Cyclohexane, 1,4-bis(ethoxymethyl)-

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study according to current guidelines and under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

GLP compliance:

yes (incl. QA statement)

Remarks:

BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Test material

Test material information

Constituent 1

Reference substance name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

EC Number:

700-868-7

Cas Number:

54889-63-3

Molecular formula:

C12 H24 O2

IUPAC Name:

(1r,4r)-1,4-bis(ethoxymethyl)cyclohexane; (1s,4s)-1,4-bis(ethoxymethyl)cyclohexane

Details on test material:

- Name of test material (as cited in study report): Cyclohexane, 1,4-bis(ethoxymethyl)-
- Physical state: Liquid
- Analytical purity: 99.6 corrected area-%
- Lot/batch No.: 10474-11-115
- Stability under test conditions: The stability under storage conditions over the study period was guaranteed by the sponsor, and the sponsor holds this responsibility.
- Homogeneity: homogeneous by visual inspection
- pH-value: ca. 5 (undiluted test substance)

Test animals / tissue source

Species:

other: not applicable

Strain:

other: not applicable

Details on test animals or tissues and environmental conditions:

not applicable

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Amount / concentration applied:

750 μL of the undiluted test substance

Duration of treatment / exposure:

10 minutes followed by a 2-hours post-incubation period

Observation period (in vivo):

10 minutes followed by a 2-hours post-incubation period

Number of animals or in vitro replicates:

not applicable

Details on study design:

Preparation of the bovine corneas and measurement of initial corneal opacity:
Corneas were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour.
Afterwards, the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups.

Application of the test substance and washing:
Each treatment group (test substance, NC and PC) consisted of 3 corneas.
Before application the medium in the anterior chamber was removed using a syringe.
750 μL of the undiluted liquid test substance was applied into the anterior chamber using a pipette.
Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in deionized water (positive control, PC) using a pipette.
The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes. The test substance, NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red).

Post-exposure incubation for liquid test substances and surfactants:
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Measurement of final corneal opacity:
Before measurement, each cornea was observed visually and observations were recorded.
Final corneal opacity readings were taken for each cornea with an opacitometer.

Determination of permeability
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

Results and discussion

In vivo

Results

Irritation parameter:

other: IVIS

Basis:

mean

Time point:

other: 10 minutes followed by a 2-hours post-incubation period

Score:

5.7

Reversibility:

other: not applicable

Any other information on results incl. tables

The mean IVIS of the negative and positive controls was -0.2 and 151.8, respectively.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive or severely irritating.

Conclusions:

Based on the observed results and applying the evaluation criteria it was concluded, that Cyclohexane, 1,4-bis(ethoxymethyl)- does not cause ocular corrosion or severe irritation in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.
The test method according to the regulatory accepted protocol at the time of reporting does not allow for the evaluation of eye irritation. The result does not exclude an irritation potential of the test substance. For final assignment of a risk phrase at present, results from another study would be needed.