3,5-DICARBOXYPHENYLBORONIC ACID

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

5-(dihydroxyboranyl)benzene-1,3-dicarboxylic acid

Cas Number:

881302-73-4

Molecular formula:

C8H7BO6

IUPAC Name:

5-(dihydroxyboranyl)benzene-1,3-dicarboxylic acid

Test material form:

solid

Test animals / tissue source

Species:

chicken

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Source, collection and transport of chicken eyes:
The eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption have been used for this assay.
The age and weight of the chickens used in this test method are that of spring chickens traditionally processed by a poultry slaughterhouse (i.e., approximately 7 weeks old, 1.5 - 2.5 kg).
Heads have been removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. The heads have been collected on 20 July 2020 at 8:20 am.
Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with
physiological saline.
The eyes were enucleated at Phycher on 20 July 2020 at 9:40 am.

Preparation of the eyes:
The eyelids were carefully excised, taking care not to damage the cornea. Then, the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 31.9°C and 32.0°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged during the dissection procedure. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these
criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.
Once all eyes had been examined and approved (see table in appendix 4), the eyes were incubated between 55 and 75 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (i.e., time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg

Duration of treatment / exposure:

10 seconds to the cornea such that the entire surface of the cornea was evenly covered with the test item.
Then the eyes were rinsed twice with 10 mL of physiological saline at ambient temperature.

Duration of post- treatment incubation (in vitro):

Treated corneas were evaluated pretreatment and starting at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

Number of animals or in vitro replicates:

3 / test item
1 / negative control
3 / positive control

Details on study design:

All observations of the cornea and measurement of corneal thickness were performed using a Haag-Streit BP900 slit-lamp microscope with depth-measuring device no. I. For the measurement of corneal thickness, the slit-width was set at 9½, equalling 0.095 mm. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects (e.g., pitting or loosening of the epithelium). All of the endpoints, with the exception of fluorescein retention (which was determined only at pretreatment and 30 minutes after test item exposure) were determined at each of the above time points.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Remarks:

mean of three eyes

Run / experiment:

eye n°7-8-9

Value:

ca. 0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

fluorescein retention score

Remarks:

mean of three eyes

Run / experiment:

eye n°7-8-9

Value:

ca. 1.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Results 3

Irritation parameter:

corneal swelling 

Remarks:

mean of three eyes

Run / experiment:

eye n°7-8-9

Value:

ca. 16

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

No morphological effects were noted, whatever the examination time.

The ocular reactions observed in eyes treated with the test item were:
- maximal mean score of corneal opacity: 0.2, corresponding to ICE class I;
- mean score of fluorescein retention: 1.7, corresponding to ICE class III;
- maximal mean corneal swelling: 16%, corresponding to ICE class II.
The combination of the three endpoints for the test item 3,5-Dicarboxybenzeneboronic acid was 1 x III, 1 x II, 1 x I.
The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.
The combination of the three endpoints for the negative control, physiological saline, was 3 x I.
Therefore, the negative control is classified as “No Category”, as expected.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item 3,5-Dicarboxybenzeneboronic acid is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.

Executive summary:

The aim of the study was to evaluate the possible ocular corrosive or severe irritating effects of the test item after administration on enucleated chicken eyes.

The test item 3,5-Dicarboxybenzeneboronic acid was applied, as supplied, at the dose of 30 mg, to 3 enucleated chicken eyes, during 10 seconds. Then the eyes were rinsed twice with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control and one eye with a negative control. Damages by the test item were assessed by determination of corneal swelling, opacity, and fluorescein retention at 30, 75, 120, 180 and 240 minutes post-dose.

The experimental protocol was established in accordance with the O.E.C.D. Test Guideline No. 438 adopted 28 June 2018.

The ocular reactions observed in eyes treated with the test item were:

- maximal mean score of corneal opacity: 0.2, corresponding to ICE class I;

- mean score of fluorescein retention: 1.7, corresponding to ICE class III;

- maximal mean corneal swelling: 16%, corresponding to ICE class II.

The combination of the three endpoints for the test item 3,5-Dicarboxybenzeneboronic acid was 1 x III, 1 x II, 1 x I.

The combination of the three endpoints for the positive control, Sodium hydroxide, was 3 x IV. Therefore, the positive control is classified as “Corrosive/Severe Irritant”, as expected.

The combination of the three endpoints for the negative control, physiological saline, was 3 x I. Therefore, the negative control is classified as “No Category”, as expected.

In accordance with the Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions lead to the category “no prediction can be made”, as defined by the OECD guideline No.438. Therefore, the test item 3,5-Dicarboxybenzeneboronic acid is not predicted as causing serious eye damage (Category 1) or as not classified for eye irritation/serious eye damage (No category) with the Isolated Chicken Eye test method. Additional testing (in vitro and/or in vivo) are required to establish a definitive classification.