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Diss Factsheets

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:
2017
Report date:
2017

Materials and methods

Test guidelineopen allclose all
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:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2010
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 4-morpholin-1-ylethylsulphonate
EC Number:
275-203-2
EC Name:
Sodium 4-morpholin-1-ylethylsulphonate
Cas Number:
71119-23-8
Molecular formula:
C6H13NO4S.Na
IUPAC Name:
sodium 4-morpholin-1-ylethylsulphonate
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg per eye)

CONTROLS
- Amount(s) applied (volume or weight with unit): 30 mg imidazole; 30 µL NaCl (in saline)
Duration of treatment / exposure:
After 10 seconds the cornea surface was rinsed thoroughly with approx. 20 mL saline solution at ambient temperature
Observation period (in vivo):
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.
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:
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 (19.3 ºC to 20.2 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
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 fluroescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was ingood 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
The prepared eye was placed in a stell clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), again avoiding too mucg pressure on the eye. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp 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 approx. 3 to 5 drops/min.The door of the chmber was closed, excpet for manipulations and examination, to maintain temperature and humidity.
Eyes selected for testing were again examined with the slit lamp microscope. Eyes with a high baseline flurescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl in saline (9 g/L) (n=1)

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED: Imidazole (n=3)

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g per eye for 10 seconds

OBSERVATION PERIOD: 4 hours

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Thorough rinsing with approx. 20 mL saline solution at ambient temperature
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity scores ranging from 0 (no opacity) to 4 (complete opacity; iris invisible) according to the test guideline were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention scores raning from 0 - 3 according to the test guideline were measured at 0 and 30 min.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Cornea thickness was measured using the depth measauring device on the slit lamp microscope (Haag-Strei BQ 900) with the slit-width set at 9.5, i.e. 0.095mm.
- Macroscopic morphological damage to the surface: not applicable
- Others (e.g, histopathology): not performed

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

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
1 (240 min)
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
2 %
Positive controls validity:
valid
Remarks:
30 %
Irritation parameter:
cornea opacity score
Run / experiment:
1 (240 min)
Value:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.5
Positive controls validity:
valid
Remarks:
4.0
Irritation parameter:
fluorescein leakage
Run / experiment:
1 (240 min)
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
3.0
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an experimental study according to OECD TG 438 under GLP conditions, the test substance scored into ICE Class I for corneal opacity and swelling and into ICE Class II for flurescein retention. With the overall ICE score of 2xI and 1xII, the substance is classified as "No Category".
Executive summary:

A study was conducted according OECD TG 438. The purpose of this Isolated Chicken Eye Test (ICET) was to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. This test identifies chemicals either inducing "serious eye damage" (category 1 of the Globally Harmonised System for the Classification and Labelling of Chemicals (GHS)), or not requiring classification for eye irritation or serious eye damage according to the GHS. The test item was applied in a single dose (30 mg/eye) onto the cornea of isolated chicken eyes and rinsed after 10 seconds with saline. 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 Imidazole (positive control) was ground before use in the study. The test item and positive control were applied in an amount of 30 mg/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. With the overall ICE score of 2xI and 1xII, the substance is classified as "No Category". Positive and negative controls showed the expected results. The experiment was considered to be valid.