Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2017 - 13 September 2017
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 guideline
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Willing New Material Technology Co.,Ltd.; 23161211201
- Expiration date of the lot/batch: 10 December 2017
- Purity: 97%


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 ºC)

Test animals / tissue source

Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: TARAVIS KFT. (Address: 9600 Sárvár, Rábasömjéni út. 129., Hungary)

Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.

After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within 2 hours of collection in each experiment.

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
Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
3 animals
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 2% (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluoresceintreated cornea was examined with a 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 to 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 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 avoid too much pressure on the eye by the clamp. 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/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. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological 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, 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 it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during thenacclimatization and treatment periods.

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% within the -45 min and the zero time. No changes in thickness (0.0%) were observed in the eyes of this study. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
30 μLPhysiological saline (0.9% (w/v) NaCl)

POSITIVE CONTROL USED
30 mg Imidazole

APPLICATION DOSE AND EXPOSURE TIME
10 seconds

OBSERVATION PERIOD
The control eyes 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 approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
The time of application was observed, then after an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline at ambient temperature, taking care not to damage the cornea but attempting to remove all residual of the test item if possible.

METHODS FOR MEASURED ENDPOINTS:
Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit BP 900® slit-lamp microscope was used for the measurements.

- Macroscopic morphological damage to the surface:
Morphological effects include “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective according to the interpretation of the Investigator.

SCORING SYSTEM:
- Mean corneal swelling (%):Refer to Section 3.8.1 of the report
- Mean maximum opacity score: Refer to Section 3.8.2 of the report
- Mean fluorescein retention score at 30 minutes post-treatment: Refer to Section 3.8.3 of the report

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
percent corneal swelling
Remarks:
75 mins
Run / experiment:
TiBTD Expt 1
Value:
1.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
240 mins
Run / experiment:
TiBTD Expt 1
Value:
2.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
TiBTD Expt 1
Value:
0.83
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class II
Irritation parameter:
fluorescein retention score
Run / experiment:
TiBTD Expt 1
Value:
0.33
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
75 mins
Run / experiment:
TiBTD Expt 2
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
240 mins
Run / experiment:
TiBTD Expt 2
Value:
1.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
cornea opacity score
Run / experiment:
TiBTD Expt 2
Value:
0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
TiBTD Expt 2
Value:
0.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
percent corneal swelling
Remarks:
75 mins
Run / experiment:
Positive control Expt 2
Value:
10.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class II
Irritation parameter:
percent corneal swelling
Remarks:
240 mins
Run / experiment:
Positive control Expt 2
Value:
28.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class III
Irritation parameter:
cornea opacity score
Run / experiment:
Positive control Expt 2
Value:
4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
fluorescein retention score
Run / experiment:
Positive control Expt 2
Value:
3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
other: All parameters
Run / experiment:
Negative control Expt 1 & 2
Value:
0
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Other effects / acceptance of results:
The results from Positive Control Experiement 1 were:
Mean maximum corneal swelling at up to 75 min - 8.0% ICE Class II
Mean maximum corneal swelling at up to 240 min - 24.1% ICE Class III
Mean maximum corneal opacity - 4.00 ICE Class IV
Mean fluorescein retention - 3.00 ICE Class IV

OTHER EFFECTS:
- Visible damage on test system:

Test item Expt 1: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were cleared at 120 minutes after the post-treatment rinse.

Test item Expt 2: Test item was stuck on all cornea surfaces after the post-treatment rinse. The one cornea surface (1/3) was cleared at 30 minutes, and two cornea surfaces (2/3) were cleared at 75 minutes after the post-treatment rinse.

Positive control: Imidazole was stuck on all cornea surfaces after the posttreatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Positive control: Imidazole was stuck on all cornea surfaces after the posttreatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

ACCEPTANCE OF RESULTS:
The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in each experiments. This study was considered to be valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation assays in isolated chicken eyes with TiBTD, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

In an in vitro eye irritation test in isolated chicken eyes (ICE) assay (17_053-038CS), isolated chicken eyes were exposed to TiBTD for 10 seconds in 2 independent experiments. Physiological saline (0.9% (w/v) NaCl) was used for the negative control and imidazole was used for the positive control. The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse.

The positive and negative controls gave the appropriate responses. In experiment I, no significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. Slight cornea opacity change (severity 0.5 or 1) was observed on three eyes. No significant fluorescein retention change (severity 0.5) was noted on two eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. All cornea surfaces were cleared at 120 minutes after the post-treatment rinse. In experiment II, no significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on the test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on all three eyes. No significant fluorescein retention change (severity 0.5) was noted on all three eyes. Test item was stuck on all cornea surfaces after the post-treatment rinse. The one cornea surface was cleared at 30 minutes and two cornea surfaces were cleared at 75 minutes after the post-treatment rinse.

The ICE classes for TiBTD in experiment 1 were: 1xI (mean corneal swelling) 1xII (mean corneal opacity) 1xI (mean fluorescein retention).  TiBTD did not meet any of the criteria for Eye damage – Category 1 but did meet all the criteria for not classified. The ICE classes for TiBTD in experiment 2 were: 1xI (mean corneal swelling) 1xI (mean corneal opacity) 1xI (mean fluorescein retention). TiBTD did not meet any of the criteria for Eye damage – Category 1 but did meet all the criteria for not classified. TiBTD is not irritating to the eye based on the results from this experiment.

This in vitro eye irritation test in isolated chicken eyes (ICE) assay is acceptable and satisfies the guideline requirement for an OECD 438 study.