Zinc glycinate sulfate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2022-10-28 to 2022-10-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EU) No 2017/735 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 (Reach).

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Bovine Corneal Opacity and Permeability (BCOP) Assay, SOP of Microbiological Associates Ltd., UK, Procedure Details, April 1997.

Version / remarks:

April 1997

Test guideline 3

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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:

26th June 2020

Test guideline 4

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Version / remarks:

09 Dec 2010

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2‐aminoacetate, hydron, zinc(2+) sulfate hydrate

Molecular formula:

C2H7NO7SZn

IUPAC Name:

2‐aminoacetate, hydron, zinc(2+) sulfate hydrate

Test material form:

solid

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals (e.g. age, sex, weight): The slaughterhouse did not confirm the age of the donor cattle despite repeated requests.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Freshly isolated bovine eyes of donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were stored in HBSS (Hank’s Balanced Salt Solution) containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter house and during transportation on the same morning to the laboratory using a Styrofoam box.
- Time interval prior to initiating testing: The corneas were isolated on the same day after delivery of the eyes.
- Indication of any existing defects or lesions in ocular tissue samples:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.
- Indication of any antibiotics used: 100 units penicillin per mL medium, and 100 µg streptomycin per mL medium)
- Selection and preparation of corneas: Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneas in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the medium was changed before basal opacity measurement (t0).
- Quality check of the isolated corneas: Only corneas with a value of the basal opacity < 7 were used.

Test system

Vehicle:

physiological saline

Remarks:

(0.9% NaCl in deionised water)

Controls:

yes

yes, concurrent vehicle

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Concentration: The test item was tested as a 20% solution (w/v) in saline.
VEHICLE
- Amount(s) applied: 0.75 mL

Duration of treatment / exposure:

The incubation period was 240 minutes.

Duration of post- treatment incubation (in vitro):

The anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.

Number of animals or in vitro replicates:

Number of Corneas:
15 corneas where used for the experiment (three per group):
Negative Control I: 3
Negative Control II: 3
Positive Control I: 3
Positive Control II: 3
Test Item: 3

Details on study design:

NUMBER OF REPLICATES: 3
NEGATIVE CONTROL USED: deionised water
SOLVENT CONTROL USED: Saline (0.9% NaCl in deionised water)
POSITIVE CONTROL USED
Positive Control 1: 10% (w/v) Benzalkonium chloride (purity not indicated by the producer) in saline using sonication.
Positive Control 2: 20 % (w/v) Imidazole in saline
APPLICATION DOSE AND EXPOSURE TIME
The test item was tested as a 20% solution (w/v) in saline.
The incubation period was 240 minutes.
TREATMENT METHOD: closed chamber
REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
After exposure, the test item or the control items, respectively, were each rinsed off from the according application sides with EMEM containing phenol red for at least three times or more until phenol red was still discoloured (yellow or purple), or the test item was still visible. Once the medium was free of the test item the corneas were given a final rinse with cMEM without phenol red. Fresh cMEM was added into both compartments and opacity was measured (t240).
- POST-EXPOSURE INCUBATION:
After the final opacity measurement was performed, the incubation medium was removed from both chambers. The posterior chamber was filled with fresh cMEM first. Then the anterior compartment was filled with 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneas were incubated again in a horizontal position for 90 ± 5 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment was removed, well mixed and transferred into a 96 well plate.
METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer determines changes in the light transmission passing through the corneas and displays a numerical opacity value. The opacitometer (OP_KiT opacitometer (Electro Design, 63-Riom, France)) was calibrated as described in the manual and the opacity of each of the corneas was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490).
SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
IVIS GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
> 55 Category 1
DECISION CRITERIA: according to guideline

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1 Experiment with five groups (two negative controls, two positive controls, one test item)

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Any other information on results incl. tables

 

Test Group	Opacity value = Difference (t240-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Standard Deviation IVIS	Proposed Category
		Mean		Mean
Negative Control (Saline)	0	0.33	0.071	0.067	1.07	1.34	0.58	No Category
	1		0.067		2.01
	0		0.064		0.96
Negative Control (deionised water)	1		0.065		1.98	1.29	0.60	No Category
	0		0.060		0.90
	0		0.066		0.99
Positive Control (10 % Benzalkonium chloride)	76.67*		0.768*		88.18	91.70	3.05	Category 1
	82.67*		0.710*		93.31
	85.67*		0.529*		93.60
Positive Control (20 % Imidazole)	88.67*		0.769*		100.20	101.15	4.74	Category 1
	80.67*		1.086*		96.95
	87.67*		1.242*		106.29
Test Item	1.67*		0.00**		1.67	1.00	0.58	No Category
	0.67*		0.00**		0.67
	0.67*		0.00**		0.67

* corrected values

** Value was set to zero since the calculated value was negative

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, the test item is not categorized (EU CLP/GHS No Category).

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Zinc Monoglycinate Sulfate Hydrate by means of the BCOP assay using fresh bovine corneas (OECD 437, 2013-07-26).
After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) solution in saline (0.9% (w/v) NaCl in deionised water) of the test item Zinc Monoglycinate Sulfate Hydrate as well as the positive and the negative controls were each applied to different corneas fixed in an incubation chamber in horizontal position and incubated for 240 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneas and opacity was measured again (t240).
After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
With the negative controls saline and deionised water, neither an increase of opacity nor permeability of the corneas could be observed (saline mean IVIS = 1.34; deionised water mean IVIS = 1.29).
The positive control (10% (w/v) Benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 91.70) corresponding to a classification as serious eye damaging (EU CLP/GHS (Category 1) as well as the positive control imidazole (mean IVIS = 101.15).

Based on these results, zinc monoglycinate sulfate dihydrate does not require classification according to Regulation (EC) No. 1272/2008 (CLP) or the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) with respect to eye irritation.