Zinc bis(dihydrogen phosphate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Feb - June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(dihydrogen phosphate)

EC Number:

237-067-2

EC Name:

Zinc bis(dihydrogen phosphate)

Cas Number:

13598-37-3

Molecular formula:

Zn(H2PO4)2

IUPAC Name:

zinc bis(dihydrogen phosphate)

Test material form:

solid: crystalline

Remarks:

White crystals

Details on test material:

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

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.

Test animals / tissue source

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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 mg of zinc 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

Results and discussion

In vitro

Results

Results 1

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

Results 2

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.

Results 3

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.

Results 4

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

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.