Zinc di(acetate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 January 2013 - 28 March 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD Guideline 437, GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc di(acetate)

EC Number:

209-170-2

EC Name:

Zinc di(acetate)

Cas Number:

557-34-6

Molecular formula:

C2H4O2.1/2Zn

IUPAC Name:

zinc diacetate

Details on test material:

- Name of test material (as cited in study report): Zinc acetate
- Physical state: white powder
- Analytical purity: 97.3%
- Lot/batch No.: 20111127
- Expiration date of the lot/batch: 27 November 2013
- Storage condition of test material: Room temperature

Test animals / tissue source

Species:

other: Bovine cattle (in-vitro test)

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes were obtained from freshly slaughtered cattle at the abattoir. EVA, Saint-Pierre-sur-Dives - France.
- Age at study initiation: Up to 12 months old.

TRANSPORT FROM SUPPLIER TO CITOXLAB: The eyes were transported in a specific container to avoid corneal damage, at ambient temperature, immerged in buffered Hanks medium containing an antibiotic.

Upon arrival, the selection and preparation of corneas was performed as soon as possible.

Test system

Vehicle:

water

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL ± 8 µL
- Concentration (if solution): 20% (w/v) in water solution.

VEHICLE
- Drinkingn water treated by reverse osmosis using a ELIX 5 apparatus.

Duration of treatment / exposure:

4 hours ± 5 min.

Number of animals or in vitro replicates:

3 corneas were used for each treated series: test item 20% in water, vehicle control (water), positive control 20% imidazole solution in 0.9% NaCl.

Details on study design:

STUDY DESIGN:
Corneas (without defects) were mounted in corneal holders. Both chambers of the corneal holders were filled with complemented MEM culture medium (cMEM) and pre-incubated for 1 hour and 5 minutes ± minutes at 32 ºC. Before the treatment, the eyes were macroscopically observed and a first opacity measurement was performed using a opacitometer (OPT0). Then, the of the anterior chamber was removed and the test item applied using a micropipette onto the epithelium of the cornea for 4 hours. At the completion, the test item was removed from the opening of the anterior chamber and the epithelium was rinsed with cMEM (32 ºC). A second opacity measurement was then performed (OPT2). Afterwards, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically in water for 90 minutes at 32 ºC. At the end of the incubation, the optical density at a wavelength of 490 nm of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Then, each cornea was observed for opaque spots and other irregularities.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes
- Time after start of exposure: 4 hours

SCORING SYSTEM:
Opacity: Change in opacity = OPT2 - OPT0, corrected with the change of the vehicle control.
Permeability: Corrected permeability = OD490 nm - control OD490 nm
In-vitro irritancy score: IVIS = corrected opacity + (15 x cOD490)
IVIS >= 55.1 = ocular corrosive/severe irritant.
IVIS < 55.1 = not ocular corrosive or severe irritant

TOOL USED TO ASSESS SCORE: lamp, opacitometer, fluorescein

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS

Basis:

mean

Time point:

other: 4 h

Score:

69.5

Irritant / corrosive response data:

Test item treatment: The in-vitro irritancy score (IVIS) was 69.5.

Other effects:

No notable opaque spots or irregularities were observed on vehicle control corneas following the treatment, while opacity, residual test item and fluorescein fixation were observed on the corneas treated with the test item.

Any other information on results incl. tables

Results:

GROUP		OPACITY				PERMEABILITY		SCORE
Vehicle control	Holder	OPT0	OPT2	OPT2-OPT0		OD490 nm
	11 15 28	2 2 2	1 9 7	-1 7 5		0.020 0.021 0.020
	Mean SD			3.7 4.2		0.020 0.001
Test item	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490 nm
	32 4 17	0 1 1	87 65 70	87 64 69	83.3 60.3 65.3	0.008 0.012 0.006	-0.012 -0.008 -0.014	83.1 60.2 65.1
	Mean SD				69.7 12.1		-0.012 0.003	69.5 12.1
Positive control	Holder	OPT0	OPT2	OPT2-OPT0	cOPT	OD490 nm
	3 44 37	1 1 3	71 61 89	70.0 60.0 86.0	66.3 56.3 82.3	1.464 1.652 3.508	1.444 1.632 3.488	88.0 80.0 134.6
	Mean SD				68.3 13.1		2.188 1.130	101.1 29.2

As the test item induces an IVIS > 55.1, it was considered to be an ocular corrosive or a severe irritant.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye)

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be corrosive or severely irritant to the eye.

Executive summary:

A Bovine Corneal Opacity and Permeability Test was performed with the test item according to OECD Guideline 437 in order to assess whether the substance is an ocular corrosives or severe irritant. The test item was evaluated using a treatment time of 4 hours. Three corneas per test (test item, vehicle control and positive control) were used in the present assay. Corneas were exposed to 20% of aqueous solution of test item for 4 hours. Then the corneas were rinsed. The opacity of the corneas were measured using a opacitometer before and after treatment in order to assess the change of opacity. After treatment, the corneas were exposed to a fluorescein solution of 5 mg/ml and the permeability determination was performed by measuring the density of the incubation solution media at a wavelength of 490 nm. Afterwards the in-vitro irritancy scores (IVIS) were calculated. No notable opaque spots or irregularities were observed on vehicle control corneas following the treatment, while opacity, residual test item and fluorescein fixation were observed on the corneas treated with the test item. The IVIS for the test item was 69.5. Since the IVIS was >= 55.1, it was considered to be an ocular corrosive or a severe irritant.