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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:
2016-06-23
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:
26 July 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:
08 December 2010
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(cyclohexylamino)ethanesulphonic acid
EC Number:
203-115-6
EC Name:
2-(cyclohexylamino)ethanesulphonic acid
Cas Number:
103-47-9
Molecular formula:
C8H17NO3S
IUPAC Name:
2-(cyclohexylamino)ethanesulfonic acid
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Chicken heads for obtained from TARAVIS KFT, 9600 Sárvár, Rábasömjéni út 129, Hungary
- Age at study initiation: eyes for approx. 2 hours old (after head removal)
- Weight at study initiation: not relevant


SOURCE OF COLLECTED EYES
- Source: 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: within 2 hours from collection at ambient temperature of 19.4 ºC to 20.3 ºC, heads were 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: not specified
- indication of any existing defects or lesions in ocular tissue samples: No. 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.
- Indication of any antibiotics used: not specified

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
- Amount applied: 0.03g Imidazole (positive control) or 30 µL NaCl (in saline; negative control) per eye
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
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 isotonic saline. Then the fluorescein-treated cornea was examined with 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 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 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 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 5 drops/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, 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, equalling 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.
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.

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 and cornea opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and 30 minutes after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 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 approximately 20 mL 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.
Imidazole and test item were 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.
The gentle rinsing with 20 mL saline was performed in all (three eyes) test item treated eyes after the 30 and 75 of observation. All test item treated eyes were totally cleared at 120 minutes after the post-treatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: was performed
- Damage to epithelium based on fluorescein retention: 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 isotonic saline. Then the fluorescein-treated cornea was examined with hand-held slit lamp or 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: was performed
- Others: no histopathology evaluation was performed

SCORING SYSTEM:
based on the OECD guideline.

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

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Run / experiment:
mean up to 75 and 240 min
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0 and 0
Positive controls validity:
valid
Remarks:
23 and 26
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.0
Positive controls validity:
valid
Remarks:
4.0
Irritation parameter:
fluorescein leakage
Run / experiment:
mean
Value:
0.5
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: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: -

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

Any other information on results incl. tables

Table 1: Results for the test item

 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

0.2

I

  Mean fluoresin retention

0.5

I

 Other Observations

None    

Overall ICE class

3xI

 

Table 2: Results for positive control Imidazole

 Observation

  Value

  ICE Class

 Mean maximum corneal swelling at up to 75 min

23%

III

 Mean maximum corneal swelling at up to 240 min

26%

III

 Mean maximum corneal opacity

4.0

IV

  Mean fluoresin retention

3.0

IV

 Other Observations

Cornea opacity score 4 was observed in three eyes at 75 minutes after the post-treatment rinse.

Overall ICE class

1xIII, 2xIV

The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1. 

 

Table 3: Results for the negative control NaCl (9 g/L saline)

 Observation

  Value

  ICE Class

 Mean maximum corneal swelling at up to 75 min

0% 

 Mean maximum corneal swelling at up to 240 min

 0%

 Mean maximum corneal opacity

 0.0

I

  Mean fluoresin retention

 0.0

 I

 Other Observations

None    

Overall ICE class

3xI  

 

Based on the overall ICE Class the negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.

Positive and negative control values were within the corresponding historical control data ranges.

 

Table 4: Historical control data of Positive control (Period of 2011 - 2015)

IMIDAZOLE HISTORICAL CONTROL Dose level: 30 mg / eye

n=168

Relative
obobservation time
(m(min)

Corneal thickness

Corneal opacity score

Fluorescein retention

30

75

120

180

240

 

 30

75

120

180

240

 

 ∆FR

Maximium

swelling (%):

34

45

49

54

55

Max. OS:

4.0

4.0

4.0

4.0

4.0

Max. FR:

3.0

Minimum swelling (%):

3

9

12

14

15

Min. OS:

2.8

3.3

3.5

3.5

3.5

Min. FR:

2.7

Average:

19

27

31

35

37

Average:

3.7

3.9

3.9

3.9

3.9

Average:

3.0

 

Table 5: Historical control data of Negative control (Period of 2011 - 2015)

NaCl (9 g/L saline) SOLUTION HISTORICAL CONTROL Dose level: 30 µL / eye

n=120

Relative
obobservation time
(m (min)

Corneal thickness

Corneal opacity score

Fluorescein retention

30

75

120

180

240

 

30

75

120

180

240

 

 ∆FR

Maximum

swelling (%):

3

3

3

4

3

Max. OS:

0.5

0.5

0.5

0.5

0.5

Max. FR:

0.5

Minimum swelling (%):

0

0

0

0

0

Min. OS:

0

0

0

0

0

Min. FR:

0.0

Average:

0.1

0.3

0.3

0.3

0.3

Average:

0.0

0.0

0.0

0.0

0.0

Average:

0.0

Remark:

n = number of examined eyes

∆FR = Difference between fluorescein retention and fluorescein retention reference value

OS = Opacity score

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed.
Executive summary:

An Isolated Chicken Eye Test (ICET) according to OECD 438 was used to evaluate the potential ocular corrosivity and irritancy of the test item by its ability to induce toxicity in enucleated chicken eyes. 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. The overall ICE class was 3xI.

Positive and negative controls showed the expected results. The experiment was considered to be valid.

In this in vitro eye corrosive and severe irritant study, using the Isolated Chicken Eye model with the test item, no ocular corrosion or severe irritation potential was observed. The overall ICE score was 3xI.