Phenoxymethylpenicillin potassium

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 July 2019 - 12 November 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

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:

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:

April 2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Phenoxymethylpenicillin potassium

EC Number:

205-086-5

EC Name:

Phenoxymethylpenicillin potassium

Cas Number:

132-98-9

Molecular formula:

C16H17KN2O5S

IUPAC Name:

potassium (2S,5R,6R)-3,3-dimethyl-7-oxo-6-[(2-phenoxyacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Name: Pen V Potassium
Lot No.: B519322
Appearance:white, solid powder
Expiry date: 30 April 2024
Storage condition: at room temperature, protected from light

Specific details on test material used for the study:

Test Item name: Pen V Potassium
Lot No.B519322
Appearancewhite, solid powder
Expiry date 30 April 2024
Storage room temperature, protected from light

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

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 (19.3 ºC to 20.4 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 4 drops/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate numbers of eyes were 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

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. Changes in thickness were not observed in the 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 Pleuromulin was applied onto the centre of the cornea. The Pleuromulin 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. The cornea surface of Imidazole and test item treated eyes 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

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 classes were one II (based on the corneal swelling of 13% within 240 minutes), once III (based on the fluorescein retention of 1.8) and once IV(based on the corneal opacity score of 3.3).

Other effects / acceptance of results:

The test substance did not cause ocular corrosion or severe irritation in the enucleated chicken eyes.
The negative control NaCl (9 g/L saline) had no significant effects on the chicken eye in this study.
The positive control Imidazole was classed as corrosive/severely irritating, UN GHS Classification: Category 1.

Any other information on results incl. tables

Test Item: Pen V Potassium

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	8%	II
Mean maximum corneal swelling at up to 240 min	13%	II
Mean maximum corneal opacity	3.3	IV
Mean fluorescein retention	1.8	III
Other Observations	None
Overall ICE Class	1xII, 1xIII, 1xIV

 

Positive Control: Imidazole

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	26%	III
Mean maximum corneal swelling at up to 240 min	34%	IV
Mean maximum corneal opacity	4.0	IV
Mean fluorescein retention	3.0	IV
Other Observations	Corneal opacity score 4was observed in two eyes and score 3 was seen in one eye at 30 minutes after the post-treatment rinse.
Overall ICE Class	3xIV

The positive control Imidazole was classed as corrosive/severely irritating, UNGHS Classification: Category 1.

Negative Control: NaCl (9 g/L saline)

Observation	Value	ICE class
Mean maximum corneal swelling at up to 75 min	2%	I
Mean maximum corneal swelling at up to 240 min	2%	I
Mean maximum corneal opacity	0.5	I
Mean fluorescein retention	0.0	I
Other Observations	None
Overall ICE Class	3xI

The negative control NaCl (9g/L saline) had no significant effects on the chicken eye in this study.

 

 

 

Applicant's summary and conclusion

Interpretation of results:

other: Inconclusive because no classification as Category I nor as No Category can be set.

Conclusions:

No ocular corrosion or severe irritation potential was observed. The overall ICE classes were one II (based on the corneal swelling of 13% within 240 minutes), once III (based on the fluorescein retention of 1.8) and once IV(based on the corneal opacity score of 3.3). According to the guideline OECD 438, the test item overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.

Executive summary:

An in vitro chicken eye (ICE) test was performed according to OECD438 Guideline. 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 classes were one II (based on the corneal swelling of 13% within 240 minutes), once III (based on the fluorescein retention of 1.8) and once IV(based on the corneal opacity score of 3.3). According to the guideline OECD 438, the test item overall in vitro classification is neither UN GHS Classification Category I (an ocular corrosive or severe eye irritant) nor No Category. Thus, according to the guideline OECD 438, test item has been categorized as “No prediction can be made”.