Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-06-21

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

08 December 2010

Deviations:

no

Test guideline 2

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate

EC Number:

278-169-7

EC Name:

Sodium 4-(2-hydroxyethyl)piperazin-1-ylethanesulphonate

Cas Number:

75277-39-3

Molecular formula:

C8H18N2O4S.Na

IUPAC Name:

sodium 1-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonate

Test material form:

liquid

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughter house, TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
- Number of animals: not specified
- Characteristics of donor animals: not specified
- Storage, temperature and transport conditions of ocular tissue: heads were transported approximately within 2 hours from collection, 19.3 to 20.2 ºC, heads were wrapped with paper moistened with saline, placed in a plastic box that can be closed (4-5 heads/box)
- Indication of any existing defects or lesions in ocular tissue samples: Eyes with defects before treatment were rejected.
- Indication of any antibiotics used: not specified
- Selection and preparation of corneas: Non-damaged eyes were removed from the heads. The nictitating membrane was cut away with other connective tissue.
- Quality check of the isolated corneas: Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount applied: 0.03 g (per eye)

CONTROLS
- Amounts applied: 0.03 g imidazole; 30 µL NaCl (in saline)

Duration of treatment / exposure:

10 seconds

Duration of post- treatment incubation (in vitro):

4 hours

Number of animals or in vitro replicates:

3 eyes (test substance and positive control)
1 eye (negative control)

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
With the eyes still in the chicken heads, eyelids were carefully cut away with scissors from the chicken eyes. One small drop of fluorescein solution 2 % (w/v) was applied onto the cornea surface for a few seconds and then rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a 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, which may lead to 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 to maintain the appropriate humidity.

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. Changes in thickness were not observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effects after treatment. The location of any minor findings was marked on the record sheet as a drawing, if applicable. If any eye was considered to be unsuitable following baseline assessment, it was discarded.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: After 10 seconds the cornea surface was rinsed thoroughly with approx. 20 mL saline solution at ambient temperature. Imidazole was stuck on the corneas’ surface in all eyes at 30 minutes after the post-treatment rinse. The gentle rinsing with 20 mL saline was performed in all Imidazole treated eyes after the 30, 75, 120 and 180 minutes of observation, but cornea surfaces were not totally cleared at 240 minutes after the post-treatment rinse.

OBSERVATION PERIOD
30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable)
Cornea thickness and opacity were measured at all time points. Flurescein retention was determined at baseline (t=0) and at 30 min.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: yes, using a slit lamp microscope
- Damage to epithelium based on fluorescein retention: yes, using a slit lamp microscope
- Swelling: depth measuring device on the slit lamp microscope (Haag-Streit BQ 900) with the slit-width set at 9½, equalling 0.095 mm
- Macroscopic morphological damage to the surface: yes, using a slit lamp microscope

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

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

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

mean maximum up to 240 min

Value:

2

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

2

Positive controls validity:

valid

Remarks:

30

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean maximum

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0.5

Positive controls validity:

valid

Remarks:

4.0

Results 3

Irritation parameter:

fluorescein leakage

Run / experiment:

mean maximum

Value:

1

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

0

Positive controls validity:

valid

Remarks:

3

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline: not applicable

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was determined to have no eye irritation potential.

Executive summary:

The purpose of this Isolated Chicken Eye Test (ICET) according to OECD 438 was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. The test compound was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken. 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. Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 0.03 g/eye by powdering the entire surface of the cornea attempting to cover the cornea surface uniformly with the test substance or positive control. Three test item treated eyes and three positive control eyes were used in this study. One negative control eye was treated with 30 μL saline solution. After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with approximately 20 mL saline solution at ambient temperature and this procedure was repeated for each eye. In this ICET, the test item did not cause ocular corrosion or severe irritation in the enucleated chicken eyes. The overall ICE class was 2xI, 1xII. Positive and negative controls showed the expected results. The experiment was considered to be valid.