Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals / tissue source

Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Licensed slaughterhouse, i.e. Zakład Przemysłu Drobiarskiego JAS-DROP in Krzyżowice.
- Number of animals:
- Characteristics of donor animals: 1.5 to 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
After sedation of the chickens by electric shock and incision of the neck for bleeding, their heads were transported to the laboratory in a plastic container at ambient temperature. During the transport, the heads were humidified with a physiological salt solution by placing moistened paper towels inside the container. The time interval between the collection of the chickens’ heads and the use of their eyeballs in the ICE test was 35 minutes. All eyeballs used in the test came from the same group of eyes collected on a specific day.
- indication of any existing defects or lesions in ocular tissue samples: None.
- Indication of any antibiotics used: None.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL and POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.03 g.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 0.03 mL

Duration of treatment / exposure:
10 seconds.
Number of animals or in vitro replicates:
Three eyeballs per run.
Two runs were conducted.
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
After a careful excision of the eyelids so as not to damage the cornea, the eyeballs were checked for the presence of the damage. The surface of the eyeballs was treated with a 2% fluorescein solution (w/v) for a few seconds in order to evaluate the corneal integrity. After the removal of the dye by rinsing the corneal surface with a physiological salt solution, fluorescein retention and corneal opacity scores were determined using the slit-lamp microscope BP 900 LED to ensure that the cornea was undamaged. Only eyeballs without damage were dissected. The enucleated eyeball was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to the superfusion apparatus so that the entire cornea was supplied with the physiological saline drip. After the insertion of the eyeballs to the clamp, another evaluation of fluorescein retention, corneal opacity and corneal swelling was performed. Results of corneal opacity and fluorescein retention for all examined eyeballs were less than 0.5. Deviation of corneal thickness for all examined eyeballs was less than 10%.
The corneal thickness was determined using of the depth measuring device no. 1 on the slit-lamp microscope and using of SP-100 pachymeter. The slit-width on slit-lamp microscope was set at 0.095 mm. The pachymeter measures the period of time from emitting ultrasounds to receiving the same ultrasound, reflected by corneal posterior part. Applying a calculation formula shown below, corneal thickness was calculated.
L=V x t/2, where
L- corneal thickness;
V-ultrasound velocity trough cornea;
t-measured time.

EQUILIBRATION AND BASELINE RECORDINGS
Prior to the application of the test item and the control items, all examined and approved eyeballs were incubated at temperature of 32°C ± 1.5°C for 45-60 minutes in order to equilibrate them to the test system. During the incubation, the eyeballs were continuously supplied with physiological saline at constant temperature of 33 ± 0.5°C and in the average volume of 0.10-0.15 mL/min.
After that, a zero reference measurement for corneal thickness and opacity was recorded. It served as a baseline (i.e. time = 0). The fluorescein score determined at dissection of eyeball served as the baseline measurement for that endpoint.

NUMBER OF REPLICATES
Three eyeballs per run.
Two runs were conducted.

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
Imidazole

APPLICATION DOSE AND EXPOSURE TIME
0.03 g for test material and positive control (solids) and 0.03 mL for negative control.
The test item and the control items were uniformly applied to the corneal surface for 10 seconds.

OBSERVATION PERIOD
240 min.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Eyeballs were rinsed from the eye with 20 mL of physiological saline at ambient temperature.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal opacity was determined by assigning appropriate values to opaque areas. The mean corneal opacity value for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal opacity, as observed at any time point, the final category score for the test item and the control items was given.
- Damage to epithelium based on fluorescein retention:The mean fluorescein retention value for all test eyeballs was calculated on the grounds of the observations made 30 minutes after the end of the exposure. This value was used to determine the general category score for the test item and the control items.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: 0.095 mm.
- Macroscopic morphological damage to the surface: Gross evaluation was performed to to determine whether any morphological effects, e.g. pitting of corneal epithelial cells, roughening of the corneal surface, and sticking of the test item to the cornea were visible.
- Others: HIstopathological evaluation was conducted for the first run.

SCORING SYSTEM:
- Mean corneal swelling (%)
The degree of corneal swelling was determined by measuring corneal thickness using a slit-lamp microscope and a pachymeter. It was expressed as a percentage.

- Mean maximum opacity score
The mean percentage of corneal swelling for all test eyeballs was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, the final category score for the test item and the control items was given.

Corneal opacity scores:
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

Fluorescein retention scores:
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: The ICE classes for each of the three endpoints was assigned according to the guideline. The classification criteria as indicated in the TG was used.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
fluorescein retention score
Run / experiment:
1
Value:
1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of three eyeballs. It corresponds to an ICE class III.
Irritation parameter:
cornea opacity score
Run / experiment:
1
Value:
2.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum average value at any time point. It corresponds to an ICE class IV.
Irritation parameter:
percent corneal swelling
Run / experiment:
1
Value:
30.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum average value at any time point. It corresponds to an ICE class III.
Irritation parameter:
fluorescein retention score
Run / experiment:
2
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Average of three eyeballs. It corresponds to an ICE class III.
Irritation parameter:
cornea opacity score
Run / experiment:
2
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: maximum average value at any time point. It corresponds to an ICE class III.
Irritation parameter:
percent corneal swelling
Run / experiment:
2
Value:
18.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Maximum average value at any time point. It corresponds to an ICE class III.
Other effects / acceptance of results:
Gross evaluation of the treated corneas:
First run:
During the gross examination of the positive control treated and the eyeballs treated with the test item, the roughening of the corneal surface and deposits of the item were observed. The negative control eyeballs did not exhibit any changes of the corneal surface.

Second run:
During the gross examination of the positive control treated, the roughening of the corneal surface and deposits of the item were observed. were observed. The negative control eyeballs did not exhibit any changes of the corneal surface. The deposits of the test item were observed on the cornea surface treated with test item.


Histopathological evaluation of the treated corneas
The histopathological evaluation was performed only for the first run. The following changes were found in the group exposed to the test item – the treated group (three eyeballs):

− in eyeball no. 1: dissection, vacuolation, and necrosis of the anterior corneal epithelium, dissection of the corneal stroma;
− in eyeball no. 2: vacuolation, slight erosions, and necrosis of the anterior corneal epithelium, dissection of the corneal stroma;
− in eyeball no. 3: dissection, erosions, and necrosis of the anterior corneal epithelium, dissection of the corneal stroma.

The following changes were found in the group exposed to imidazole – the positive control (three eyeballs):

− in the eyeball no. 4: vacuolation, detachment, erosions and necrosis of the anterior corneal epithelium;
− in the eyeball no. 5: detachment, erosions and necrosis of the anterior corneal epithelium;
− in the eyeball no. 6: detachment, erosions and necrosis of the anterior corneal epithelium. These changes confirmed corrosive properties of imidazole.

The following changes were found in the group exposed to physiological saline – the negative control (three eyeballs):
− in the eyeball no. 7: very slight erosions of the superficial layer of the anterior corneal epithelium, dissection of the corneal stroma;
− in the eyeball no. 8: no lesions;
− in the eyeball no. 9: erosions of the superficial layer of the anterior corneal epithelium, dissection of the corneal stroma.

All changes which were observed in eyeball no. 7 and no. 9 are associated with physiological process of replacement of the epithelial cells, and / or changes in ocular pressure during the experiment, and / or technical processing of the material.

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

Any other information on results incl. tables

Table 5. Summary of results (the first run)

  Maximal ICE Class  
Fluorescein retention Corneal opacity Corneal swelling UN GHS Classification
Tazobactam III IV III No prediction can be made
Imidazole IV IV IV Category 1
Physiological saline I   I No Category

Table 6. Summary of results (the second run)

  Maximal ICE Class  
Fluorescein retention Corneal opacity Corneal swelling UN GHS Classification
Tazobactam III III III No prediction can be made
Imidazole IV IV IV Category 1
Physiological saline I   I No Category

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
The substance cannot be classified as severe irritant or corrosive to the eyes and it is not possible to state that the test item is not irritant to the eyes according to the UN GHS classification which is in accordance with the CLP criteria. According to the histopathological findings the test can be regarded as moderately irritating to the eyes.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the substance tazobactam 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 0.03 g of the test item, the same amount of imidazole and 0.03 mL of physiological saline were applied to other eyeballs as positive and negative controls. Three eyeballs were used for the test item and for each control per run. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438 recomendations, histopathological evaluation of the corneal layers was conducted for the first run. Because no conclusion could be made based on the first run, a second run of three eyes for the test item was performed, concurrent controls were included. According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints were 2xIII and 1xVII in the first run and 3xIII in the second run, however taking into account the histopathological findings, the test can be considered as moderately irritating.