Phenoxymethylpenicillin potassium

Endpoint summary

Description of key information

The test item is not irritating (or corrosive) to skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2019 - 7 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

28 July 2015.

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 131 (09 June 2012): EpiSkinTM Skin Irritation Test 15 min – 42 hours

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

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

Test system:

human skin model

Remarks:

Supplier: SKINETHIC Laboratories 4, rue Alexander Fleming, 69366 Lyon Cedex 07 - France Batch No.:19-EKIN-025 Expiry date: 24 June 2019

Source species:

human

Cell type:

non-transformed keratinocytes

Details on animal used as source of test system:

EpiSkinTM Small Model (EpiSkinTMSM), manufactured by EPISKIN SNC Lyon, France, is a three-dimensional human epidermis model. Adult human-derived epidermal keratinocytes are seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after 13-day culture period comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum (Tinois et al., 1994). Its use for skin irritation testing involves topical application of test materials to the surface of the epidermis, and the subsequent assessment of their effects on cell viability.

Justification for test system used:

The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and no- skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiSkinTMSM KIT Contents
Units: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport. Plate:12-well assay platePunch: EpiSkinTMSM biopsy punch for easy sampling of epidermis Medium: A flask of sterile “Maintenance Medium” for incubations.(Batch No.: 19-MAIN3-027; Exp. Date: 26 June 2019) A flask of sterile “Assay Medium” for use in MTT assays.(Batch No.: 19-ESSC-026; Exp. Date: 26 June 2019)
EpiSkinTMSM KIT Reception ProcedureThe colour of the agar medium used for transport was checked for its pH: - orange colour = good - yellow or violet colour = not acceptable The colour of the temperature indicator was inspected to verify that the kit has not been exposed to a temperature above 40 °C: - the indicator changes from white to grey at 40 °C The kit was found to be in good order at reception.
EpiSkinTMSM KIT Storage ProcedureThe EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8 °C.
Indicator for potential false viabilityOptical properties of the test material or its chemical action on MTT may interfere with the assay leading to a false estimate of viability. This may occur when the test item is not completely removed from the tissue by rinsing or when it penetrates the epidermis. If the test material acts directly on MTT (MTT-reducer), is naturally coloured, or becomes coloured during tissue treatment, additional controls should be used to detect and correct for test substance interference with the viability measurement.
Check-method for possible direct MTT reduction with test itemApproximately 10 mg test item was added to 2 mL MTT 0.3 mg/mL solution and mixed. The mixture was incubated for three hours at 37±1 °C protected from light and then any colour change observed: - Test items which do not interact with MTT:yellow- Test items interacting with MTT: blue or purple If the MTT solution colour becomes blue or purple, the test substance interacts with the MTT. It is then necessary to evaluate the part of optical density (OD) due to the non-specific reduction of the MTT (i.e. by using killed epidermis).
Check-method to detect the colouring potential of test item Prior to treatment, the test item was evaluated for its intrinsic colour or ability to become coloured in contact with water (simulating a tissue humid environment). Approximately 10 mg test item was added to 90μL of water (prepared in Toxi-Coop ZRT. by MILLIPORE Synergy UV HF ASTM 1: F8JA80461C) and mixed. The mixture was shaken for 15 minutes at room temperature and then colour checked (unaided eye assessment).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

approximately 10 mg

Duration of treatment / exposure:

15 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

ca. 90

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

2

Value:

86

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3

Value:

ca. 79

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

The results obtained from this in vitro skin irritation test, using the EPISKIN model, indicated that the test item reveals no skin irritation potential under the utilised testing conditions. The test item Pen V Potassium is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).

Executive summary:

Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes(± 0.5 min) at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. Epidermis units were then incubated at 37±1 °C for 42 hours (± 1 h) in an incubator with 5±1 % CO2, ≥95 % humidified atmosphere. The viability of each disk was assessed by incubating the tissues for 3 hours (± 5 min) with MTT solution at 37±1 °C in 5±1 % CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5 % aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50 % of the negative control. In this in vitro skin irritation test using the EPISKIN model, the test item Pen V Potassiumdid not show significantly reduced cell viability in comparison to the negative control (mean value: 85 %). All obtained test item viability results were far above 50 % when compared to the viability values obtained from the negative control. Therefore the test item was considered to be non-irritant to skin. Positive and negative controls showed the expected ODs, cell viability values were within acceptable limits and the standard deviations of all calculated viability values (positive and negative control, test item) were below 18. The experiment was considered to be valid.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

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

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

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)

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

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.

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.

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.

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.

 

 

 

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”.

Reference 2

Endpoint:

eye irritation, other

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2021

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model, but not (completely) falling into its applicability domain, and documentation / justification is limited

Justification for type of information:

1. SOFTWARE: OECD Toolbox 4.4.1.
2. MODEL: Eye irritation/corrosion Exclusion rules by BfR - Scheme type Linear - Scheme nature: EndpointSpecific - Version: 2.0 - December 2016.
3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: [K+].CC1(C)S[C@@H]2[C@@H](NC(=O)COc3ccccc3)C(=O)N2[C@H]1C([O-])=O
4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL Developer: National Institute for Public Health and the Environment (RIVM), Netherlands Donator: National Institute for Public Health and the Environment (RIVM), Netherlands. The aim of this profiler is to classify chemicals with respect to its eye irritation or corrosion potential. Unlike most profilers, it determines compounds that do not exhibit an irritating / corrosive effect, namely those that are not characterized by below mentioned Risk Phrases according to Annex III of European Union Directive 67/548/EEC: Nature of special risks attributed to dangerous substances and preparations:
R34: Causes burns
R35: Causes severe burns
R36: Irritating to eyes (moderate eye irritation reversible within ≤ 21 days)
R41: Risk of serious damage to eyes (moderate but persistent eye lesions, eye corrosion)
Definition of structural classes for which separate skin irritation exclusion rules have been defined:
1. Class C - Structure contains only Carbon (C), hydrogen (H) and oxygen (O) atoms – Formula CxHyOz
2. Class CN - Structure contains only C, H, O and Nitrogen (N) atoms – Formula CxHyOzNa
3. Class CNHal - Structure contains only C, H, O, N and Halogen (Hal) atoms – Formula CxHyOzNaHalb
4. Class CNS - Structure contains only C, H, O, N and Sulphur (S) atoms – Formula CxHyOzNaSb
5. Class CHal - Structure contains only C, H, O and Hal – Formula CxHyOzHalb
Tsakovska I, Netzeva T, Worth A., (2005), Evaluation of (Q)SARs for the prediction of Eye Irritation/Corrosion Potential. Physico-chemical exclusion rules. Report for the European Chemicals Bureau, available at:
https://eurl-ecvam.jrc.ec.europa.eu/laboratories-research/predictive_toxicology/doc/Evaluation_of_Eye_Irritation_QSARs.pdf

5. APPLICABILITY DOMAIN The exclusion rules for eye irritation/corrosion are based on physico-chemical cut-off values to identify chemicals that do not exhibit eye irritation or corrosion potential. The parameters used for defining eye irritation rules are: Lipid Solubility, Octanol Water partition coefficient, Aqueous Solubility, Melting Point and Molecular Weight.

Qualifier:

no guideline required

Specific details on test material used for the study:

[K+].CC1(C)S[C@@H]2[C@@H](NC(=O)COc3ccccc3)C(=O)N2[C@H]1C([O-])=O

Irritation parameter:

other: (Q)SAR prediction

Remarks on result:

no indication of irritation

Interpretation of results:

other: not irritating

Conclusions:

The structural and parametric boundaries used to define the chemical categories, in “Eye irritation/corrosion Exclusion rules by BfR” profiler, confirmed that the structure of Penicillin V Potassium do not exhibit an eye irritation or corrosion potential.

Executive summary:

According to the “Eye irritation/corrosion Exclusion rules by BfR” profiler, Penicillin V Potassium do not exhibit an eye irritation or corrosion potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

No classification for skin and eye irritation is derived from the results.