Bis(2-chloroethyl) ether

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 15-sept-2015 to 21-mar-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye 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:

Bis(2-chloroethyl) ether

EC Number:

203-870-1

EC Name:

Bis(2-chloroethyl) ether

Cas Number:

111-44-4

Molecular formula:

C4H8Cl2O

IUPAC Name:

1-chloro-2-(2-chloroethoxy)ethane

Test material form:

liquid

Specific details on test material used for the study:

TEST MATERIAL
- Name (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Batch No 20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%). Protected from light

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: TARAVIS KFT.
- Age at study initiation: 7 weeks old

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 30µL
- Concentration: 100% w/w

Duration of treatment / exposure:

10s

Observation period (in vivo):

30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse

Number of animals or in vitro replicates:

3 replicates

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. One small drop of 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.

NUMBER OF REPLICATES
Each treatment group and concurrent positive control consisted of three eyes.

NEGATIVE CONTROL USED
Negative control eye was treated with 30 μL of physiological saline

POSITIVE CONTROL USED
Positive control eyes were treated with 30 μL 5% (w/v) Benzalkonium chloride solution.

APPLICATION DOSE AND EXPOSURE TIME
30 μL of test item was applied to the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance. The exposure time was 10s.

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual the test item if possible
- Indicate any deviation from test procedure in the Guideline: no deviation

SCORING SYSTEM:
- Mean corneal swelling (%)
- Mean maximum opacity score
- Mean fluorescein retention score at 30 minutes post-treatment

DECISION CRITERIA: The decision criteria as indicated in the TG was used.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

at up to 75 min

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 4

Irritation parameter:

cornea opacity score

Value:

0.17

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Based on an in vitro eye irritation study on isolated chicken’s eyes performed according to according to the OECD Guideline No. 438 under GLP, Bis(2-chloroethyl) ether did not induce significant eye irritation. The substance is not classified as irritating to the eye according to GHS criteria.

Executive summary:

An in vitro eye irritation study was performed in isolated chicken’s eyes on Bis(2-chloroethyl) ether according to the OECD Guideline No. 438 under GLP.

After the zero reference measurements, the eye was held in horizontal position and 30 μL of test item was applied onto the centre of the cornea such that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. The positive control eyes were treated with 30 μL benzalkonium chloride solution. The negative control eye was treated with 30 μL of physiological saline (9% (w/v) NaCl solution). In the study, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were in good correlation with the historical control data. Thus, the experiment was considered to be valid.

No significant corneal swelling was observed during the four-hour observation period on test item treated eyes. Very slight corneal opacity change (severity 0.5) was noted on one eye. Fluorescein retention change (severity 0.5) was noted on one eye. No other corneal effect was observed. Based on this in vitro eye irritation in the isolated chicken eyes test with 2,2`- Dichlorodiethyl ether, the test item is not irritant to the eye.