Sodium (2-butoxyethoxy)acetate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May 2018

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 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:

09 October 2017

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:

28 April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium (2-butoxyethoxy)acetate

EC Number:

268-040-3

EC Name:

Sodium (2-butoxyethoxy)acetate

Cas Number:

67990-17-4

Molecular formula:

C8H16O4.Na

IUPAC Name:

sodium (2-butoxyethoxy)acetate

Test material form:

liquid: viscous

Details on test material:

Batch Number: P59564
Appearance: white-yellow, low melting point paste
Purity: 100 %
Expiration date: 31.12.2018
Storage: room temperature (ca. 20°C)

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): approximately 7 weeks old, 1.5 - 2.5 kg
- Storage, temperature and transport conditions of ocular tissue: Head collection was performed by a slaughter house technician. Heads were removed immediately after sedation of the chickens (sedation was happened 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 (19.2ºC to 20.3 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 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).
- Time interval prior to initiating testing: approx. 2 h
- indication of any existing defects or lesions in ocular tissue samples: none

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: approx. 30 µL

Duration of treatment / exposure:

10 sec

Duration of post- treatment incubation (in vitro):

The control and test item treated eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.

Number of animals or in vitro replicates:

3

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 fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL saline solution. Then the fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5). 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. The nictitating membrane and other connective tissue were cut away. The prepared eyes were kept on wet papers in a closed box to maintain an appropriate humidity. The treatment group and the concurrent positive control consisted of three eyes. The negative control group consisted of one eye. The enucleated 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, too much pressure on the eye by the clamp was avoided. 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 saline solution dripping from a stainless steel tube, at a rate of approximately 3 to 5 drops/minute. 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, the 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 saline solution which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or a high 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 the eyes, or eyes that showed any other signs of damage, were rejected and replaced. If the selected eyes were appropriate for the test, acclimatisation started and was conducted for approximately 45 to 60 minutes. The temperature of the circulating water was verified to ensure that the temperature in all chambers was in the range of 32 ± 1.5 °C during the acclimatisation and treatment periods.

EQUILIBRATION AND BASELINE RECORDINGS
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and fluorescein retention 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 minutes before the start of application. Slight thinning up to 7 % is considered acceptable when maintaining enucleated eyes. Any minor corneal opacity was noted. Minor observations or deviations from the above was considered acceptable provided they are not observed in all eyes of the test item group, otherwise the eyes were replaced. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl (9 g/L) Saline

POSITIVE CONTROL USED: Acetic acid 10% (v/v)

APPLICATION DOSE AND EXPOSURE TIME: 30 µL, 10 sec

OBSERVATION PERIOD: 30, 75, 120, 180 and 240 minutes after the post-treatment rinse

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The time of application was monitored. After an exposure period of 10 seconds the cornea surface was rinsed thoroughly with 20 mL saline solution at ambient temperature, while taking care not to damage the cornea but attempting to remove the entire 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 a minimum.

METHODS FOR MEASURED ENDPOINTS:
- Damage to epithelium based on fluorescein retention: The fluorescein-treated cornea was examined with a slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged (i.e., fluorescein retention ≤ 0.5).
- Swelling: 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.

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

DECISION CRITERIA: Results from corneal opacity, swelling, and fluorescein retention are evaluated separately to generate an ICE (Isolated Chicken Eye) class for each endpoint. The ICE classes for each endpoint are then combined to generate an Irritancy Classification for each test substance The decision criteria as indicated in the TG was used (see table 1 of material and methods)

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Remarks:

mean

Run / experiment:

1st experiment

Value:

38

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

cornea opacity score

Remarks:

mean

Run / experiment:

1st experiment

Value:

4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

1st experiment

Value:

3

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No pitting of corneal epithelial cells, loosening of epithelium and roughening of the corneal surface were noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Prior to routine use of the method Toxi-Coop ZRT demonstrated the technical proficiency in a separate study (study no.: 392.549.3229) using the ten Proficiency Chemicals according to OECD Test Guideline No. 438.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes. Thw mean fluorescein retention was slightly lower than the actual historical control data range (Actual fluorescein retention range: 0.2-2.8). The slightly lower counts were evaluated as acceptable without any influence on the final conclusion of the study.

Any other information on results incl. tables

Table 1 Result of the test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	33%	IV
Mean maximum corneal swelling at up to 240 min	38%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4 was observed in (all) three eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	3xIV

Table 2 Result of positive control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	28%	IV
Mean maximum corneal swelling at up to 240 min	30%	III
Mean maximum corneal opacity	3.8	IV
Mean fluorescein retention	0.0*	I
Other Observations	Corneal opacity score 4 was observed in two eyes at 30 minutes after the post-treatment rinse.
Overall ICE Class^1	1xI, 2xIV

*: Mean fluorescein retention was slightly lower than the actual historical control data range (Actual fluorescein retention range: 0.2-2.8). The slightly lower counts were evaluated as acceptable without any influence on the final conclusion of the study.

Table 3 Result of negative control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0%	I
Mean maximum corneal swelling at up to 240 min	0%	I
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class^1	3xI

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In this ICET, sodium (2-butoxyethoxy) acetate caused ocular corrosion or severe irritation in the enucleated chicken eyes.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item sodium (2-butoxyethoxy) acetate by its ability to induce toxicity in enucleated chicken eyes. The test compound 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 substance exposure) were determined at each of the above time points.

The test item sodium (2-butoxyethoxy) acetate, positive control (acetic acid 10% (v/v) solution), and 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, sodium (2-butoxyethoxy) acetate caused ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE classes were 3xIV (based on corneal swelling of 38% within 240 min, opacity score of 4.0 and based on a fluorescein retention of 3.0).Positive and negative controls showed the expected results. The experiment was considered to be valid.