1,2-Ethandiylbis(oxy-2,1-ethandiyl)-dimethansulfonat

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

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)

Version / remarks:

From 25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Version / remarks:

Amendment Commission Regulation (EC) No 2017/735, dated 14 February 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

Cas Number:

80322-82-3

Molecular formula:

C8H18O8S2

IUPAC Name:

2-[2-(2-hydroxyethoxy)ethoxy]ethanol,methanesulfonic acid

impurity 1

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

impurity 2

Reference substance name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

IUPAC Name:

1,2-Ethandiylbis(oxy-2,1-ethandiyl)-monomethanesulfonat

Test material form:

liquid

Details on test material:

Appearance: Dark yellow liquid
Batch No.: 01110008
Storage: -5 – 40 °C, container tightly closed, protected from light

Specific details on test material used for the study:

Batch No.: 01110008
Storage: At room temperature (max 40 °C), protected from light

Test animals / tissue source

Species:

chicken

Strain:

other: COBB 500

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni utca 129. Hungary
- Storage, temperature and transport conditions of ocular tissue: Only the eyes of healthy animals considered suitable for entry into the human food chain were used. Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was performed by electric current). The heads were transported to Toxi-Coop ZRT. at the earliest convenience for use approximately within 2 hours from collection. The ambient temperature was optimal (20.3 ºC to 21.2 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
After collection, the heads were visually inspected for appropriate quality and wrapped with paper moistened with saline, then placed in a plastic box that can be closed (4-5 heads/box).
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. Cornea integrity was checked by applying one small drop of fluorescein 2 % (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL

POSITIVE AND NEGATIVE CONTROL
- Positive control: 30 µL
- Negative control: 30 µL

Duration of treatment / exposure:

10 seconds

Number of animals or in vitro replicates:

Test item and positive control samples: Three replicates
Negative control samples: One replicate

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
- Eyes selection
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. Cornea integrity was checked by applying one small drop of fluorescein 2 % (w/v) solution onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL isotonic saline. Then the fluorescein-treated cornea was examined with 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.

- Preparation of eyes
The eyeball was carefully removed from the orbit by holding the nictitating membrane with surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye 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.

- Eyes examination and acclimatization time
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, avoiding too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were selected and after being placed in the superfusion apparatus, the selected eyes were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the isotonic 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 using the depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equaling 0.095 mm. 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 was conducted for approximately 45 to 60 minutes. The temperature was verified to be in the range of 32 ± 1.5 °C in all chambers during the acclimatization and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than ± 5-7 % within approximately 45 to 60 minutes before the start of application. Slight changes in thickness (0 % to 2 %) were observed in the eyes, finding considered as normal when maintaining enucleated eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. None of the eyes were discarded as no eye was considered unsuitable after the baseline assessment.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position and 30 µL of test item was applied from micropipette onto the center of the cornea, taking care not to damage or touch the cornea with the application equipment. This procedure was repeated with the remaining two test item treated eyes.
The three positive control eyes were treated with (30 µL/eye) benzalkonium chloride solution (5 %) and one negative control eye was treated with 30 µL isotonic saline [NaCl (9 g/L saline)] according to the above procedure.

OBSERVATION PERIOD
The control 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 ± 5 minutes were considered acceptable.
The cornea thickness was measured and cornea opacity and possible morphological effects were examined at all time points. Fluorescein retention was determined at baseline (t = 0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
The time of application was monitored, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL isotonic saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove all the residual test item if possible. The eye in the holder was then returned to its chamber. The time while the eye was out of the chamber was limited to the minimum.
The cornea surface of negative and positive control treated corneas was also rinsed thoroughly after an exposure period of 10 seconds with 20 mL isotonic saline solution at ambient temperature, while taking care not to damage the corneas.

DECISION CRITERIA:
The conclusion on eye irritancy was based on the criteria given in OECD 438 guideline on quantitative assessments.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum corneal swelling up to 75 min

Run / experiment:

mean

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Maximum corneal swelling at up to 75 min: 0 %

Positive controls validity:

valid

Remarks:

Mean maximum corneal swelling at up to 75 min: 25 %

Remarks on result:

other: ICE Class I

Results 2

Irritation parameter:

percent corneal swelling

Remarks:

mean maximum corneal swelling up to 240 min

Run / experiment:

mean

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Maximum corneal swelling at up to 240 min: 0 %

Positive controls validity:

valid

Remarks:

Mean maximum corneal swelling at up to 240 min: 42%

Remarks on result:

other: ICE Class I

Results 3

Irritation parameter:

cornea opacity score

Remarks:

mean maximum corneal opacity

Run / experiment:

mean

Value:

0.2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Maximum corneal opacity: 0.0

Positive controls validity:

valid

Remarks:

Mean maximum corneal opacity: 4.0

Remarks on result:

other: ICE Class I

Results 4

Irritation parameter:

fluorescein retention score

Remarks:

Mean fluorescein retention

Run / experiment:

mean

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

Fluorescein retention: 0.0

Positive controls validity:

valid

Remarks:

Mean fluorescein retention: 3.0

Remarks on result:

other: ICE Class I

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
- Acceptance criteria met for positive control: The positive control Benzalkonium chloride solution 5 % (w/w) solution was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Applicant's summary and conclusion

Interpretation of results:

other: No Category

Conclusions:

In this ICET, the overall ICE classes were thrice I. According to the guideline OECD 438, Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is categorized as “No Category”.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item Triethylene glycol dimethanesulfonate (CAS 80322-82-3) by its ability to induce toxicity in enucleated chicken eyes. The test item was applied in a single dose (30 μL/eye) onto the cornea of isolated chicken eyes in order to potentially classify the test compound as either 1: causing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or 2: not requiring classification for eye irritation or serious eye damage according to the GHS. Tested corneas were evaluated pre-treatment and at approximately 30, 75, 120, 180, and 240 minutes after the post-treatment rinse. The endpoints evaluated were corneal opacity, swelling, fluorescein retention, and morphological effects. All of the endpoints, with the exception of fluorescein retention (which was determined only at pre-treatment and 30 minutes after test item exposure) were determined at each of the above time points.

The test item Triethylene glycol dimethanesulfonate, the positive control [Benzalkonium chloride solution (5 %)], and the negative control (NaCl, 9 g/L saline) were applied in a volume of 30 μL/eye, in such a way to evenly cover the whole cornea surface of each tested eye. Three test item treated eyes, three positive control eyes and one negative control eye were used in this study.

After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with ~20 mL saline solution at ambient temperature and this procedure was repeated for each eye.

 

In this ICET, the overall ICE classes of the test item were thrice I (based on the corneal swelling of 2 % within 240 minutes, based on the corneal opacity score of 0.2 and based on the fluorescein retention of 0.5).

The positive control was classed as corrosive/severely irritating, UN GHS Classification: Category 1 and the negative control had no significant effects on the chicken eye in this study. Furthermore, the three endpoints of the positive and the negative control were in the historical control range. So, the positive and negative controls showed the expected results. The experiment was considered to be valid.

In this ICET, the overall ICE classes were thrice I. According to the guideline OECD 438, Triethylene glycol dimethanesulfonate (CAS 80322-82-3) is categorized as “No Category”.