(6R,7R)-7-[[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetyl]amino]-3-[(E)-[(3'R)-1'-[(1,1-dimethylethoxy)carbonyl]-2-oxo-[1,3'-bipyrrolidin]-3-ylidene]methyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid diphenylmethyl ester

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2017 to 26 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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:

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: OECD Guidelines for the Testing of Chemicals No. 438 (26 July 2013).

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl (3E,3'R)-3-{[(6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-2-[(diphenylmethoxy)carbonyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methylidene}-2-oxo-[1,3'-bipyrrolidine]-1'-carboxylate

EC Number:

609-463-9

Cas Number:

376653-41-7

Molecular formula:

C57H54N8O8S2

IUPAC Name:

tert-butyl (3E,3'R)-3-{[(6R,7R)-7-[(2Z)-2-(5-amino-1,2,4-thiadiazol-3-yl)-2-[(triphenylmethoxy)imino]acetamido]-2-[(diphenylmethoxy)carbonyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methylidene}-2-oxo-[1,3'-bipyrrolidine]-1'-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: BAL0001026
CAS number: 376653-41-7
Batch/Lot number: 012
Appearance: Off-white powder
Purity: Content by mass balance: 84.9% w/w
Expiry date: 30 June 2020
Storage conditions: Under inert gas, protected from light and humidity (tight closed container), frozen (≤-15 °C)
Safety precautions: Enhanced safety precautions were applied considering the supplied safety datasheet to assure personnel health and safety.

Specific details on test material used for the study:

No further details specified in the study report.

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

Strain of chicken: ROSS 308
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) used for human consumption. Heads were collected by a slaughter house technician and were transported to Citoxlab Hungary Ltd. at ambient temperature.
After collection, the heads were inspected for 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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

30 mg of the test item was applied
30 µL of physiological saline (negative control)
30 mg powdered Imidazole (positive control)

Duration of treatment / exposure:

exposure period of 10 seconds

Duration of post- treatment incubation (in vitro):

240 minutes (approximately ± 5 minutes)

Number of animals or in vitro replicates:

3 replicates per experiment and test group

Details on study design:

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 2% (w/v) fluorescein solution was applied onto the corneal surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated 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.

Preparation of eyes
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 according to the instructions given in the OECD 438 guideline (plastic boxes humidified with tissues moistened with isotonic saline).

Eyes examination and acclimatization time
Each prepared eye was placed firmly (without excessive restraint pressure) in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head when the chicken was standing). 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 closed 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 remained closed except for manipulations and examinations, to maintain temperature (32±1.5°C) 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 corneal 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. The selected eyes were appropriate for the test, which were acclimatized to the superfusion chamber for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5°C) during the acclimatization and treatment periods.
Identification
The eyes were identified by chamber number, marked on the door of the chamber.

THE BASELINE ASSESSMENTS
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (t=0) for each individual eye. The corneal 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 in each experiment. 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 suitable for the assay.

TEST PROCEDURE
Treatment
After the zero (t=0) reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test material was applied onto the centre of the cornea. In each experiment, 30 mg of the test item was applied onto the entire surface of the cornea attempting to cover the corneal surface uniformly with the test item, taking care not to damage or touch the cornea.
In each experiment negative control eye was treated with 30 µL of physiological saline; positive control eyes were treated with 30 mg powdered Imidazole.
One eye was treated with physiological saline, three eyes with the test item and another three with the powdered Imidazole positive control in each experiment.

Test item removal
The time of application was noted, then after an exposure period of 10 seconds from the end of the application the corneal surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test material.
Additional gentle rinsing with 20 mL saline was performed at each time point when test item or positive control material remained on the cornea.

OBSERVATION
Observation and assessment of corneal effects
The control eyes and test eyes were evaluated pre-treatment and at nominal time points of 30, 75, 120, 180 and 240 minutes (approximately ± 5 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. A Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment I

Value:

0.33

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all corneal surfaces after the post-treatment rinse.

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Experiment II

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all corneal surfaces after the post-treatment rinse.

Other effects / acceptance of results:

TEST ITEM
The test item BAL0001026 showed no significant corneal effect in the first experiment (Experiment I) and was therefore considered to be negative. As the test item was solid, the negative results were confirmed by a second experiment (Experiment II) according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes, the test item (BAL0001026) was non-irritant, UN GHS Classification: No Category.

POSITIVE CONTROL
The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1

NEGATIVE CONTROL
The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified

MORPHOLOGICAL EFFECTS
In each experiments test item was stuck on all corneal surfaces after the post-treatment rinse. In Experiment I, one corneal surface was cleared at the 240 minutes timepoint after the post-treatment rinse. Minimal amount of test item was observed on the corneal surfaces in two eyes at the 240 minutes timepoint after the post-treatment rinse. In Experiment II, two corneal surfaces were cleared at the 240 minutes timepoint after the post-treatment rinse. Minimal amount of test item was observed on the corneal surfaces in one eye at the 240 minutes timepoint after the post-treatment rinse.
In each experiment the positive control material was stuck on all corneal surfaces after the post-treatment rinse, the corneal surfaces were not cleared at 240 minutes after the post-treatment rinse.
No other morphological effects were observed in the study.

Any other information on results incl. tables

Table of test item for Classification (Experiment I)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.6%	I
Mean maximum corneal swelling at up to 240 min	1.7 %	I
Mean maximum corneal opacity	0.33	I
Mean fluorescein retention	0.50	I
Other Observations	Test item was stuck on all corneal surfaces after the post-treatment rinse. The corneal surface (1/3) was cleared at 240 minutes after the post-treatment rinse. Minimal amount of test item was observed on the corneal surfaces (2/3) in two eyes at 240 minutes after the post-treatment rinse.
Overall ICE Class	3xI

 

Table of test item for Classification (Experiment II)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	3.3%	I
Mean maximum corneal swelling at up to 240 min	3.8 %	I
Mean maximum corneal opacity	0.50	I
Mean fluorescein retention	0.00	I
Other Observations	Test item was stuck on all corneal surfaces after the post-treatment rinse. The corneal surfaces (2/3) was cleared at 240 minutes after the post-treatment rinse. Minimal amount of test item was observed on the corneal surface (1/3) in one eye at 240 minutes after the post-treatment rinse.
Overall ICE Class	3xI

 

Table of positive control for Classification (Experiment I)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	12.7%	II
Mean maximum corneal swelling at up to 240 min	28.0%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all corneal surfaces after the post-treatment rinse. The corneal surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

Table of positive control for Classification (Experiment II)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	11.5%	II
Mean maximum corneal swelling at up to 240 min	28.0%	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all corneal surfaces after the post-treatment rinse. The corneal surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII 2xIV

 

Table of negative control for Classification (Experiment I)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

Table of negative control for Classification (Experiment II)

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0%	I
Mean maximum corneal swelling at up to 240 min	0.0%	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.00	I
Other Observations	None
Overall ICE Class	3xI

 

Validity of the test

The results from all eyes used met the quality control standards. The negative and positive control results were within the historical data range in each of the experiments and therefore this study was considered to be valid.

Historical Control data (updated on 23 January 2018):

Negative Control: Physiological Saline

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2 %	3.4 %
Maximum corneal swelling at up to 240 min	-4.8 %	3.4 %
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of studies	358

 

Positive Control: Imidazole

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-6.6 %	25.0 %
Maximum corneal swelling at up to 240 min	-15.9 %	36.7 %
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00
Number of studies	157

 

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 BAL0001026, the test item was non-irritant, UN GHS Classification: No Category.

Executive summary:

An in vitro eye irritation study was performed in isolated chicken’s eyes on the test item, BAL0001024. The irritant effects of the test item were evaluated according to the method described in OECD No. 438 guideline (26 July 2013).

 

The study was conducted in 2 stages; the first experiment (Experiment I) provided an initial assessment of the irritation potential of the test item which was confirmed in a second experiment (Experiment II). In each experiment, after the time zero reference measurements, isolated eyes were held in a horizontal position and 30 mg of the test item or positive control item were applied directly onto the centre of the cornea in such a way to cover the entire surface of the cornea. After 10 seconds, the eyes were rinsed with physiological saline (0.9% (w/v) NaCl solution). The positive control eyes were treated with 30 mg powdered Imidazole and the negative control eye was treated with 30 µL of physiological saline. In each experiment, three test item treated eyes, three positive control treated eyes and one negative control treated eye were examined. The eyes were examined for corneal swelling, opacity or damage (based on fluorescein staining).

 

The results from all eyes used in the study met the quality control standards. The negative and positive control results were within the historical control data ranges in each experiment, thus, the study was considered valid.

 

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No cornea opacity change (severity 0.5 on two eyes and no cornea opacity change on one eye) was observed on test item treated eyes. No significant fluorescein retention change (severity 0.5) was noted on test item treated eyes. Test item was stuck on all corneal surfaces after the post-treatment rinse. One corneal surface was cleared at 240 minutes after the post-treatment rinse. Minimal amount of test item was observed on the corneal surfaces in two eyes at 240 minutes after post-treatment rinse.

 

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No significant cornea opacity change (severity 0.5) was observed on test item treated eyes. No significant fluorescein retention change was noted on test item treated eyes. The test item was stuck on all corneal surfaces after the post-treatment rinse. The two corneal surfaces were cleared at 240 minuntes post-treatment rinse. Minimal amount of test item was observed on the corneal surface in one eye at 240 minutes post-treatment rinse.

 

Based on these in vitro eye irritation assay in isolated chicken eyes with BAL0001026, the test item was non-irritant: UN GHS Classification: No Category.

SUMMARY TABLE FOR UN GHS CLASSIFICATION

EXPERIMENT I AND EXPERIMENT II

 

Criteria for “No category” (all true)
3 endpoints classed as I or 2 endpoints classed as I and 1 endpoint classed as II:	True
No severe corneal morphological changes:	True
Test item was not stuck to the cornea at 240 minutes after the post-treatment rinse:	True

 

Criteria for “Category 1” (one or more true)
2 or more endpoints classed as IV:	False
Corneal opacity ≥ 3 at 30 min (in at least 2 eyes):	False
Corneal opacity = 4 at any time point (in at least 2 eyes):	False
Severe loosening of epithelium (in at least 1 eye):	False

 

Criteria for “No prediction can be made” (one or two true)
Based on the endpoints not classifiable for No Category, or for Category 1:	False
Particles of test item were stuck to the cornea and could not be washed off during the study:	False