Dioctyl maleate, branched

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 October 2017 to 05 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 437 (adopted 26 July 2013) and Method B47 of Council Regulation (EC) No 440/2008.

Deviations:

no

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)

Version / remarks:

The study was conducted to meet the known requirements of OECD Guidelines for Testing of Chemicals Method 437 (adopted 26 July 2013) and Method B47 of Council Regulation (EC) No 440/2008.

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Dioctyl maleate, branched

EC Number:

287-673-6

EC Name:

Dioctyl maleate, branched

Cas Number:

85566-63-8

Molecular formula:

C20H36O4

IUPAC Name:

1,4-bis(octan-2-yl) (2Z)-but-2-enedioate

Specific details on test material used for the study:

The test article, a clear colourless liquid, was identified as Bernel Ester DCM and was received at Covance as follows:
Test Article: Bernel Ester DCM
CAS Number: 85566-63-8
Storage: 15-25oC, protected from light
Purity: 92.78%, assumed 100% for testing

A certificate of analysis for the test article was provided by the Sponsor and is presented in the Attachments.

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System and Study Design

Specification
Corneas from bovine eyes were obtained from a local abattoir. The eyes were removed after slaughter, completely immersed in Hanks’ Balanced Salt Solution (containing penicillin at 100 IU/mL and streptomycin at 100 µg/mL) in a suitably sized container and transported on the same day to the testing facility.

Assessment on Arrival
On arrival at the test facility the eyes were carefully examined for defects including increased opacity, scratches and neovascularisation. Only corneas free from such defects were used.

Excision and Preparation of Corneas
Upon arrival at the test facility, the corneas were excised from the eyes and loaded onto the specifically designed holders. Both chambers of each holder were filled with pre-warmed Minimal Essential Medium (MEM), with the posterior chamber filled first, ensuring that no bubbles were formed. The holders were incubated at 32±1 °C for at least 1 hour. After the incubation, the media was removed from both the anterior and posterior chambers. Fresh media was added to the posterior chamber first and then the anterior chamber (this media replacement order ensured the cornea retained its natural curvature as much as possible). The opacity of each cornea was measured using an opacitometer. Any corneas found to have scratches or increased neovascularization or an opacity of >7 opacity units when examined prior to treatment were discarded.

Test system

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

750 µl or sufficient to completely cover the cornea

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Opacity: 2 hours
Permeability: 1 hour 25 minutes

Number of animals or in vitro replicates:

3

Details on study design:

Treatment of Corneas
A volume of 750 µL of the test article (or enough test article to completely cover the cornea) was applied to each of three corneas followed by a ten minute incubation at 32 °C. After this incubation, each cornea was washed with media containing phenol red (as a pH indicator) until this indicator showed no pH effect occurring (and demonstrating that the test article had been removed successfully). The corneas were then washed once in media without phenol red before being incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied. For the permeability endpoint, 1 mL of sodium fluorescein (4 mg/mL solution) was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour and 25 minutes. Following this period, the media in the posterior chamber was removed and held in a labelled tube. Three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD490) using a spectrophotometer.
A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas. These groups were subject to the procedures detailed above.

Terminal Procedures
At the end of the assay all corneas were preserved in 10% Neutral Buffered Formalin. However, the Sponsor confirmed that microscopic examination was not required. The corneas were discarded on finalisation of the study report.

Data Evaluation
The In Vitro Irritation Score (IVIS) was determined as follows:
IVIS = mean opacity value + (15 x mean permeability value).
The opacity and mean permeability (OD490) values are corrected for background opacity and the negative control OD490 values.

Decision Criteria
The test article is concluded as inducing serious eye damage if the IVIS is >55.
The test article is concluded as not requiring classification for eye irritation if the IVIS is ≤3.
No prediction can be made if the IVIS is >3 but ≤55.

Assay Acceptance Criteria
The assay was considered valid if the following criteria are met:
The positive control yields an IVIS within 2 standard deviations of the historical control mean.
The negative control yields opacity and permeability values that are less than the established upper limits for these endpoints for bovine corneas as treated at the testing facility.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean value

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

opacity reading of 0.0

Positive controls validity:

valid

Remarks:

opacity reading of 58.7

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

fluorescein leakage

Run / experiment:

Mean value

Value:

0.002

Vehicle controls validity:

valid

Negative controls validity:

valid

Remarks:

The mean group corrected optical density for the negative control was 0.000

Positive controls validity:

valid

Remarks:

The mean group corrected optical density for the positive control was 0.277.

Remarks on result:

no indication of irritation

Any other information on results incl. tables

Corneal Opacity

Results are presented in Table 8.1.

The mean corrected opacity reading for the test article was -1.0.

The mean corrected opacity reading for the negative control was 0.0.

The mean corrected opacity reading for the positive control was 58.7.

The corneas treated with the test article were noted to be clear and unchanged following treatment. The corneas treated with the positive control were noted to be cloudy and wrinkled following treatment.

Corneal Permeability

Results are presented in Table 8.2.

The mean group corrected optical density for the test article was 0.002.

The mean group corrected optical density for the negative control was 0.000.

The mean group corrected optical density for the positive control was 0.277.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that Bernel Ester DCM, produced an IVIS score of -0.97 and therefore does not require classification for eye irritation.
The assay was considered valid as the assay acceptance criteria were met.

Executive summary:

This study was conducted to determine whether the test article, Bernel Ester DCM, causes serious eye damage or does not require classifying for eye irritation, using the bovine corneal opacity and permeability (BCOP) assay.

A volume of 750 µL of the undiluted test articlewas applied to each of three corneas followed by a 10 minute incubation at 32±1°C. After this incubation, each cornea was washed with media containing phenol red followed by media without phenol red. The corneas were then incubated (horizontally) for 2 hours after which, corneal opacity was measured and then the anterior chamber emptied.

For the permeability endpoint, sodium fluorescein solution was added into the anterior chamber and the corneas were incubated in the vertical position for 1 hour 25 minutes at 32±1°C. Following this period, the media in the posterior chamber was removed and three 350 μL aliquots of this media (per cornea) were analysed for optical density at 490 nanometers (OD₄₉₀).

A volume of 750 µL of the negative or positive control was similarly applied to further groups of three corneas. These corneas were subject to the procedures detailed above.

The test article, Bernel Ester DCM, produced an IVIS score of -0.97 and therefore does not require classification for eye irritation.