (20 times). Any remaining liquid were removed onto an absorbent paper.
- Observable damage in the tissue due to washing: not specified
- Modifications to validated SOP: none

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: 570nm
The proper status of the instrument was verified by measuring a Verification plate (Manufacturer: Thermo Fisher Scientific, Catalogue Number: 240 72800, Serial Number: 0920-14, Date of calibration: 14 October 2020, calibration is valid until October 2022) at the required wavelength on each day before use.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability:
- Barrier function:
- Morphology:
- Contamination:
- Reproducibility:

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
Red colour was observed after one hour of incubation of the test item in MTT medium, 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 (NSCliving%). 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 NSCliving % value for the test item was calculated as 0.3%. This value was below 5%, therefore additional data calculation 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:

3

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:

89.8

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 hydroxide, the mean cell viability was 89.8% 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.

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro Epiderm model test with zinc hydroxide, 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 hydroxide, the mean cell viability was 89.8% 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 hydroxide, 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 vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

see reference

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

64 mg ZnO (volume of 0.1 mL)

Duration of treatment / exposure:

24 hours

Observation period (in vivo):

1, 24, 48 and 72 hours after exposure

Number of animals or in vitro replicates:

3 males

Details on study design:

see reference

Irritation parameter:

conjunctivae score

Basis:

mean

Time point:

24/48/72 h

Score:

>= 0.7 - <= 1

Max. score:

3

Reversibility:

fully reversible

Remarks:

in 72h

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

2

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Max. score:

4

Remarks on result:

no indication of irritation

Irritant / corrosive response data:

No symptoms of systemic toxicity were observed and no mortality occurred. Slight iridial irritation (grade 1) was observed in one animal, at 1 hour only. Slight irritation of the conjuctivae (grade 1-2) was seen as redness (mean scores over 24-72 hours 0.7, 1 and 1), which had completely resolved at 72 hours in all animals. Chemosis (grade 2) and discharge (grade 1) were also observed in all animals, but at 1 hour only. No corneal opacity or epithelial damage was observed in any of the animals

Other effects:

none

Interpretation of results:

GHS criteria not met

Conclusions:

ZnO not irritating in an vivo rabbit study.

Executive summary:

In an eye irritation/corrosion well-performed study according to Directive 92/69/EEC B.5 and OECD guideline 405, three male New Zealand White rabbits were treated by instillation of approximately 64 mg of zinc oxide (a volume of about 0.1 ml) into the conjuctival sac of one eye. The other eye remained untreated and served as control. After 24 hours, both eyes of two animals were rinsed with water. The eyes were examined at 1, 24, 48 and 72 hours after instillation. No symptoms of systemic toxicity were observed and no mortality occurred. Slight iridial irritation (grade 1) was observed in one animal, at 1 hour only. Slight irritation of the conjuctivae (grade 1-2) was seen as redness (mean scores over 24-72 hours 0.7, 1 and 1), which had completely resolved at 72 hours in all animals. Chemosis (grade 2) and discharge (grade 1) were also observed in all animals, but at 1 hour only. No corneal opacity or epithelial damage was observed in any of the animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on the available information, the slightly soluble zinc oxide, zinc phosphate, zinc metal and insoluble zinc sulphide are not eye irritants and therefore zinc carbonate and zinc hydroxide are also expected to be not irritating to eyes. None of the slightly soluble or insoluble zinc compounds appear to cause respiratory tract irritation.

The slightly soluble and insoluble zinc compounds (zinc oxide, zinc hydroxide, zinc phosphate, zinc carbonate, zinc metal and zinc sulphide) are not corrosive based on the available irritation data.

Justification for classification or non-classification

Skin irritation:

Based upon OECD 439 in vitro skin irritation data, zinc hydroxide does not require classification as a skin irritant

Eye irritation:

The slightly soluble and insoluble zinc compounds ( zinc oxide, zinc hydroxide, zinc phosphate, zinc carbonate, zinc metal and zinc sulphide)

are not irritating or corrosive based on the available irritation data and therefore no classification is required according to EC criteria either for irritation or corrosion.