Zinc bis(benzenesulphinate)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 21 to 22, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

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:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Zinc bis(benzenesulphinate)

EC Number:

246-148-1

EC Name:

Zinc bis(benzenesulphinate)

Cas Number:

24308-84-7

Molecular formula:

C12H10O4S2Zn

IUPAC Name:

zinc bis(benzenesulphinate)

Test material form:

solid: particulate/powder

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

Bovine eyes source: breeding service CHOVSERVIS a.s., division TORO® Hlavečník, Hradec Králové, Czech Republic.
Eyes were collected by slaughterhouse employees. The eyes were enucleated as soon as possible after death. No detergent was used. Only healthy animals (12 to 30 months old) considered suitable for entry into the human food chain were used as a source of corneas for use in the BCOP test. The risk of contamination was minimised (e.g. by keeping the container containing the eyes on ice, by adding antibiotics to the HBSS used to store the eyes during transport, e.g., penicillin at 100 IU/ml and streptomycin at 100 μg/ml).
The time interval between collection of the eyes and use of corneas in the BCOP was minimised (typically collected and used on the same day). The results were based on the selection criteria for the eyes, as well as the positive and negative control responses. All eyes used in the assay were from the same group of eyes collected on a specific day.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

Test substance was tested as solution of test substance at 20 % concentration in a 0.9 % sodium chloride solution. 2 g of test substance was suspended in 10 ml of 0.9 % sodium chloride solution.
Closed-chamber method was used because 10 % solution was applicable by micropipette.

Duration of treatment / exposure:

4 h

Duration of post- treatment incubation (in vitro):

Incubation for 1.5 h at 32 ± 1 °C with 5 mg/ml of sodium fluorescein, before measurement of absorbance at 490 nm.

Number of animals or in vitro replicates:

3 corneas per negative control
3 corneas per positive control
3 corneas per test substance

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The eyes, once they arrive at the laboratory, were carefully examined for defects including scratched, and neovascularisation. Only corneas from eyes free of such defects were used.
The isolated corneas, after achieve normal metabolic activity (inductive incubation at 32 ± 1°C for one hour), were examined again. The corneas that show macroscopic tissue damage (e.g., scratches, pigmentation, neovascularization) or a baseline opacity >7 opacity units were discarded.
From 20 eyes, 1 eye was eliminated after inductive incubation, because cornea was damaged cornea during installation of cornea to holder. Nine corneas were used for the study (corneas no. 1, 2, 3, 4, 5, 7, 8, 9 and 10), 7 eyes was superfluous and remaining 3 were used for testing of another substance.

NEGATIVE CONTROL USED: 0.9 % NaCl

POSITIVE CONTROL USED: 20 % imidazole

APPLICATION DOSE AND EXPOSURE TIME: 2 g of test substance was suspended in 10 ml of 0.9 % NaCl solution; after application, incubation was continued for 4 h.

TREATMENT METHOD: closed chamber, test item was applicable by micropipette.

POST-EXPOSURE:
After the exposure period, negative control and positive control substance was removed from the anterior chamber with EMEM (containing phenol red); corneas were given a final rinse with EMEM (without phenol red). EMEM (without phenol red) was used as a final rinse to ensure removal of phenol red from the anterior chamber prior to opacity measurement. The anterior chamber was then refilled with fresh EMEM without phenol red. Opacity and permeability of each cornea were recorded.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: measured quantitatively with the aid of an opacitometer; resulting values of illuminance was converted to opacity values as:
opacity = I0/Ix - 0.9894 / 0.0251
where
I0 = value of illuminance - holders with class windows and liquid without cornea
Ix - measured illuminance - holders with class windows and liquid, with cornea

- Corneal permeability: the amount of sodium fluorescein dye that penetrates all corneal cell layers (i.e., the epithelium on the outer cornea surface through the endothelium on the inner cornea surface) measured indirectly using visible light spectrophotometry. 1 ml sodium fluorescein solution (5 mg/ml) was added to the anterior chamber of the corneal holder, which interfaced with the epithelial side of the cornea, while the posterior chamber, which interfaced with the endothelial side of the cornea, is filled with fresh EMEM. The holder was incubated in horizontal position for 1.5 hours at 32 ± 1 ºC.
The amount of sodium fluorescein that crosses into the posterior chamber was quantitatively measured with the aid of UV/VIS spectrophotometry (Spectrophotometer GENESYSTM 10 UV/VIS Scanning). The values of absorbance measured at 490 nm were recorded as optical density (OD490) values. This term was used because the measuring is performed with visible light spectrophotometer using a standard 1 cm path length.

- Mean opacity: opacity values of treated corneas were corrected by subtracting individual background opacity values and the mean opacity is calculated.

- Mean permeability: mean OD value of treated corneas was corrected by subtracting the mean OD value of negative control and the mean permeability is calculated.

SCORING SYSTEM:
IVIS calculation: resulting mean opacity and OD490 values for each treatment group was combined in an empirically-derived formula to calculate an in vitro irritancy score (IVIS) for each treatment group as follows:
IVIS = mean opacity value + (15 × mean permeability OD490 value)

DECISION CRITERIA:
The IVIS cut-off value for identifying test item as including serious eye damage (UN GHS Category 1) and test item not requiring classification for eye irritation or serious damage (UN GHS No Category) will be given hereafter:

IVIS UN GHS
≤ 3 no category (UN GHS: not classified)
> 3; ≤ 55 no prediction can be made
> 55 category 1 (UN GHS: “serious eye damage”)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

mean (3 corneas) - corrected by negative control value

Value:

2.35

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

other: permeability

Run / experiment:

mean (3 corneas) corrected by negative control value

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

mean (3 corneas)

Value:

2.35

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
Appearance of corneas was observed before and after application of test item, negative and positive control.
No macroscopic damage was observed on corneas before application.
Corneal opacity was observed on corneas treated by positive control. Corneas treated by negative control and test item were without macroscopic damage.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: opacity and permeability values are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative or solvent/vehicle control.
- Acceptance criteria met for positive control: IVIS falls within one standard deviations of the current historical mean, which is to be updated at least every three months.

IVIS – historical value for positive control
mean standard deviation upper limit lower limit
77.3 4.55 81.93 72.84
Notes:
lower limit = mean - one standard deviation of the current historical mean
upper limit = mean + one standard deviation of the current historical mean
historical means of IVIS are updated at least every three months.

The value of IVIS for positive control (20 % imidazole) during the study was 80.60, thus the study is considered acceptable.


Opacity and permeability – historical values for negative control
value mean standard deviation upper limit
opacity 2.68 0.92 3.60
permeability 0.0295 0.0127 0.0422
Notes: upper limit = mean + one standard deviation of the current historical mean
Historical means of opacity and permeability are updated at least every three months.

Opacity for negative control (0.9 % NaCl) obtained during the study was 2.03 and value of permeability was 0.031, thus the study is considered acceptable.

Any other information on results incl. tables

Opacity values

group	cornea no.	opacity after treatment	baseline opacity	opacity difference	mean opacity difference	mean opacity (corrected)
NC (0.9 % NaCl)	1	4.94	4.27	0.67	2.03	-
	2	5.47	2.14	3.33
	3	5.48	3.39	2.09
PC (20 % imidazole in 0.9 % NaCl)	4	58.70	1.44	57.26	54.23	52.20
	5	55.49	2.36	53.13
	7	55.04	2.75	52.29
test substance	8	5.89	1.86	4.03	4.38	2.35
	9	7.12	2.15	4.97
	10	7.43	3.28	4.15

Values of permeability (optical density values)

group	cornea no.	values of permeability (optical density at 490 nm)	mean permeability	mean permeability (corrected)
NC (0.9 % NaCl)	1	0.061	0.031	-
	2	0.021
	3	0.012
PC (20 % imidazole in 0.9 % NaCl)	4	1.965	1.924	1.893
	5	2.047
	7	1.761
test substance	8	0.012	0.005	-0.026 (0 used for calculation)
	9	0.003
	10	0.001

IVIS = mean opacity value + 15 × mean permeability OD490

group	calculation	results
NC (0.9 % NaCl)	2.03 + 15 × 0.031	2.50
PC (20 % imidazole in 0.9 % NaCl)	52.20 + 15 × 1.893	80.60
test substance	2.35 + 15 × 0	2.35

Applicant's summary and conclusion

Interpretation of results:

other: not irritant according to the CLP Regulation (EC 1272/2008)

Conclusions:

Not irritant.

Executive summary:

Method

Evaluation of the potential for ocular corrosivity or severe irritancy was measured as the ability of test substance to induce opacity and increased permeability in an isolated bovine cornea.

Test was performed according to OECD guideline 437.  

A total of 9 corneas was used, divided in 3 groups, i.e. a treatment group, a positive control group with 20 % imidazole and a negative control group with 0.9 % NaCl.

Test item was tested as suspension prepared from test item at 20 % concentration in a 0.9 % NaCl solution.

The closed-chamber method was used, because test item was applicable by micropipette. The opacity and permeability of each cornea were measured. In Vitro Irritancy Score (IVIS) was calculated from values of opacity and permeability as:

IVIS = mean opacity value + 15 × mean permeability OD490 value

Results 

IVIS for test substance was 2.35. Corneas treated by test item were without macroscopic damage.