Phenoxymethylpenicillin potassium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

5 July 2019 - 22 August 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 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

29 July 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

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
Chemical name:Phenoximethylpenicillin Potassium
Structural formula: C16H17KN2O5S
Active component: ≥ 99 %
Batch No.: B519322
Appearance:White solid powder, weakly odorous
Date of production: April 2019
Expiry date: April 2024
Storage: Room temperature (15-25°C, humidity 50 ± 10%), protected from light.

Method

Target gene:

The method is based on the detection of mutations (either induced or spontaneously generated) in the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) located on the X chromosome.HPRT is a cellular enzyme that allows cells to salvage hypoxanthine and guanine from surrounding medium for use in DNA synthesis. If a toxic base analog 6-thioguanine (6-TG) is present in the medium, then the analogue will be phosphorylated via the HPRT pathway and incorporated into the nucleic acid. Thus, the cells die unless the enzyme is rendered inactive by mutation.

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

Sub-line (K1) of Chinese hamster ovary cell line CHO

Details on mammalian cell type (if applicable):

The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line and was purchased from ECACC (European Collection of Cell Cultures). The cell stocks are kept in liquid nitrogen. Each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary; results were fully documented within the raw data file. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks. The CHO K1 cells for this study were grown in Ham's F12 medium(F12-10) supplemented with 1 % antibiotic-antimycotic solution (containing 10000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %). During the 5 treatments with the test item, solvent (negative control) and positive controls, the serum content was reduced to 5 % (F12-5). The selection medium for TG resistant mutants contained 3.4 μg/mL 6-thioguanine (6-TG) (EX-CELL® CD CHO Serum-Free Medium for CHO Cells-SEL).

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

Test concentrations with justification for top dose:

200 - 500 - 1000 - 2000 μg/mL.

Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity [a non GLP Preliminary Solubility Test was performed May 29, 2019. The test item was dissolved in Ham's F12 medium: A clear solution was obtained up to a concentration of 100 mg/mL.] During the pre-test on toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes (tissue cultures quality: TC sterile) at 5x106 cells each and incubated with culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in the absence or presence of S9 mix (50 μL/mL) andincubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration was adjusted to 40 cells/mL with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days. Colonies were then fixed with methanol and stained with Giemsa and the colonies werecounted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative control. Precipitation of the test item was visually examined at beginning and end of the treatments. The results of the pre-test on cell toxicity were used for dose selection of the test item concentrations used in the main mutation assay. Four concentrations were selected.

Vehicle / solvent:

Name: Ham's F12 medium
Supplier: Sigma-Aldrich, Germany
Lot Number: RNBH3381, RNBH0847
Expiry date: February 2020, October 2019
Storage condition: At 2-8 °C

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Mutation Assay Experimental Design
A 5-hour treatment in the presence and absence of S9-mix was performed. For the 5-hour treatment, 5 x106 cells were each placed in sterile dishes and incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2. Duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix. On the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item. Following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours. After the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber. Solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment. In samples where sufficient cells survived, cell number was adjusted to 105 cells/mL. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

Measurement of pH and Osmolality
In the pre-test on toxicity (dose selection) and in the main mutation assays the pH of each treatment solution were measured at the start of the treatment. In the pre-test on toxicity (dose selection) the osmolality was not be measured, only in the main mutation assay.

Plating for Survival
A total of 5 mL (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies. Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.

Expression of the Mutant Phenotype
During the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x106 cells were taken on days 1, 3, 6 and evaluated on day 8.

Selection of the Mutant Phenotype
At the end of the expression period, cultures from each dose level were adjusted to 2 x 105 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 μg/mL of thioguanine (6-TG).

Plating for Viability
At the end of the expression period cell number in the samples was adjusted to 2 × 105 cells/mL. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.

Fixation and Staining of Colonies After the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.

Rationale for test conditions:

Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity (A non GLP Preliminary Solubility Test was performed May 29, 2019. The test item was dissolved in Ham's F12 medium. A clear solution was obtained up to a concentration of 100 mg/mL). During the pre-test on toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes (tissue cultures quality: TC sterile) at 5x106 cells each and incubated with culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in the absence or presence of S9 mix (50 μL/mL) and incubated at 37 °C for 5 hours. For each concentration of test solution or control solution, 5 mL was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm). The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for colony growing. Colonies were then fixed with methanol and stained with Giemsa and the colonies werecounted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control. Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments. In addition, pH was considered for dose level selection. The results of the pre-test on cell toxicity were used for dose selection of the test item concentrations used in the main mutation assay. Four concentrations were selected for the treatment without and with metabolic activation system, respectively.

Evaluation criteria:

The assay was considered valid if:
•The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
•The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
•Adequate number of cells and concentrations were analysable.
•Two experimental conditions with and without metabolic activation were tested.
•The highest concentration is adequate.
•The cloning efficiency of the negative controls is between the range of 60 % to 140 % [Day 1] and 70% to 130 % on Day 8.
Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
•at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
•any of the results are outside the distribution of the laboratory historical negative control data (based 95% control limit),
•the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
•none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
•there is no concentration-related increase when evaluated with an appropriate trend test,
•all results are inside the distribution of the historical negative control data (based 95% control limit). The test chemical is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:•mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups. •mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups •The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis by Microsoft Excel software.

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Applicant's summary and conclusion

Conclusions:

Pen V Potassium tested up to the maximum recommended concentration (2000 μg/mL) with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevantincreases in mutant frequency. It is concluded that the test item, Pen V Potassium, was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.

Executive summary:

The test item Pen V Potassium, dissolved in Ham's F12 medium, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver: Mutation Assay 5-hour treatment period without S9-mix: 250, 500, 1000 and 2000 μg/mL Mutation Assay 5-hour treatment period with S9-mix: 250, 500, 1000 and 2000 μg/mLIn the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for lower-cytotoxic substances is 2000 μg/mL (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure. There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.In both experimental parts, there were no increases in mutation frequency when compared to the concurrent solvent control and the laboratory historical control data at any concentration tested in the absence and presence of metabolic activation. All results were inside the range of the historical negative control data (based 95% control limit). The mutation frequency found in the solvent controls was in the 95% confidence interval of the historical control data. The concurrent positive controls ethyl methanesulfonate (1.0 μL/mL) and 7, 12-dimethyl benzanthracene (20 μg/mL) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid. Pen V Potassium tested up to the maximum recommended concentration (2000 μg/mL) with and without metabolic activation over a 5 hour treatment period did not induce statistically significant and biologically relevantincreases in mutant frequency. It is concluded that the test item, Pen V Potassium, was not mutagenic in this in vitro mammalian cell gene mutation test performed with Chinese hamster ovary cells.