Zinc bis(dihydrogen phosphate)

Endpoint summary

Description of key information

Skin irritation/corrosion:

An in vitro skin irritation study and in vitro skin corrosion study were performed with an EpiDerm model with test item zinc bis(dihydrogen phosphate) (Piehl, 2010). In this in vitro Epiderm model test with zinc bis(dihydrogen phosphate), the results indicate that the test item is non-irritant and not corrosive to the skin.

 

Eye irritation:

The irritating potential of the test item was assessed in a HET-CAM test (Kremer and Kreutz, 2006). The aim of the study being to evaluate a substance's ability to induce toxicity in the chorioallantoic membrane of a hen. The results showed mildly irritating effects of the test item.
A further in vitro EpiOcular test was conducted and showed that zinc bis(dihydrogen phosphate), dihydrate had eye irritating properties (Tóth-Gönczöl,  2022)  
An in vitro eye irritation study was performed in isolated chicken’s eyes with zinc bis(dihydrogen phosphate) (Gorbay-Nagy, 2022) to assess whether the test substance could be classified as a severe irritant (category 1) or not. The irritation effects of the test item were evaluated according to the OECD No. 438 guideline (2018). The test item showed light irritating effects. The test item was not classified as a severe irritant and not classified as non-irritant. So irritating properties were once again confirmed but the test item is not a severe irritant.

Therefore, zinc bis(dihydrogen phosphate), dihydrate is classified as category 2 for eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

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)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

physiological saline

Details on test system:

TEST SITE
- Area of exposure: after moistening the EpiDerm tissue with 25µl PBS before dosing, 25 mg of the test article was added into the Millicell atop the tissue

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffered saline (PBS)
- Time after start of exposure: test article remained in contact with the EpiDerm tissue for 60 minutes.

SCORING SYSTEM:
After washing EpiDerm tissue was transferred to a 24 well plate contaiing 300µl MTT solution. Afterwards the tissues were returned to the incubator for a three-hour MTT incubation period. Following MTT incubation period, each EpiDerm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbancy of an aliquot of the extracted MTT formazan was measured at 540nm using a microplate reader.

Analysis of data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability= 100x(OD sample/OD negative control)

Quality controls: negative controls meets the acceptance criterion if the mean OD of the 2 tissues each time point is greater than or equal to 1.0 and smaller than or equal to 2.5. The positive controls meets the acceptance criterion if the mean relative tissue viability, expressed as percentage of the negative control tissues is less than or equal to 20%
the SD calculated from individual percent tissue viabilities of the three identically treated replicates is less than 18%

Amount/concentration applied:

25 mg

Duration of treatment / exposure:

60 min

Irritation / corrosion parameter:

% tissue viability

Value:

92.1

Remarks on result:

other:

Remarks:

Basis: mean. Time point: 60min. Remarks: recovery period: 42h. (migrated information)

Interpretation of results:

GHS criteria not met

Conclusions:

Following exposure with Zinc bis(dihydrogen phosphate), the mean treated skin value was 92.1% and therefore non irritant. All validity criteria were within acceptable limits and therefore the study can be considered as valid.

In this in vitro skin irritation test in the EPISKIN model with Zinc bis(dihydrogen phosphate) the results indicated that the test item is Non Irritant (NI) [UN GHS: No Category].

Executive summary:

A study was conducted to predict dermal irritation potential of Zinc bis(dihydrogen phosphate) in the context of identification and classification of skin irritation hazard according to the EU classification (R38 or no label) and GHS classification system (category 2 and non-irritants) by using a three-dimensional human epidermis model (OECD 439). MatTek EpiDerm tissue samples were treated in triplicate with the test article, negative control and positive control for 60 minutes. Following treatment and subsequent incubation time, the viability of the tissues was determined using MTT uptake and conversion, and the absorbance of each sample was measured at 540nm. The viability was then expressed as a percent of control values.

In this in vitro EPIDERM model test with Zinc bis(dihydrogen phosphate), the results indicate that the test item is non irritant.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

skin obtained from plastic surgery from multiple donors

Vehicle:

water

Details on test system:

TEST SITE
- Area of exposure: approximately 25 mg of the test article + 25 µl tissue culture water (negative control) were applied on the top of each EpiDerm tissue (9mm in diameter)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffered saline (PBS)
- Time after start of exposure: test article remained in contact with the EpiDerm tissue for 3 and 60 minutes.

SCORING SYSTEM:
After washing EpiDerm tissue was transferred to a 24 well plate contaiing 300µl MTT solution. Afterwards the tissues were returned to the incubator for a three-hour MTT incubation period. Following MTT incubation period, each EpiDerm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbancy of an aliquot of the extracted MTT formazan was measured at 540nm using a microplate reader.

Analysis of data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability= 100x(OD sample/OD negative control)

Quality controls: negative controls meets the acceptance criterion if the mean OD of the 2 tissues each time point is greater than or equal to 0.8. The positive controls meets the acceptance criterion if the mean relative tissue viability at the 3 minute time point is less than or equal to 30%
Inter-tissue viability meets the acceptance criterion if the difference between two identically treated tissues is no greater than 30%

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg of the test article, plus 25 ul tissue culture water were applied to the top of each EpiDerm tissue

Duration of treatment / exposure:

3 and 60 min

Details on study design:

TEST SITE
- Area of exposure: approximately 25 mg of the test article + 25 µl tissue culture water (negative control) were applied on the top of each EpiDerm tissue (9mm in diameter)

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of the exposure period, each EpiDerm tissue was rinsed with phosphate buffered saline (PBS)
- Time after start of exposure: test article remained in contact with the EpiDerm tissue for 3 and 60 minutes.

SCORING SYSTEM:
After washing EpiDerm tissue was transferred to a 24 well plate contaiing 300µl MTT solution. Afterwards the tissues were returned to the incubator for a three-hour MTT incubation period. Following MTT incubation period, each EpiDerm tissue was rinsed with PBS and then treated overnight with 2.0 ml of extractant solution (isopropanol) per well. The absorbancy of an aliquot of the extracted MTT formazan was measured at 540nm using a microplate reader.

Analysis of data:
The mean absorbance value for each time point was calculated from the optical density (OD) of the duplicate samples and expressed as percent viability for each sample using the following formula:
% viability= 100x(OD sample/OD negative control)

Quality controls: negative controls meets the acceptance criterion if the mean OD of the 2 tissues each time point is greater than or equal to 0.8. The positive controls meets the acceptance criterion if the mean relative tissue viability at the 3 minute time point is less than or equal to 30%
Inter-tissue viability meets the acceptance criterion if the difference between two identically treated tissues is no greater than 30%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 3 min

Value:

95.6

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

after 60 min

Value:

88.2

Interpretation of results:

GHS criteria not met

Conclusions:

In this in vitro EPIDERM model test with Zinc bis(dihydrogen phosphate), the results indicate that the test item is not a skin corrosive.

Executive summary:

A study was conducted to predict and classify the skin corrosivity potential of Zinc bis(dihydrogen phosphate) by using a three-dimensional human epidermis model (OECD 431). MatTek EpiDerm tissue samples were treated in duplicate with the test article, negative control and positive control for 3 minutes and 60 minutes. Following treatment, the viability of the tissues was determined using MTT uptake and conversion, and the absorbance of each sample was measured at 540nm. The viability was then expressed as a percent of control values.

In this in vitro EPIDERM model test with Zinc bis(dihydrogen phosphate), the results indicate that the test item is not a skin corrosive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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:

- Name: Zinc bis(dihydrogen phosphate), dihydrate
- Chemical name: Zinc bis(dihydrogen phosphate), dihydrate
- CAS number: 13986-21-5
- Batch/Lot number: 9000054373
- Description: White crystals
- Purity: Considered as 100%
- Manufacturer/supplier: Budenheim Ibérica S.L.U.
- Expiry date: 01 February 2023
- Stability of Bulk Test Item: The test item is considered stable when stored under appropriate storage conditions: controlled room temperature (15-25°C, ≤70% relative humidity). The test item was 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 Routine safety precautions (gloves, goggles, face mask, lab coat) for unknown materials were applied to ensure personnel health and safety.
- Preparation of Formulation: The test item was applied in its original form. Although it was not a fine powder, so the test item was ground finely in a mortar and pestle.

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Eyes collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old, mean weight: 2.5 kg) which are used 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 bis(dihydrogen phosphate), dihydrate 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

Number of animals or in vitro replicates:

Three test item treated eyes, three positive control treated eyes and one negative control eye 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 was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5% within the -45 min and the zero time No significant corneal thickness changes (-1.6% in one eye) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
: 3

NEGATIVE CONTROL USED
- yes, Physiological saline (Salsol solution, 0.9% (w/v) NaCl)

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 bis(dihydrogen phosphate), dihydrate
-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.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material if possible. Additional gentle rinsing with 60 mL saline was performed at each time point when the test material or positive control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- 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 was focused such that the physiological saline solution appeared as a visible, clear (sharp) image as it moved across the cornea surface.
- Damage to epithelium based on fluorescein retention: The fluorescein retention determination the settings of the slit lamp microscope was the same as for opacity assessment, but the green light filter was used.
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 observable
- Others (e.g, histopathology): not performed

SCORING SYSTEM:
- Mean corneal swelling (%)
: according to ICE classification criteria for corneal thickness
- Mean maximum opacity score:
according to ICE classification criteria
- Mean fluorescein retention score at 30 minutes post-treatment
: according to ICE classification criteria

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. - yes

Irritation parameter:

corneal swelling 

Run / experiment:

75min

Value:

10.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

ICE class II

Irritation parameter:

corneal swelling 

Run / experiment:

240

Value:

12.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Slight corneal swelling (mean= 12.5%) was observed during the four-hour observation period on all test item treated eyes.

Irritation parameter:

cornea opacity score

Run / experiment:

1

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Slight cornea opacity change (severity 1 on both eyes) was observed.

Irritation parameter:

fluorescein retention score

Run / experiment:

1

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

Value given is for the mean fluorescein retention score. Slight fluorescein retention change (severity 1 on both eyes) was observed corresponding to ICE class II.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no severe corneal morphological changes

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

Results

The mean values of the treated eyes for maximum corneal thickness change, corneal opacity change and fluorescein retention change are given below. The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity change and the mean fluorescein retention change ICE classes are used GHS classification.

Test item:

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10.7%	II
Mean maximum corneal swelling at up to 240min	12.5%	II
Mean maximum corneal opacity change	1.0	II
Mean fluorescein retention change	1.0	II
Other Observations	Minimal amount of test item was stuck on the cornea surface in two eyes (#15) (#16) after 240 minutes post treatment rinse.
Overall ICE Class	3xII

Based on this in vitro eye irritation study in isolated chicken eyes with zinc bis(dihydrogen phosphate), dihydrate, the test item is not classified as a severe irritant and not classified as non-irritant (no prediction can be made). 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	23.8%	III
Mean maximum corneal swelling at up to 240min	34.3%	IV
Mean maximum corneal opacity change	4.0	IV
Mean fluorescein retention change	3.0	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
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240min	1.8%	I
Mean maximum corneal opacity change	0.00	I
Mean fluorescein retention change	0.00	I
Other Observations	None
Overall ICE Class	3xI

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

 

Summary for UN GHS Classification

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:	False
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:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False
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

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.

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on this in vitro eye irritation assay in isolated chicken eyes with zinc bis(dihydrogen phosphate), dihydrate, the test item was not classified as a severe irritant and not classified as non-irritant (no prediction can be made). It is concluded that further information is required for classification.

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 zinc bis(dihydrogen phosphate), dihydrate. The three positive control eyes were treated in a similar way with 30 mg powdered imidazole and the negative control eye was treated with 30 µL of physiological saline (0.9% (w/v) NaCl solution. Corneal thickness, corneal opacity and fluorescein retention were measured and any morphological effects (e.g. pitting or loosening of the epithelium) were evaluated.

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

Slight corneal swelling (mean= 12.5%) was observed during the four-hour observation period on all test item treated eyes. Slight cornea opacity change (severity 1 on both eyes) was observed. Slight fluorescein retention change (severity 1 on both eyes) was observed. No other morphological effect was observed. Based on this in vitro eye irritation study in isolated chicken eyes with zinc bis(dihydrogen phosphate), dihydrate, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

No other corneal effect was observed.

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:	False
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:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False
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

Based on this in vitro eye irritation assay in isolated chicken eyes with
zinc bis(dihydrogen phosphate), dihydrate, the test item was not classified as a severe irritant and not classified as non-irritant (no prediction can be made). It is concluded that further information is required for classification.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

March - June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

Test design of this assay is based on the protocol published by MatTek Corporation: “EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals” (adopted version 2017), but study design will follow OECD Guideline No. 492.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Name: Zinc bis(dihydrogen phosphate), dihydrate
Chemical name: Zinc bis(dihydrogen phosphate), dihydrate
CAS number: 13986-21-5
Batch/Lot number: 9000054373
Description: White crystals
Purity: Considered as 100%
Manufacturer/supplier: Budenheim Ibérica S.L.U.
Expiry date: 01 February 2023

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability of Bulk Test Item: 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.
- 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 will be applied to ensure personnel health and safety.
- Preparation of Formulation
The test item was applied in its original form, however the test item was ground to fine powder.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 mg

POSITIVE and NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µl

Duration of treatment / exposure:

6h +/- 15min

Observation period (in vivo):

not applicable

Duration of post- treatment incubation (in vitro):

18h +/- 15min

Number of animals or in vitro replicates:

In this assay, two replicates were used for the test item. Two negative controls and two positive controls were also run in the assay. Furthermore, as the test item was coloured, two additional test item-treated living tissues were used for the non-specific OD evaluation.

Details on study design:

- Details of the test procedure used: Test design of this assay is based on the protocol published by MatTek Corporation: “EpiOcularTM Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals” (adopted October 2017), which is in agreement with OECD Guideline No. 492. (adopted 18 June 2019).
- RhCE tissue construct used, including batch number: The EpiOcularTM model consists of an airlifted, living, multilayered ocular tissue construction (surface 0.60 cm2), reconstructed from normal (non-transformed) human-derived keratinocytes. This is a non-keratinized epithelium which models the cornea epithelium with progressively stratified, but not cornified cells. The 3D tissue consists of highly organized cell layers similar to that found in the cornea. The model features a normal ultra-structure and is functionally equivalent to human in vivo tissue.

Species: Reconstructed Human cornea-like epithelium (tissues).
Supplier: MatTek, Bratislava, Slovak Republic.
Selection: At receipt, the tissues will be inspected for obvious defects prior to use.
Expiry date: The living EpiOcular tissues can be used within 72 hours of their production.
Batch No.: 34960

Doses of test chemical and control substances used:
- TEST MATERIAL: Amount(s) applied (volume or weight with unit): 50 mg
- POSITIVE and NEGATIVE CONTROL: Amount(s) applied (volume or weight with unit): 50 µl

- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable):
The test item and both negative and positive controls were applied topically on duplicate tissues and incubated at +37°C for 6 hours. At the end of the treatment period, each tissue was rinsed with D PBS, incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2 in a >95% humidified atmosphere.

- Number of tissue replicates used per test chemical and controls (positive control, negative control): 2

- Description of the method used to quantify MTT formazan: MTT medium (300 μL/well) will be added to each well below the tissues units (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 (±10 minutes). After the 3-hour MTT incubation period is complete, MTT will be gently aspirated from all wells (e.g. using a suction pump), then wells will be refilled with PBS and aspirated. The rinsing will be repeated twice and made sure that tissues are dry after the last aspiration. Transfer inserts 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 either overnight (2-8°C in the dark) without shaking or alternatively, 2-3 hours with shaking (~120 rpm) at room temperature. After the extraction period is complete, the inserts will not be pieced and allowed the extract to run into the well from which the insert was taken. Afterwards the insert can be discarded. A blank sample containing 2 mL of extracting solution (MTT-100-EXT) will be processed in parallel.

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1

Value:

1.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

The mean cell viability was 1.4% compared to the negative control (after adjustment for non-specific colour control). This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None observed

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Check-method for possible direct MTT reduction with test item
50 mg test item was added to 1 mL freshly prepared MTT solution (1.0 mg/mL) and mixed. The mixture was incubated at 37°C in an incubator with 5 % CO2, in a >95% humidified atmosphere for 3 hours (±5 minutes) protected from light, and then any colour change was recorded:
• Test item which directly reacts with MTT: blue or purple
• Test item which does not react with MTT: other colours
After three hours of incubation, yellow colour of the mixture was detected in the test tube. Thus, the test item did not react with MTT, and additional controls were not necessary.

Colouring potential of test item
The test item is white colour therefore NSCliving control was used in this study.
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.
Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment).
50 mg of test items were added to 2 mL of water and incubated for at one hour at +37°C, 5% CO2. No colour change was observed in the mixture.

Additional Controls

As the test item was coloured (white), 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 the table below. The mean value for optical density (measured at 570 nm) was 0.005, and the NSC_living % value for the test item was calculated as 0.2%. Calculation with this value was necessary.

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

Additional Control	Optical Density			NSC % (living)
		Measured	Blank corrected
Treated with test item	1	0.050	0.004	0.2
	2	0.051	0.005
	mean	-	0.005

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 Zinc bis(dihydrogen phosphate), dihydrate treated samples showed 1.4% relative viability compared to the negative control (after adjustment for non-specific colour controls).

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

Substance	Optical Density (OD)			TODTT	Viability (%RV)
		Measured	Blank corrected
Negative control: distilled water	1	2.215	2.169	-	102.4
	2	2.114	2.068	-	97.6
	mean	-	2.119	-	100.0
Positive control: Methyl Acetate	1	0.538	0.492	-	23.2
	2	0.578	0.533	-	25.1
	mean	-	0.512	-	24.2
Test item	1	0.082	0.036	0.032	1.5
	2	0.077	0.032	0.027	1.3
	mean	-	0.034	0.029	1.4

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).


3. TOD_TT: the measured values were corrected for non-specific controls (NSC_living value was 0.005, therefore the final correction was 0.029).

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

In this in vitro EpiOcular™ model test with Zinc bis(dihydrogen phosphate), dihydrate (Batch/Lot number: 9000054373), the results indicate that the test item is probably irritant to eyes (UN GHS Classification: Category 2 or Category 1).

Executive summary:

The purpose of this study was to evaluate the eye hazard potential of the test item, Zinc bis(dihydrogen phosphate), dihydrate, based on its ability to induce cytotoxicity in the EpiOcular^TM cornea epithelial model, as measured by the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1] assay.

Disks of EpiOcular™ (two units) were treated with the test item and incubated at 37°C, 5% CO₂ in a >95% humidified atmosphere for 6 hours. At the end of the treatment period, each tissue was rinsed with Dulbecco’s Phosphate Buffered Saline (D-PBS), incubated for 25 minutes at room temperature to remove any remaining test item from the tissue, blotted on absorbent material, and then incubated for another 18 hours at 37°C, 5% CO2 in a >95% humidified atmosphere. After the incubation, MTT solution was added to the units and incubated for a further 3 hours to determine cell viability. The precipitated formazan crystals were then extracted using isopropanol and quantified spectrophotometrically at 570 nm.

Distilled water and Methyl acetate treated epithelium tissues were used as negative and positive controls, respectively (two 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 % relative to the negative control. If the mean relative viability is less or equal (≤) to 60% of the negative control, the test item is considered to be irritant to Reconstructed human Cornea-like Epithelium.

Following exposure with test item, the mean cell viability was 1.4% compared to the negative control (after adjustment for non-specific colour control). This is below the threshold of 60%, therefore the test item was considered as being irritant to Reconstructed human Cornea-like Epithelium. The experiment met the validity criteria, therefore the study was considered to be valid.

In conclusion, in this in vitro EpiOcular™ model test with Zinc bis(dihydrogen phosphate), dihydrate (Batch/Lot number: 9000054373), the results indicate that the test item is probably irritant to eyes (UN GHS Classification: Category 2 or Category 1).

Reference 3

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Shortly documented, scientifically good

Qualifier:

equivalent or similar to guideline

Guideline:

other: Hen's Egg test on the Chorio-Allantoic Membrane

Principles of method if other than guideline:

Hen´s Egg Test Chorioallantoic Membrane (HET-CAM) test permits the identification of irritative reactions which appear to be similar to those which occur in the eye using the standard Draize rabbit eye test. In the HET-CAM test system, three reactions are determined: haemorrhage, lysis and coagulation of the chorio-allantoic membrane at the ninth day of embryonation when nerve tissue and pain perception have not yet developed.

GLP compliance:

yes

Species:

other: hen's eggs

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: eggs treated with reference substance

Amount / concentration applied:

0.3ml of the 10% mixture of the test article (1g of test article was mixed with distilled water to a total volume of 10ml)

Duration of treatment / exposure:

5 minutes

Observation period (in vivo):

5 minutes

Number of animals or in vitro replicates:

6 White Leghorn eggs per test substance

Irritation parameter:

other: relative irritation potential

Value:

0.99

Positive controls validity:

valid

Substance (undiluted)	Conc (% free H3PO4)	SAT (intern batch nr)	relativ irritation potential (Q)	SD  ±	Assessment
phosphorsäure	4.5	060236	0.96	0.13	moderately irritating *
zinkphospat-lösung 2 (zink-bis-dihydrogenphosphat, 14.6%)	4.5	060237	0.99	0.05	moderately irritating *
zinkphospat-lösung 2, verdünnt (faktor 1.72 mit wasser) (zink-bis-dihydrogenphosphat, 14.6%)	2.6	060252	0.99	0.09	moderately irritating *
referenzsubstanz 'Texapon ASV'	5% AS	050359	1.00	0.04	moderately irritating

* extravasale Koagulationsnachweis: positiv

Interpretation of results:

moderately irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Test substance, zinkphospat-lösung 2 (zink-bis-dihydrogenphosphat, 14.6%), as undiluted, is moderately irritating. Slight extravasale coagulation is assessed.

Executive summary:

The aim of this study is to evaluate the potential ocular irritancy of a test substance as measured by its ability to induce toxicity in the chorioallantoic membrane of a hen. Effects are measured by the onset of: (1) hemorrhage; (2) coagulation; and (3) vessel lysis. These assessments are considered individually and then combined to derive a score, which is used to classify the irritancy level of the test substance.

Based on the relative irritation potential (Q), the test substance, as undiluted, is moderately irritating

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation/corrosion:

The in vitro skin irritation and corrosion test in the EpiDerm model (OECD 431; OECD 439) (Piehl, 2010) with zinc bis(dihydrogen phosphate) indicates that the test item is not a skin irritant and not-corrosive. Following exposure to zinc bis(dihydrogen phosphate), the mean cell viability after 60min was 92.1% and 88.2% in the skin irritation and skin corrosion test respectively, therefore under the condition of this assay the test item was considered as being non-irritant and not-corrosive to skin.

Eye irritation:

Three in vitro eye irritation tests were conducted with zinc bis(dihydrogen phosphate):

- The irritating potential of the test item was assessed in a HET-CAM test. The aim of the study being to evaluate a substance's ability to induce toxicity in the chorioallantoic membrane of a hen. The results showed mildly irritating effects of the test item.
- An in vitro EpiOcular test was also conducted and showed that zinc bis(dihydrogen phosphate), dihydrate had eye irritating properties. 
- An isolated chicken eye test was done to assess whether the test substance could be classified as a severe irritant (category 1) or not. The test item was not classified as a severe irritant and not classified as non-irritant. So irritating properties were once again confirmed but the test item is not a severe irritant.

Therefore, zinc bis(dihydrogen phosphate), dihydrate is classified as category 2 for eye irritation.

Justification for classification or non-classification

Skin irritation: In vitro data shows that zinc bis(dihydrogen phosphate) was not irritating to skin and therefore does not require classification as a skin irritant.

Eye irritation: In vitro data in the HET-CAM and EpiOcular tests suggest that zinc bis(dihydrogen phosphate) is irritating to the eyes. A further ICE in vitro test allows discrimination between category 1 or not irritating. Results from this ICE test show that the test item is not a category 1 irritant, so a category 2 classification is derived.