Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016
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 guidelineopen allclose all
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)
Version / remarks:
July 2013
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Name: 7-ACT
Chemical name: (6R, 7R)-7-Amino-3-(2,5-dihydro-2-methyl-5-oxo-6-hydroxy-1,2,4-triazin-3-yl) thiomethyl-ceph-3-em-4-carbonic acid
CAS No.: 58909-56-1
Supplier: Biochemie Ges.m.b.H.
Batch No.: 3201530
Purity: 99.5 % (content related to waterfree substance)
Water content: 1.5 %
Impurities: Heavy metals: < 20 ppm, by-products: < 0.2 % each
Appearance: white powder
pH: 3.97 (1 % aqueous suspension, measured by pH-meter)
Stability of the test substance: stable under conditions of storage
Disintegration: from 180 *C on
Storage: in a refrigerator in the dark
Date of handing over: July 26, 1994



Test animals / tissue source

Details on test animals or tissues and environmental conditions:
No animals but isolated chicken eyes were used.
Chicken heads collection and transport:
Strain of chicken: ROSS 308
Source: TARAVIS KFT. 9600 Sárvár, Rábasömjéni út 129. Hungary
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 (19.4 ºC to 20.3 ºC) during the transport. All eyes used in the assay were from the same groups of eyes collected on one specific day.
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).

Eyes selection
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.

Preparation of eyes
The eyeball was carefully removed from the orbit by holding the nictitating membrane with 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.

Eyes examination and acclimatization time
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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
0.03 g test item.
The test item was finely ground before application.
Duration of treatment / exposure:
Once.
Duration of post- treatment incubation (in vitro):
10 seconds. Observations 30, 75, 120, 180 and 240 minutes after the post-treatment rinse.
Number of animals or in vitro replicates:
3 replicates
Details on study design:
The baseline assessments:
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 (0% to 2%) were observed in the eyes, 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.

Test procedure
Treatment
After the zero reference measurements, one out of three test item treated eyes was taken out of its chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position over a container to catch waste, while the test item was applied onto the centre of the cornea. The test item was applied in an amount of 0.03 g by attempting to cover the cornea surface uniformly with the test substance, while taking care not to damage or touch the cornea with the application equipment. This procedure was repeated for each test item treated eye.

The three positive control eyes were treated in a similar way with 0.03 g Imidazole.
One negative control eye was treated with saline solution. The saline solution was applied in a volume of 30 μL from micropipette, in such a way that the entire surface of the cornea was covered with negative control, taking care not to damage or touch the cornea with the application equipment.

Test item removal:
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 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.
The 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 and test item 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 and assessment of corneal effects:
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.

Retention of corneas
At the end of the procedures, the corneas were carefully removed from the eyes and placed individually into labeled containers of preservative fluid (4% formaldehyde) for potential histopathology and stored.

Histopathology
After consultation with the Sponsor no histopathology evaluation was performed. Corneas are discarded 2 months after the final report.

Results and discussion

In vitro

Results
Irritation parameter:
other: Overall ICE class is 2 x I, 1 x II
Run / experiment:
Mean value
Negative controls validity:
valid
Remarks:
ICE class 3 x I
Positive controls validity:
valid
Remarks:
ICE class 3 x IV
Remarks on result:
other: The overall ICE class is 2 x I, 1 x II.

In vivo

Irritant / corrosive response data:
Observation Value ICE Class1
Mean maximum corneal swelling at up to 75 min 7% II
Mean maximum corneal swelling at up to 240 min 11% II
Mean maximum corneal opacity 0.5 I
Mean fluorescein retention 0.5 I
Other Observations None
Overall ICE Class1 2xI, 1xII

Positive Control: Imidazole
Observation Value ICE Class1
Mean maximum corneal swelling at up to 75 min 29% IV
Mean maximum corneal swelling at up to 240 min 36% IV
Mean maximum corneal opacity 4.0 IV
Mean fluorescein retention 3.0 IV
Other Observations Cornea opacity score 4 was observed in two eyes at 75 minutes after the post-treatment rinse.
Overall ICE Class1 3xIV
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Negative Control: NaCl (9 g/L saline)
Observation Value ICE Class1
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 0.5 I
Other Observations None
Overall ICE Class1 3xI
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

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No ocular corrosion or severe irritation potential was observed.
Executive summary:

An in vtro chicken eye (ICE) test was performed. Positive and negative controls showed the expected results. The experiment was considered to be valid.

No ocular corrosion or severe irritation potential was observed. The overall ICE score was 2 x I, 1 x II. According to the guideline OECD 438, the test item is categorized as "No Category".