α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 27th, 2013.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

yes

Remarks:

test item was applied dry.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

EC Number:

246-042-5

EC Name:

α-(2,4-dichlorophenyl)-1H-imidazole-1-ethanol

Cas Number:

24155-42-8

Molecular formula:

C11H10Cl2N2O

IUPAC Name:

1-(2,4-dichlorophenyl)-2-(1H-imidazol-1-yl)ethan-1-ol

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:

SOURCE OF COLLECTED EYES
- Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): 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). The heads were transported to TOXI-COOP ZRT. at the earliest convenience for use approximately within 2 hours from collection. All eyes used in the assay were from the same groups of eyes collected on one specific day.
- Time interval prior to initiating testing: 2h
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: no.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.03 g/eye.

Duration of treatment / exposure:

10 seconds.

Duration of post- treatment incubation (in vitro):

240 ± 5 minutes

Number of animals or in vitro replicates:

3 eyes for the test item and positive control, 1 eye for the 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 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 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.
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 nm. 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.

EQUILIBRATION AND BASELINE RECORDINGS: Acclimatization was conducted for approximately 45 to 60 minutes. The temperature of the circulating water 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. Slight changes in thickness (-3% to 2%) were observed in the eyes, a 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. 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.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 30 µL of 9 g/L NaCl, saline (Source: REANAL, Batch: PP/2012/10937) in ultra-pure water.

POSITIVE CONTROL USED: 0.03 g Imidazole (Source: Sigma-Aldrich, Batch: SLBC7446V)

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g test item, exposure 10 s.

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 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 all the residual test item if possible. The eye in the holder was then returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: The test item was stuck on the corneas at 30 minutes after the post-treatment rinse, so a gentle rinsing with additional 20 mL saline was performed. The cornea surfaces were not totally cleared, little volume of test item was stuck on the cornea surface at 240 min after the posttreatment rinse.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: qualitative assessment.
- Damage to epithelium based on fluorescein retention: qualitative assessment.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 nm. 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 nm.
- Macroscopic morphological damage to the surface: qualitative assessment.

SCORING SYSTEM:
- Mean corneal swelling (%):
Corneal swelling (CS) was calculated as follows: CS = [(corneal thickness at time t – corneal thickness at time t = 0)/ corneal thickness at time t = 0] x100
For the calculation of Maximum Swelling, small negative numbers for swelling (0 to -5%) following application are counted as zero. Large negative numbers (> -12 %) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect.

- Mean maximum opacity score
0 No opacity
0.5 Very faint opacity
1 Scattered or diffuse areas; details of the iris are clearly visible
2 Easily discernible translucent areas; details of the iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: as indicated in the TG.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

fluorescein retention score

Run / experiment:

mean

Value:

2.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class III.

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

mean max.

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class I.

Results 3

Irritation parameter:

percent corneal swelling

Run / experiment:

mean max. (240 min)

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: ICE Class II.

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: The test item was stuck on the cornea surface all test item treated eyes at 30 minutes after the post-treatment rinse. It could not be removed from cornea surface by extra rinse (20 mL/eye saline solution).

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes.
- Acceptance criteria met for positive control: yes.
Positive and negative control values were within the corresponding historical control data ranges (Appendix II).
- Range of historical values if different from the ones specified in the test guideline: yes.

Any other information on results incl. tables

Table 1. Summary of test item results.

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	10%	II
Mean maximum corneal swelling at up to 240 min	11%	II
Mean maximum corneal opacity	0.0	I
Mean fluorescein retention	2.5	III
Other Observations	The test item was stuck on the cornea surface all test item treated eyes at 30 minutes after the post-treatment rinse. It cannot be removed from cornea surface by extra rinse (20 mL/eye saline solution).
Overall ICE Class	1xI, 1xII, 1xIII
Conclusion	Not a severe irritant

 

Table 2. Summary of positive control (imidazole) results.

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	9%	II
Mean maximum corneal swelling at up to 240 min	15%	II
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Cornea opacity score 4 was observed in three eyes at 30 minutes after the post- treatment rinse
Overall ICE Class	1xII, 2xIV
Conclusion	Corrosive, Category 1

 

 

Table 3. Summary of negative control (saline) results.

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	3xI
Conclusion	Not classified

 

Applicant's summary and conclusion

Interpretation of results:

other: not severely irritating, based on EU criteria.

Conclusions:

Based on the available data (overall ICE class 1xI, 1xII, 1xIII), the test item is not a severe irritant.

Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either the test item, imidazole (positive control) or saline (negative control). Three eyeballs were used for the test item and the positive control, one for the negative control. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to the guideline recomendations.
According to the overall in vitro classification, no prediction can be made since the combinations of the 3 endpoints were 1xI, 1xII, 1xIII. Based on the results, the test item is not severely irritant to the eye.