1,3-dioxolane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 April 2020 - 06 April 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

1,3-dioxolane

EC Number:

211-463-5

EC Name:

1,3-dioxolane

Cas Number:

646-06-0

Molecular formula:

C3H6O2

IUPAC Name:

1,3-dioxolane

Test material form:

liquid

Details on test material:

- Name of test material (as cited in study report): 1,3-dioxolane- Physical state: liquid- Analytical purity: 99.9986%- Lot/batch No.: 2003031300R

Specific details on test material used for the study:

Clear colourless liquid

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: Lambiotte & Cie S.A. 2003031300R
- Expiration date of the lot/batch: 03 March 2022
- Storage condition of test material: Room temperature in the dark

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Local abattoir; excised by an abattoir employee after slaughter
- Characteristics of donor animals (e.g. age, sex, weight): Adult cattle (typically 12 to 60 months old)
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics; transported to the test facility over ice packs on the same day of slaughter
- Time interval prior to initiating testing: corneas were prepared approximately 1 hour after arrival
- Indication of any antibiotics used: penicillin at 100 IU/mL and streptomycin at 100 µg/mL

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Three

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
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 65 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
NUMBER OF REPLICATES
3
NEGATIVE CONTROL USED
Sodium chloride (CAS 7647-14-5) 0.9% w/v in water
Purity: 0.9%
SOLVENT CONTROL USED (if applicable)
Not applicable
POSITIVE CONTROL USED
Ethanol (CAS 64-17-5)
Purity: > 99.8%
APPLICATION DOSE AND EXPOSURE TIME
0.75 mL - 10 minutes
TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

POST-EXPOSURE INCUBATION: 120 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: calibrated opacitometer
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD492)
- Corneal Epithelium Condition

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: For an acceptable test the following positive control criterion should be achieved:
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 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

Irritation parameter:

in vitro irritation score

Value:

ca. 115.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS: Visible damage on test system: The corneas treated with the test item were cloudy 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control responses should result in opacity and permeability values that are less than or equal to the upper limits established from the previous 12 months historical data set. When testing liquids the negative control upper limit for opacity should be ≤ 0.7 and for permeability ≤ 0.021.
- Acceptance criteria met for positive control: The assay will be accepted if the positive control produces an IVIS which falls within two standard deviations of the current historical mean. For liquids, the IVIS should fall within the range 36.4 to 61.5
- Outliers: positive control: When testing liquids, outliers are identified as having an IVIS ≤ 30.1 or ≥ 67.8; negative control: When testing liquids, outliers are identified as having an opacity ≥ 1.0 and/or a permeability ≥ 0.029

Any other information on results incl. tables

Treatment	In Vitro Irritancy Score
Test item	115.7
Negative Control	0.1
Positive Control	41.1

Individual and Mean Corneal Opacity and Permeability Measurements

Treatment	Cornea Number	Opacity					Permeability (OD492)		In Vitro Irritancy Score
		Pre-Treatment	Post-Treatment	Post Incubation	Post-Incubation - Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	2	5	4	5	0		0.001
	3	5	5	5	0		0.009
	6	6	6	6	0		0.001
					0.0*		0.004¨		0.1
Positive Control	9	7	35	34	27	27.0	1.087	1.083
	11	5	32	32	27	27.0	0.713	0.709
	12	4	29	32	28	28.0	0.958	0.954
						27.3·		0.916·	41.1
Test Item	14	5	96	112	107	107.0	0.987	0.983
	15	5	95	111	106	106.0	1.218	1.214
	16	5	74	89	84	84.0	1.153	1.149
						99.0·		1.116·	115.7

 

OD = Optical density          * = Mean of the post-incubation - pre‑treatment values         ¨ = Mean permeability                            · = Mean corrected value

 

Corneal Epithelium Condition Post Treatment and Post Incubation

Treatment	Cornea Number	Observation
		Post Treatment	Post Incubation
Negative Control	2	Clear	Clear
	3	Clear	Clear
	6	Clear	Clear
Positive Control	9	Cloudy	Cloudy
	11	Cloudy	Cloudy
	12	Cloudy	Cloudy
Test Item	14	Cloudy	Cloudy
	15	Cloudy	Cloudy
	16	Cloudy	Cloudy

Applicant's summary and conclusion

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

According to UN GHS Classification, 1,3-Dioxolane causes serious eye damage and is classified as Category 1 under the conditions of the test.

Executive summary:

Introduction

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.

Method

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).

Data Interpretation

The test item is classified according to the prediction model as follows:

IVIS	UN GHS
≤ 3	No Category
>3; ≤ 55	No prediction can be made
> 55	Category 1

Results

The In Vitro irritancy scores are summarized as follows:

Treatment	In Vitro Irritancy Score
Test Item	115.7
Negative Control	0.1
Positive Control	41.1