2-(4-chlorobenzoyl)benzoic acid

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 01, 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

equivalent or similar 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)

Deviations:

yes

Remarks:

see 'Principle of method if other than guideline'

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-(4-chlorobenzoyl)benzoic acid

EC Number:

201-615-9

EC Name:

2-(4-chlorobenzoyl)benzoic acid

Cas Number:

85-56-3

Molecular formula:

C14H9ClO3

IUPAC Name:

2-(4-chlorobenzoyl)benzoic acid

Test material form:

solid

Details on test material:

Purity >99.1 %; Appearance: white powder

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Bovine eyes
Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint-Pierre-sur-Dives, France.

Age: bovine cattle were up to 12 months old (typically, 5 to 8 months old).
Transport from Supplier to laboratory: the eyes were transported at ambient temperature, immerged in buffered Hanks medium containing an antibiotic Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 μg/mL final). A container with smooth internal surfaces was used for the transport to avoid physical damage to the corneas.

Preparation of the corneas
Upon arrival the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them. Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.

Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 μg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.
Storage of the corneas: as the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium (Sigma-Aldrich Ref. M3769, Medium 199 Modified, with Earle's Salts, without L-glutamine, NaHCO3, and phenol red) containing 5% dextran, plus penicillin/streptomycin, at +4 °C, for a maximum of 24 hours before use.

Test system

Vehicle:

other: paraffin oil

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

The test substance was tested at the concentration of 20 % (w/v) in parafin oil.

Duration of treatment / exposure:

4 hours (± 5 minutes)

Number of animals or in vitro replicates:

3 replicates for the test substance, positive and vehicle control

Details on study design:

Assembly of the corneas and the holders
The corneas were carefully examined macroscopically before their assembly in the holders, in order to detect the presence of any defects. Any corneas with defects were discarded. The corneas were then mounted in the corneal holders with the endothelial side against the O-ring of the posterior chamber. Each cornea was identified with the corresponding holder number.

Pre-incubation
For pre-incubation, both chambers of the corneal holder were filled to overflowing with MEM culture media supplemented with 1 % fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. The posterior chamber was always filled first to maintain the natural concave shape of the cornea. After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32 °C (± 1 °C). At the end of the pre-incubation period, the medium was removed from both chambers of the holder using a metal gavage tube attached to a vacuum pump to ensure complete evacuation. They were refilled with fresh cMEM (previously heated to +32 °C), starting with the posterior chamber and taking care that no air bubbles were present. The chambers were re-sealed and the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

Allocation of the corneas
The test substance formulation, the vehicle and positive controls were tested on three corneas each. The corneas were distributed as follows:
- the median value of the OPT0 values of all pre-incubated corneas (with OPT0 ≤ 7) was calculated,
- three corneas with opacity values close to the median value were selected as vehicle control corneas,
- the remaining corneas were shared out between test substance formulation and positive control-treated series using a manual distribution procedure.
To rigorously respect the treatment and incubation times, the three corneas from the same series were always processed in the same order at each step.

Treatment of corneas
The medium of the anterior chamber was removed and the test substance positive and vehicle controls were applied onto the epithelium of the cornea. The treatment time of each series of three corneas was carefully measured with a chronometer, starting from the beginning of treatment of the first cornea of each series. Then each further operation (rinsing, measurement, etc.) was carried out in the same order for the three corneas of each series. After application of the substances, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32 °C (± 1 °C), for the selected treatment time.

Rinsing of the corneas
The purpose of rinsing was to eliminate as much as possible residue of the test substance, the vehicle or the positive control, while taking care not to physically damage the cornea. On completion of the treatment period, the test substance formulation was removed from the front opening of the anterior chamber (open-chamber method) and the epithelium was rinsed as follows:
- any test substance formulation adhering to the walls of the anterior chamber was removed with a cotton bud and using a pipette of heated cMEM (+32 °C),
- the corneas were rinsed three times with pre-warmed cMEM containing phenol red (i.e. until the test substance formulation had been completely removed from the chamber or until the phenol red was not discolored). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.
The rinsing efficiency was visually confirmed by observing the transparency and the color changing of the rinsing medium (containing phenol red). Some difficulties were encountered during the rinsing since the test substance formulation adhered to the walls of the anterior chamber, but at the end of the rinsing procedure, it was successfully removed.
The anterior chamber was refilled with fresh pre-warmed cMEM without phenol red. The front cover was replaced. Care was taken to make sure that no air bubbles were present within the holders (by ensuring that each chamber was filled to overflowing with pre-warmed cMEM).
Following the 4-hour treatment, the medium of both chambers was renewed with pre-warmed cMEM (+32 °C) and the second opacity measurement (OPT2) was performed directly.

Opacity measurements
An opacitometer was used to measure light transmission (i.e. the level of opacity) through the center of each mounted cornea. A numerical opacity measurement (arbitrary unit) was displayed and recorded.
Corneal opacity was determined by reading each holder in the right hand chamber of the calibrated opacitometer, versus an empty holder (without cMEM, cornea and glasses), placed in the left hand chamber.

Permeability determination
After the second opacity measurement, the medium of the anterior chamber was removed and the anterior chamber received 1 mL of a fluorescein solution. As the test substance is a non-surfactant solid, the concentration of the fluorescein solution was 5 mg/mL.
Before use, the fluorescein solution was validated. For each series of three corneas, a chronometer started from the fluorescein solution application time of the first cornea of the series. The holders were incubated vertically (cornea positioned horizontally with the fluorescein-treated side uppermost) in a water bath at +32 °C (± 1 °C) for 90 minutes (± 5 minutes). At the end of incubation, the maximum volume of cMEM recoverable from the posterior chamber of each holder was transferred into an identified tube. The medium was homogenized prior to determination of OD490 nm, using single-use cuvettes (1 cm path length) and a spectrophotometer (cMEM used as the blank).

Macroscopic examination
After permeability determination, the corneas were removed from the holders and observed for opaque spots, other irregularities and any separation of the epithelium. Then, the corneas were discarded.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Opacity measurements

Value:

1.7

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 2

Irritation parameter:

in vitro irritation score

Value:

96

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

MACROSCOPIC OBSERVATIONS

- Fluorescein fixation was observed on vehicle control-treated corneas 3/3.
- Thickening of the corneas and fluorescein fixation were observed on the corneas treated with the test substance formulation 3/3.
- Opacity, fluorescein fixation and thickening of the corneas were observed on those treated with the positive control 3/3.

IN VITRO IRRITANCY SCORE

The individual and mean opacity values:
positive control: 105
vehicle control: 2
test substance: 1.7

Permeability values:
positive control: 140
vehicle control: 0.065
test substance: 96

With one exception (mean OD490 nm of the vehicle control corneas at 0.065 instead of below 0.0296), all acceptance criteria were fulfilled. The study was therefore considered as valid.
The mean In Vitro Irritancy Score (IVIS) of corneas treated with the test substance formulation was 96.
As the mean IVIS was > 55, the test substance was considered as a test chemical inducing serious eye damage (UN GHS Category 1).

Any other information on results incl. tables

EVALUATION

The measuring principle of an opacitometer and a photometer is basically the same one. The reduced transmission through the cornea is measured by the photometer. Transmission is the inverse of the opacity. The photometer gives the inverse value of the transmission as absorbance which is the common logarithm of the opacity. Based on these correlations, the opacity was calculated.

 

Calculation of Opacity Value

Opacity was calculated from the measured absorbance at 570 nm following

O = 10^A O = 1 / (10^(-E))

with

O = Opacity

A = Absorbance opacitometer at 570 nm

E = Absorbance photometer at 570 nm

The difference between opacity before and after exposition is used for further evaluation.

 

Correction of Measured Absorbance at 490 nm

As cuvettes with a path length of 0.2 cm are used in the measurement of the Fluorescein-Na solution in the spectral photometer, the path length must be corrected to 1 cm. Coefficient: 1/0.2 = 5; all absorbances were multiplied with this coefficient.

 

Calculation of IVIS (In Vitro Irritancy Score)

The IVIS of each replicate of the negative control was calculated from the following equation:

IVIS = opacity difference + (15 * corrected OD490 value)

The IVIS of each replicate of the positive control and of the test substance were calculated from the following equation:

IVIS = (opacity difference – mean opacity difference of the negative controls) + [15 * (corr. OD490 – mean corr. OD490 of the negative controls)]

Note: All calculations are performed with unrounded values. Therefore, re-calculation with rounded values may lead to slightly different results.

Applicant's summary and conclusion

Interpretation of results:

other: CLP

Remarks:

Category 1 based on CLP criteria

Conclusions:

Under the study conditions, the test substance was identified as inducing serious eye damage (UN GHS Category 1).

Executive summary:

A study was conducted to determine the corneal damage potential using a method based on OECD Guideline 437: Bovine Corneal Opacity and Permeability Test Method, in compliance with GLP. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes at 32°C. A single experiment was performed using three corneas for each treated series (test substance formulation, positive control and vehicle control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test substance formulation was applied at the concentration of 20% (w/v) in paraffin oil, in a single experiment using a treatment time of 4 hours and using the open-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. At the completion of the treatment period, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. A second opacity measurement was then performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the Optical Density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. Macroscopic examination thickening of the corneas and fluorescein fixation were observed on all the corneas treated with the test substance formulation.In vitroIrritancy Score with one exception (mean OD 490 nm of the vehicle control), all acceptance criteria were fulfilled. The study was therefore considered as valid. The meanIn vitroIrritancy Score (IVIS) of corneas treated with the test substance formulation was 96. As the mean IVIS was > 55, the test substance was considered as inducing serious eye damage (UN GHS Category 1) (Richez, 2016).