Trizinc bis(orthophosphate)

Skin irritation / corrosion

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

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)

Test material

Test material information

Constituent 1

Reference substance name:

Trizinc bis(orthophosphate)

EC Number:

231-944-3

EC Name:

Trizinc bis(orthophosphate)

Cas Number:

7779-90-0

Molecular formula:

H3O4P.3/2Zn

IUPAC Name:

trizinc bis(orthophosphate)

Test material form:

solid: particulate/powder

Details on test material:

Chemical name: Trizinc bis(orthophosphate)
CAS number: 7779-90-0
Batch/Lot number: 12007405-0
Description: White powder
Purity: Considered as 100%
Manufacturer/supplier: Heubach GmbH
Manufacturing date: 21 February 2020
Expiry date: 02 February 2024
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.

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 was provided by Heubach GmbH. Batch/Lot number: 12007405-0
- Purity, including information on contaminants, isomers, etc.: considered as 100%
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.
- Manufacturing date: 21 February 2020
- Expiry date: 02 February 2024

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. Routine safety precautions (gloves, goggles, face mask, 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

In vitro test system

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
- Start of experiment: 1 March 2022

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: 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.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
25 mg test item was added to 1 mL MTT medium 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:

3

Test system

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).

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

93.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Remarks:

Following exposure to trizinc bis(orthophosphate), the mean cell viability was 93.5% 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:

- OTHER EFFECTS:
- Direct-MTT reduction: 25 mg test item was added to 1 mL MTT medium 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

Any other information on results incl. tables

 

Results

Additional controls:
Red change 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 (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	0.055	0.010	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 93.5% relative viability compared to the negative control.

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	1.910	1.865	94.0
	2	1.845	1.800	90.7
	3	1.950	1.905	95.7
	mean	--	1.857	93.5	2.7

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).

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

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro Epiderm model test with trizinc bis(orthophosphate), 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 trizinc bis(orthophosphate), the mean cell viability was 93.5% 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 trizinc bis(orthophosphate), the results indicate that the test item is non-irritant to skin.