Phenoxymethylpenicillin potassium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 July 2019 - 17 September 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

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

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: Pen V Potassium
CAS No.:132-98-9
Lot No.: B519322
Appearance:White solid powder
Expiry date:30 April 2024
Purity:99.0 %
Storage: Room temperature, protected from light
Molecular mass:388.48 g/mol

In chemico test system

Details on the study design:

The test item was formulated in a non-GLP trial forming as follows: the solubility of the test item was evaluated at the concentration of 100 mM. Acetonitrile did not dissolve the test item, it sank to the bottom of the test tube even after vortexing. Therefore, the test item was tried to be dissolved in ultrapure water and a homogenous, clear and transparent solution was formed after about 3 minutes of vortexing at 100 mM. The formulation with ultrapure water was tried to be mixed with the buffers of the test system (phosphate and acetate buffer) in a ratio corresponding to the reaction sample assembly in order to observe the compatibility of the formulation with the buffers of the test system. No precipitate was observed in any cases, homogenous solutions were gained. Since ultrapure water is the preferred vehicle after acetonitrile according to the guideline and the formulation complied with all obligations of the test guideline, it was chosen as the appropriate vehicle for the study and solubility in other vehicles was not evaluated. The pre-experiments on solubility of the test item was not performed in compliance with the GLP-Regulations and is excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of present study.

The reactivity of a test chemical and synthetic Cysteine or Lysine containing peptides is evaluated by combining the test chemical with a solution of the peptide and monitoring the remaining concentration of the peptide following 24 hours of interaction time at room temperature. The peptide is a custom material containing phenylalanine to aid the detection and either Cysteine (“C”) or Lysine (“K”) as the reactive center. Relative concentrations of the peptides following the 24 hour incubation are determined by high performance liquid chromatography with gradient elution and UV detection at 220 nm. Reaction samples, reference controls A, B and C, co-elution controls and positive controls are prepared and analysed in triplicates in batches of up to 26 chemicals (including controls).
Steps of the DPRA Method done in chronological order
-Solubility assessment of the test chemical – ultrapure water was used as a solvent
-Preparation of buffer solutions -Pre-weighting of test chemicals and positive control
-Pre-weighting of cysteine or lysine peptide for the stock solution
-Test chemical and positive control solution preparation
-Peptide stock solution preparation -Serial dilution of standards
-Assembling of standards, reaction samples, positive controls, reference controls (A, B and C) and co-elution controls. For each set of control/sample replicates, the triplicate vials are prepared individually but from the same solutions.
-Preparation of HPLC system (column equilibration)
-HPLC analysis
-Data evaluation

HPLC system:SHIMADZU LC2030 (Prominence-i LC-2030C)
Serial number:L21445402951AE
Detector:220 nm – D2 lamp
Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
Serial number:USRY003976
Column temperature: 30°C
Sample temperature: 25°C
Injection volume: 7μL
System equilibration: 50% phase A and 50% phase B for 2 hours at 30°C and running the gradient twice before injecting the first sample
Run time:20 min
Flow conditions: gradient flow
Mobile phases for HPLC:
Mobile Phase A – 0.1 % (v/v) trifluoroacetic acid in ultra-pure water
Mobile Phase B – 0.085 % (v/v) trifluoroacetic acid in acetonitrile

Results and discussion

Positive control results:

The mean peptide depletion value for the positive control was 61.53 %

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

mean

Parameter:

other: cysteine depletion (migrated information)

Value:

4.94 %

Vehicle controls validity:

valid

Negative controls validity:

not specified

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

mean

Parameter:

other: lysine depletion (migrated information)

Value:

15.11 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on these results and thecysteine 1:10 / lysine 1:50 prediction model, the test item “Pen V Potassium” was concluded to be positive and to show low reactivity towards the synthetic peptides thus is a potential skin sensitizer under the experimental conditions of the in chemico Direct Peptide Reactivity Assay (DPRA) method.

Executive summary:

In the course of this study the skin sensitization potential of the test item “Pen V Potassium” was studied using the Direct Peptide Reactivity Assay (DPRA). For the test chemical and positive control substance, in order to derive a prediction two independent tests were conducted, one with cysteine and lysine peptides each. The results of the two valid runs were used for the classification of the test item.Peptide depletion resulted from the positive control cinnamaldehyde was 70.67 % ± 0.09 % with cysteine peptide and 52.39 % ± 0.15 % with the lysine peptide. The mean back-calculated peptide concentrations of the reference control replicates were within the expected molarity concentration range for the cysteine (0.48 – 0.51 mM) and lysine peptides (0.49 mM – 0.50 mM) and the CV % for the for the nine reference controls B and C in acetonitrile were 2.7 % and 0.4 % percentages for the cysteine and lysine peptides. For each peptide, all validity criteria were met, confirming the validity of the assay.The percent cysteine peptide depletion value of the test item was 4.94 % ± 3.83 % while the percent lysine peptide depletion was 15.11 % ± 0.48 %. The mean depletion value of the peptides was used to categorize the test chemical in one of the four classes of reactivity. No co-elution was observed with either cysteine or lysine peptides; therefore the cysteine 1:10 / lysine 1:50 prediction model was used for the discrimination between sensitizers and non-sensitizers. The mean peptide depletion of the test item was 10.03 %, which exceeded the 6.38 % threshold of the applicable prediction model and fell into the low reactivity class.