2-Propenoic acid, 2-hydroxyethyl ester, reaction products with O,O-bis(dialkyalkyl) hydrogen phosphorodithioate and dimethyl phosphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 July 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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)

Version / remarks:

09 October 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

liquid

Details on test material:

- Appearance/physical state: Light yellow liquid
- Storage conditions: Ambient temperature

Test animals / tissue source

Details on test animals or tissues and environmental conditions:

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).
- Eyes were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL test item

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three replicates

Details on study design:

PURPOSE OF THE TEST
- The Bovine Corneal Opacity and Permeability (BCOP) test is designed to identify test items that can induce serious eye damage (UN GHS Category 1) and to identify test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) using the corneas of eyes isolated from cattle. It is not recommended for the identification of test chemicals that should be classified as irritating to eyes (UN GHS Category 2 or Category 2A) or test chemicals that should be classified as mildly irritating to eyes (UN GHS Category 2B). The 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, corneal effects produced by the test item are assessed by quantitative measurements of changes in corneal opacity and permeability. Corneal opacity is measured quantitatively as the amount of light transmission through the cornea. Permeability is measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements are used to calculate an In Vitro Irritancy Score (IVIS), which is used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of a test item.
- The test method is based on the ICCVAM (2010). ICCVAM Recommended BCOP Test Method Protocol and the ICCVAM (2010). ICCVAM Test Method Evaluation Report which was initially developed from the INVITTOX protocol, 124. The INVITTOX protocol, 124 was based on the BCOP test method first reported by Gautheron et al (1992) and has been superseded by INVITTOX (2009) Protocol 127. The ocular irritancy potential of a test item is measured by its ability to induce opacity and increase permeability in an isolated bovine cornea.
- The effects are measured by decreased light transmission through the cornea (opacity), increased passage of sodium fluorescein dye through the cornea (permeability) and evaluation of fixed and sectioned cornea at the light microscopic level, if applicable.
- The opacity and permeability assessments of the cornea following exposure to a test item are considered individually, and also combined, to derive an In Vitro Irritancy Score, which is used to classify the irritancy level of the test item. Histological evaluation can be useful for identifying injury to tissue layers that do not result in significant opacity or increased permeability of the cornea or when a more complete characterization of corneal damage is needed. Collection of histological data will be conducted in accordance with current OECD guidance on the collection of eye tissues for histological evaluations and collection of data (OECD, 2018).

REFERENCE ITEM PREPARATION
- The negative control item, sodium chloride 0.9% w/v, was used as supplied.
- The positive control item, ethanol, was used as supplied.

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 °C for 65 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 (see Annex 2, attached). The average opacity for all corneas was calculated.
- Three corneas with opacity values close to the median value of all corneas were 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
- The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item 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 °C for 10 minutes.
- At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 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.
- The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
- After incubation the holders were removed from the incubator, the medium from both chambers was replaced with fresh complete EMEM and a final opacity reading was taken. Each cornea was visually observed.

APPLICATION OF SODIUM FLUORESCEIN
- Following the final 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 (4 mg/mL).
- The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes.

PERMEABILITY DETERMINATIONS
- After incubation the medium in the posterior chamber of each holder was decanted and retained.
- Media (360 μL) 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 In Vitro Irritancy Score = mean opacity value + (15 * mean permeability OD492 value)
- 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.

VISUAL OBSERVATION
- The condition of the cornea was visually assessed post-treatment and post-incubation.
CRITERIA FOR AN ACCEPTABLE TEST
- Neat ethanol 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 during the previous 12 months for the testing facility.
- Sodium chloride solution (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 the testing facility. The applicable data range is presented in Appendix 3 (attached).

MAJOR COMPUTERISED SYSTEMS
- Delta Building Monitoring System.
- Labtech LT-4500 microplate reader and LT-com software.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Main Test

Value:

10.2

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

CORNEAL OPACITY AND PERMEABILITY MEASUREMENTS
- Individual and mean corneal opacity measurements and individual and mean corneal permeability measurements are given in Appendix 1 (attached).

CORNEAL EPITHELIUM CONDITION
- The condition of each cornea is given in Appendix 2 (attached).
- The corneas treated with the test item had cloudy patches post treatment and post incubation.
- The corneas treated with the negative control item were clear post treatment and post incubation.
- The corneas treated with the positive control item were cloudy post treatment and post incubation.

IN VITRO IRRITANCY SCORE
- The In Vitro Irritancy Scores were determined to be 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control.

CRITERIA FOR AN ACCEPTABLE TEST
- Assay acceptance criteria are given in Appendix 3 (attached).
- The positive control In Vitro Irritancy Score was within the acceptance range. The positive control acceptance criterion was therefore satisfied.
- The negative control gave opacity and permeability values below the established upper limits. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

In Vitro Irritancy Scores were reported as 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control. Under the experimental conditions reported, no prediction of eye irritation could be made.

Executive summary:

GUIDELINE

The study was performed in accordance with OECD Guideline for the Testing of Chemicals No. 437 (updated 09 October 2017) “Bovine Corneal Opacity and Permeability Assay” and Method B.47 of Commission Regulation (EC) No 440/2008.

 

METHODS

The undiluted test item was applied for 10 minutes followed by an incubation period of 120 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).

 

RESULTS

In Vitro Irritancy Scores were reported as 10.2 for the test item, 0.1 for the negative control and 55.9 for the positive control.

CONCLUSION

Under the experimental conditions reported, no prediction of eye irritation could be made.