Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-01-12 to 2011-01-13

Reliability:

1 (reliable without restriction)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD Guideline for the Testing of Chemicals No. 437: “Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants” edition adopted 07 Sept. 2009

Deviations:

no

Principles of method if other than guideline:

NA

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

EC Number:

700-321-2

Molecular formula:

C30H26O6Zn, C46H42O6Zn, C62H58O6Zn

IUPAC Name:

hexazinc(2+) bis(2-hydroxy-3,5-bis[(1R)-1-phenylethyl]benzoate) bis(2-hydroxy-3,5-bis[(1S)-1-phenylethyl]benzoate) 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-(1-phenylethyl)-5-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-3-[(1R)-1-phenylethyl]benzoate 2-hydroxy-3-[(1S)-1-phenylethyl]benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1R)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-(1-phenylethyl)-3-(1-{4-[(1S)-1-phenylethyl]phenyl}ethyl)benzoate 2-hydroxy-5-[(1R)-1-phenylethyl]benzoate 2-hydroxy-5-[(1S)-1-phenylethyl]benzoate

Test animals / tissue source

Species:

other: fresh bovine corneas

Strain:

other: cattle

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
- Species: fresh bovine corneas
- Source: slaughterhouse Müller Fleisch GmbH, Enzstr. 2-4, 75217 Birkenfeld, Germany
- Age at study initiation: cattle were between 12 and 60 months old

ENVIRONMENTAL CONDITIONS
The eyes were transported to the test facility in Hank’s balanced salt solution. Then, the corneas were dissected and incubated with media at 32 ± 1°C in an incubation chamber for 1 hour.

PREPARATION
The test item is a non-surfactant solid.
Non-surfactant solids are usually tested as solutions or suspensions with 20% concentration in 0.9% sodium chloride solution, deionised water or other solvents which have no adverse effects in the test system. Since no solution of the test item was feasible, Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was ground and applied neat directly on the cornea. Specific corneal chambers were used for incubation of the eyes under test conditions.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable (negative control 0.9% NaCl)

Amount / concentration applied:

TEST MATERIAL
- 750 µl negative control solution (0.9% NaCl)
- 750 µL positive control solution (Imidazole solution, 20%)
- in average 272.3 mg of the test item were tested.

VEHICLE
- not applicable (no vehicle used)

Duration of treatment / exposure:

Exposition time on the corneas was 4 h ± 5 min. at 32 °C.

Number of animals or in vitro replicates:

3 replicates/treatment group (negative control, positive control and test item)

Details on study design:

PREPARATION
After having carefully cleaned and sterilised the corneal chambers, they were kept in the incubation chamber at 32°C.
On the day of the assay, the MEM without Phenol red was supplemented with sodium bi-carbonate, L-glutamine and 1% foetal calf serum (= complete MEM) and stored in a water bath at 32°C ± 1°C.
The same was performed with the MEM with Phenol red.
After the arrival of the corneas, they were examined and only corneas which were free from defects were used. The corneas were excised with a scalpel and cut from the globe with a 2-3 mm ring of sclera around the outside. Each cornea was transferred to a cornea holder in which pre-warmed cMEM without Phenol red was filled. The holders were then incubated for one hour in the incubation chamber at 32°C.

METHOD DESCRIPTION
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used.
The baseline opacity was measured by placing the holder with the cornea in a spectrophotometer and recording the absorption at 570 nm.
Opacity was calculated from the measured absorption at 570 nm following

O = 1 / 10(-A)
with
O = Opacity
A = Absorption at 570 nm

Correction of Measured Absorption at 490 nm
As cuvette with a path length of 0.2 cm were used in the measurement of the Fluorescein-Na solution in the spectrophotometer, the path length must be corrected to 1 cm.
Coefficient: 1/0.2 = 5: all absorptions were multiplied with this coefficient.

Calculation of IVIS (in vitro irritancy Score)
The IVIS of the negative control was calculated from the following equation:
IVIS = mean opacity value + (15 x mean corrected OD490 value)

The IVIS of the positive control and of the test item was calculated from the following equation:
IVIS = (opacity – opacity negative control) + [15 x (corr. OD490 – mean corr. OD490negative control)]


For each treatment group (negative control, positive control and test item), three replicates were used. 750 µl negative control resp. positive control solution were applied to each replicate. In average, 272.3 mg of the test item were tested.
According to the characteristics of the test item, the following treatment procedure was performed:

Open Chamber Method
The “open chamber-method” is used for non-surface-active solids and is performed predominantly with a 20% dilution or suspension of the test item dissolved in 0.9% NaCl. In order to apply the test item, the nut was unscrewed to remove the glass disc. The test item could be applied directly on the cornea now.

The following amounts of the test item were tested neat and applied directly on the cornea using a weight board:
Amounts of Test Item
Tissue Amount
1) 193.6 mg
2) 217.1 mg
3) 406.1 mg

PERFORMANCE OF THE STUDY
The test item was given on the epithelium in such a manner that as much as possible of the cornea was covered with test item.
Exposition time on the corneas was 4 h ± 5 min. at 32 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded at once (again by measurement at 570 nm). The cMEM without Phenol red was then removed from the front chamber, and 1 mL sodium fluorescein solution (concentration 5 mg/mL) was added to the front chamber. The chambers were then closed again and incubated for 90 ± 5 min at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, the permeability was measured with the spectral photometer as optical density at 490 nm.

Results and discussion

In vivo

Results

Irritation parameter:

other: in vitro irritancy scores (IVIS)

Basis:

mean

Time point:

other: 4 hours

Score:

0.595

Max. score:

0.994

Irritant / corrosive response data:

Mean opacity difference of the negative control is 0.3296.
For the permeability measurement, three replicates for each treatment group were measured.

Optical density determination at 490 nm (in triplicate each):

1.) Negative Control : 0.0221/0.0213/0.0207 -> Mean: 0.0214
2.) Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc: 0.0406/0.0244/0.0403 -> Net – Neg. Contr.: 0.0192/0.0030/0.0189
3.) Positive Control: 2.0263/1.6371/2.1760 -> Net – Neg. Contr.: 2.0049/1.6157/2.1546


A factor of 5 was taken into account when calculating the IVIS (In vitro irritancy score) to correct the path length. The net mean absorption of the negative control (0.0214) was subtracted from all individual replicates of test item and positive control.

The In vitro irritancy score (IVIS) was calculated based on the following equation
IVIS = (1.0360 – 0.3296) + [15 * (OD490 – neg. control (mean))]

whereas here -> neg. control (mean) = 0.0.214

RESULTS (IVIS):
1.) Negative Control: 0.7895/0.6403/0.5205 -> mean: 0.6500
2.) Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc: 0.9944/0.1337/0.6578 -> mean: 0.5950
3.) Positive Control: 71.9965/89.8963/122.0831 -> mean: 94.6580


CLASSIFICATION
Classification of the irritation potential:

In vitro irritancy score (IVIS) Classification
0 - 3 Non eye irritant
3.1 - 25 Mild eye irritant
25.1 - 55 Moderate eye irritant
55.1 - 80 Severe eye irritant
≥ 80.1 Very severe eye irritant

Classification was derived from Gautheron et al. (1992), refined by Vanpaarys et al. (1994) and confirmed by ICCVAM.
In the negative control, no signs of eye irritation were observed. The positive control showed very severe eye irritation.
The test item showed no eye irritation (mean value lay below negative control).

Other effects:

NA

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: other: Classification derived from Gautheron et al. (1992), refined by Vanpaarys et al. (1994), confirmed by ICCVAM

Conclusions:

Under the experimental conditions reported, the test item Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc does not possess any eye irritation potential.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc by quantitative measurements of changes in opacity and permeability in a bovine cornea.

The test item Reaction product of 2-hydroxybenzoic acid, styrene and oxozinc was brought onto the cornea of a bovine eye which previously had been incubated with cMEM without Phenol red at 32±1°C for one hour and whose opacity had been determined. The test item was incubated on the cornea for 4 hours at 32±1°C. After removal of the test item, opacity and permeability values were measured.

Physiological sodium chloride solution was used as negative control, imidazole (20% solution in 0.9% sodium chloride solution) was used as positive control. The positive control induced a very severe irritation on the cornea, mean IVIS was 94.6580.

The negative control showed no irritation, mean IVIS was 0.6500. The test item was tested pure. A mean IVIS of 0.5950 was calculated, corresponding to a classification as not eye irritant. The applied amount of test item did not show any correlation to the calculated IVIS.

No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.