1,1-diethoxyethane

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

from 2018-08-10 to 2018-08-17

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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

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

Test material information

Constituent 1

Reference substance name:

1,1-diethoxyethane

EC Number:

203-310-6

EC Name:

1,1-diethoxyethane

Cas Number:

105-57-7

Molecular formula:

C6H14O2

IUPAC Name:

1,1-diethoxyethane

Test material form:

liquid

Test animals / tissue source

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

- Source: Freshly isolated bovine eyes of cattle were collected from the slaughterhouse, Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age of the cattle: 15 - 53 months
- Corneal diameter: 25 - 28 mm

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 750 µL
- Concentration: The liquid test item (Acetal) was tested undiluted

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

Incubation for 120 minutes after treatment. For permeability determination corneas were incubated for additional 90 minutes.

Number of animals or in vitro replicates:

Three corneas were used per group (negative control, positive control or test item group).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Freshly isolated corneas were used. Excess tissue was removed from the eyes. The eyes were kept and transported in transport medium cooled on ice. The corneas were prepared immediately after delivery of the eyes to the laboratory. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (pre-warmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded. Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : 0.9 % sodium chloride solution

POSITIVE CONTROL USED : N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME : 750 µL of undiluted liquid test item for 10 minutes

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: washed at least three times with wash medium, final rinse with incubation medium
- POST-EXPOSURE INCUBATION: Incubation for 120 minutes after treatment. For permeability determination corneas were incubated for additional 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values. From the individual corrected opacity values, a mean corrected opacity value was calculated for each group.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) were
<3 No Category
>3; <55 No prediction can be made
>55 Category 1

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

1

Value:

62.8

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:

After treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.3 - 5.5). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 100.0 (study acceptance criteria range: 77.1 - 119.4). Therefore, the study fulfilled the acceptance criteria.

Any other information on results incl. tables

Table 1: The IVIS obtained after treatment

		Opacity	Permeability	IVIS
				per cornea	per group (mean value)	Standard deviation
Negative control	0.9 % sodium chloride solution	2.1	0	2.1	1.1	0.9
		0.3	-0.001	0.285
		0.9	0.002	0.93
Positive control	N,N-dimethyl-formamide	90.5	0.715	101.225	100	4.5
		74.7	1.94	103.8
		79.3	1.051	95.065
Test item	Art. W200220	33.4	1.768	59.92	62.8	3.1
		31.2	2.322	66.03
		27.2	2.359	62.585

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In a study accoring to OECD TG and EU Method the test item was determined to be eye irritating (Category 1 (irreversible effects on the eye) based on GHS criteria).

Executive summary:

To assess the ocular corrosiveness and eye irritating properties a GLP-compliant Bovine Corneal Opacity and Permeability (BCOP) test according to OECD Guideline 437 and EU method B.47 was carried out. Corneas were obtained from fresh isolated bovine eyes of cattle. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. Isolated cornea where observed visually, and pertinent observations were recorded (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns). Three corneas were used per group (negative control with 0.9% sodium chloride solution, positive control with N,N-dimethylformamide or test item group). The test item preparation, the negative and positive control preparations were introduced with the closed-chamber method through the dosing holes of the chamber. 750 µL of either the test item, negative or positive control were administered. After application, the corneas were incubated in an incubator in a horizontal position at 32 ± 1 °C for 10 minutes. Subsequently, corneal surfaces were washed at least three times with wash medium and finally with incubation medium. Fresh incubation medium was replaced in both compartments and the corneas were incubated for additional 120 minutes at 32 ± °C.

Opacity was measured by an opacitometer, permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically (OD490, microplate reader). In result, after treatment with the negative control (0.9 % sodium chloride solution) the calculated IVIS was 1.1 (study acceptance criteria range: -1.3 - 5.5). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 100.0 (study acceptance criteria range: 77.1 - 119.4). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was 62.8 and, thus higher than 55, i.e. according to OECD 437 the test item is inducing serious eye damage (UN GHS: Category 1). All validity criteria of the guidelines were fulfilled.