Diboron calcium tetraoxide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 December 2019 to 10 March 2020

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

Test guideline 1

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)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Guidance Document on ‘The Collection of Tissues for Historical Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160. Adopted July 6, 2018 Paris

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reference substance 001

Cas Number:

13701-64-9

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: E00616-912
- Expiration date of the lot/batch: 18 September 2020
- Purity: 82 %

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

TEST SYSTEM:
- Source of Bovine Eyes:
Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter (per experiment). The corneas were prepared immediately on arrival.

Test system

Vehicle:

other: Runs 1 and 2: 20 % w/v concentration in a 0.9 % sodium chloride solution; runs 3 and 4: neat solid; runs 5 and 6: 40 % w/v concentration in a 0.9 % sodium chloride solution

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied runs 1, 2, 5 and 6: 0.75 mL/cornea
- Amount(s) applied runs 3 and 4: 0.6 g/cornea
- Concentration runs 1 and 2: 20 % w/v solution in sodium chloride 0.9 % w/v
- Concentration runs 3 and 4: Not applicable (neat solid)
- Concentration runs 5 and 6: 40 % w/v solution in sodium chloride 0.9 % w/v

Duration of treatment / exposure:

4 hours

Number of animals or in vitro replicates:

Triplicates for each run

Details on study design:

STUDY DESIGN:
- Preparation of Corneas:
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used. The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for >=60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

- Selection of Corneas and Opacity Reading:
The medium from both chambers of each holder was replaced with fresh complete EMEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. Three corneas were randomly allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

- Treatment of Corneas:
For runs 1, 2, 5 and 6 the EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

For runs 3 and 4 the EMEM was removed from the anterior chamber of the BCOP holder and the test item or control items were applied to the cornea. Approximately 0.6 g of the solid test item was found to adequately cover the corneal surface. 0.75mL of each control item was applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.

At the end of the 4-hour exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed at least three times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.

- Application of Sodium Fluorescein:
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost at 32 ± 1 ºC for 90 minutes.

- Permeability Determinations:
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of media representing each cornea was dispensed into the appropriate wells of a pre-labelled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.

- Histopathology:
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10 % neutral buffered formalin.

DATA EVALUATION:
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

- Opacity Measurement:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.

- Permeability Measurement:
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.

- In Vitro Irritancy Score:
The following formula was used to determine the In Vitro Irritancy Score. Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)

- Visual Observation:
The condition of the cornea was visually assessed post treatment.

CRITERIA FOR AN ACCEPTABLE TEST:
For an acceptable test the following positive control criterion should be achieved. 20 % w/v Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during the previous 12 months for this testing facility.

For an acceptable test the following negative control criteria should be achieved. Sodium chloride 0.9 % w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values calculated from the previous 12 months data for this testing facility.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

109.9

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

large standard deviation in opacity readings

Results 2

Irritation parameter:

in vitro irritation score

Run / experiment:

2

Value:

32.2

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Results 3

Irritation parameter:

in vitro irritation score

Run / experiment:

3

Value:

4.6

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

4

Value:

2.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 5

Irritation parameter:

in vitro irritation score

Run / experiment:

5

Value:

7.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Results 6

Irritation parameter:

in vitro irritation score

Run / experiment:

6

Value:

14

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Corneal Epithelium Condition:
When the test item was applied as a 20 % w/v formulation the corneas had opaque patches/cloudiness post treatment. When the test item was applied neat the corneas had small areas of test item adherence post treatment. When the test item was applied as a slurry of 40 % w/v formulation the corneas had partial cloudiness post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes

Any other information on results incl. tables

Across the three corneas exposed to the test item a large standard deviation in opacity was noted in run 1 (74.7, 204.7, 31.7), and therefore, even though there appeared to be evidence of severe eye irritation in two of the three corneas, the discordant results could not be used as sufficient evidence to classify the test item (IVIS score 109.9). The results obtained in the second run show a lower prediction and overall lower IVIS value (IVIS score 32.2) than in the first run; however, there were still relatively large standard deviations between individual opacity readings (37.3, 16.3, 39.3) indicating that there may have been an issue with an uneven distribution of the test item when suspended at 20 % w/v in saline. Owing to the lack of reproducibility of responses over the first two BCOP runs it was difficult to make a conclusion regarding classification of the substance at this time.

Consequently, the test item was applied neat to the corneas. After applying the test item neat in runs 3 and 4 it would be expected that this method would have exhibited the most severe eye irritation response. However, the results did not align with this prediction and it was apparent that the irritation potential was reduced using this method (run 3 IVIS score 4.6 and run 4 IVIS score 2.1). However, the test item adhered to the corneas, which in turn could have affected the reliability of the opacity readings and consequently the IVIS score, although it is anticipated that this would result in a higher IVIS score. The lowered IVIS score in runs 3 and 4 implied that the test item had the potential to cause irritation with the presence of moisture (when suspended as a formulation), as seen in runs 1 and 2.

In runs 5 and 6 it became important to exhibit the irritation potential of the test item using a slurry/open chamber methodology. The application of the test item slurry to the cornea ensured that sufficient moisture was present to allow any potential chemical reaction to take place and most importantly ensure an even distribution of the formulation across the corneal surface. Also the benefit of this method was shown to prevent test item adherence at the washing phase, which in turn improved the reliability of the opacity readings (run 5 IVIS score 7.4 and run 6 IVIS score 14.0).

With the range of results obtained over the course of six BCOP runs, it was apparent that the most appropriate classification would be ‘No Prediction’ under UN GHS classification. As a neat substance, minimal irritation effects were observed (run 3 IVIS score 4.6 and run 4 IVIS score 2.1); however, the test item adhered to the corneas, potentially impacting the scores. Increased irritation effects were observed in runs 5 and 6, although these effects were close to the UN GHS IVIS classification threshold of 3. The results of runs 5 and 6 highlight potential increased irritation of the test item when in contact with moisture. Therefore, it was considered appropriate that further testing would be required to reach a classification using other appropriate testing methods.

Applicant's summary and conclusion

Interpretation of results:

other: No prediction can be made

Conclusions:

Run 1:
Inconclusive - No conclusion can be made.

Runs 2, 3, 5 and 6:
No prediction of eye irritation can be made.

Run 4:
No category. Not requiring classification to UN GHS.

Under the conditions of the test, the overall conclusion is that the test item is given No Prediction under UN GHS classification.

Executive summary:

The purpose of this test was to identify test items that can induce serious eye damage and to identify test items not requiring classification for eye irritation or serious eye damage. The Bovine Corneal Opacity and Permeability (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro. In this test method, damage by the test item is assessed by quantitative measurements of changes in corneal opacity and permeability. The test method can correctly identify test items (both chemicals and mixtures) inducing serious eye damage, as well as those not requiring classification for eye irritation or serious eye damage, as defined by the United Nations (UN) Globally Harmonized System of Classification and Labelling of Items (GHS). Test items inducing serious eye damage are classified as UN GHS Category 1. Items not classified for eye irritation or serious eye damage are defined as those that do not meet the requirements for classification as UN GHS Category 1 or 2 (2A or 2B), i.e. they are referred to as UN GHS No Category.

In runs 1 and 2 the test item was applied at a concentration of 20 % w/v in sodium chloride 0.9 % w/v for 240 minutes. In runs 3 and 4 the test item was applied neat for 240 minutes. In runs 5 and 6 the test item was applied at a concentration of 40 % w/v in sodium chloride 0.9 % w/v for 240 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability), were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

Under the conditions of the test, the overall conclusion is that the test item is given No Prediction under UN GHS classification.

Summarised below are the In Vitro Irritancy Scores and conclusions of each run:

Run 1 - 20 % saline

Treatment	In Vitro Irritancy Score
Test Item	109.9
Negative Control	0.4
Positive Control	97.3

Run 2 - 20 % saline

Treatment	In Vitro Irritancy Score
Test Item	32.2
Negative Control	0.7
Positive Control	97.3

Run 3 - Neat

Treatment	In Vitro Irritancy Score
Test Item	4.6
Negative Control	1.4
Positive Control	97.6

Run 4 - Neat

Treatment	In Vitro Irritancy Score
Test Item	2.1
Negative Control	1.8
Positive Control	98.9

Run 5 - 40 % w/v saline

Treatment	In Vitro Irritancy Score
Test Item	7.4
Negative Control	1.8
Positive Control	98.9

Run 6 - 40 % w/v saline

Treatment	In Vitro Irritancy Score
Test Item	14.0
Negative Control	1.8
Positive Control	98.9

Run 1:

Inconclusive - No conclusion can be made.

Runs 2, 3, 5 and 6:

No prediction of eye irritation can be made.

Run 4:

No category. Not requiring classification to UN GHS.