Phenoxymethylpenicillin potassium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item is not genotoxic in the Ames test, the in vitro mammalian chromosome aberration test and the in vitro mammalian cell gene mutation test (HPRT assay).

Short-term genetic toxicity test results with penicillin V and VK indicate that these drugs are not genotoxic (Dunnick JK et al., 1989).

Link to relevant study records

Reference

Reference 1

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

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

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.

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 / 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

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.

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

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.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 July 2019 - 22 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 29th July, 2016

Deviations:

yes

Remarks:

There is a deviation from the guideline regarding the using of the positive control item Ethyl methanesulfonate (EMS). EMS, a known, widely used mutagen and clastogen according to the referred literature and the Test Facilit

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

COMMISSION REGULATION (EU) 2017/735 adopted 14 February 2017

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

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.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79: Chinese hamster lung male

Metabolic activation:

with and without

Metabolic activation system:

Liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

In order to determine the treatment concentrations of test item in the main cytogenetic study a pre-test on cytotoxicity was performed. The cells were seeded into 92 x 17 mm dishes (for tissue cultures in TC sterile quality) at 5 x 105 cells each and were incubated for 24 hours in 10 mL of DME (Dulbecco’s Modified Eagle’s) medium containing 10% foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/mL) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. Cell counts were performed after 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment) using a Bürker chamber. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. At harvest the cells were trypsinised, collected and cell counts were determined. Based on the cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. The volume of culture medium was 5 mL/dish for each per group. Precipitation was evaluated at the beginning and at the end of treatment by the unaided eye. Where cytotoxicity occurs, these concentrations are a range from the maximum to little or no toxicity. The results obtained were used for dose selection of the test item used in the main chromosome aberration assays (experiments A and B). In addition, the pH and osmolality were measured in these main tests.

Vehicle / solvent:

DME (Dulbecco’s Modified Eagle’s) medium: this solvent is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary Solubility Test, and its suitability is confirmed with the available laboratory’s historical database.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent: DME (Dulbecco’s Modified Eagle’s) medium

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

The main chromosome aberration assays were conducted in two independent experiments in the presence and in the absence of S9 mix.
Duplicate cultures were used at each test item concentration and the negative control cultures as well as the positive controls for treatment without and with S9 mix. 5 x 105 cells per culture were seeded for each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the test item.
The exposure period was 3 hours.
After test item exposure the cells were washed with DME (Dulbecco’s Modified Eagle’s) medium and then growth medium was added. Sampling was made at 20 hours after start of treatment (approximately 1.5 normal cell cycles from the beginning of treatment). For concurrent measures of cytotoxicity for all treated and negative control cultures, 5 x 10^5 cells were set up.
In the cytogenetic experiment B the exposure period without metabolic activation was 20 hours.
The exposure period with metabolic activation was 3 hours. Experiment B, as experiment A, included concurrent positive and negative controls. For each group 5 x 105 cells were seeded per culture. Sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

Measurement of pH and Osmolality
In the Experimental Performance Pre-test on Toxicity (Concentration selection) and in the Chromosome Aberration Assays A and B the pH of each one treatment solution weremeasurement prior to starting the treatment. In the Experimental Performance Pre-test on Toxicity (Concentration selection) the osmolality was not be measured, only in the Chromosome Aberration Assays A and B.

Preparation of Chromosomes
Cell cultures were treated with colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.

Analysis of Methaphase Cells
All slides were independently coded before microscopic analysis and scored blind.300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

Acceptability of the Assay
The chromosome aberration assay is considered acceptable because it meets the following criteria:–the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data, –concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control, –cell proliferation in the solvent control is adequate, –adequate number of cells and concentrations are analyzable, –all requested experimental conditions were tested unless one resulted in a positive result–the criteria for the selection of top concentration are fulfilled.

Evaluation criteria:

Treatment of results–The percentage of cells with structural chromosome aberration(s) was evaluated. –Different types of structural chromosome aberrations are listed, with their numbers and frequencies for experimental and control cultures. –Gaps were recorded separately and reported, but generally not included in the total aberration frequency. –Concurrent measures of cytotoxicity for all treated and negative control cultures in the main aberration experiment (s) were recorded. –Individual culture data were summarised in tabular form. –There were no equivocal results in this study. –pH and Osmolality data were summarised in tabular form.

Statistics:

For statistical analysisCHI2 test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control. The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too.The lower and upper 95% confidence intervals of historical control were calculated with C-chart.

Interpretation of ResultsProviding 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, – the increase is dose-related when evaluated with an appropriate trend test, – any of the results are outside the distribution of the laboratory historical negative control data. Providing that all acceptability criteria are fulfilled, the 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 laboratory historical negative control data. Both biological and statistical significance should be considered together. There is no requirement for verification of a clearly positive or negative response.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

All concentrations were run in duplicates (incl. negative and positive controls) and 300 (150 per culture) well-spread metaphases were assessed.

Conclusions:

Pen V Potassium did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to the maximum recommended concentration (2000 μg/mL) in the absence and presence of metabolic activation. Thus, the test item is considered as being non- clastogenic in this system.

Executive summary:

The test item Pen V Potassium, dissolved in DME (Dulbecco’s Modified Eagle’s) medium, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:Experiment A with 3/20 h treatment/sampling timewithout S9 mix:500, 1000and 2000μg/mL test itemwith S9 mix: 500, 1000and 2000 μg/mL test itemExperiment B with 20/20 h treatment/sampling timewithout S9 mix: 500, 1000and 2000μg/mL test itemExperiment B with 20/28 h treatment/sampling timewithout S9 mix: 500, 1000and 2000μg/mL test itemExperiment B with 3/28 h treatment/sampling timewith S9 mix: 500, 1000and 2000μg/mL test itemFollowing treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 μg/mL) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.In both experimental parts no relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation. All results were inside the distribution of the historical negative control data (based 95% control limit). There were no polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation. The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 μL/mL) and cyclophosphamide (5 μg/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

AMES-Test according to the US National Toxicology Program (NTP)

Principles of method if other than guideline:

Preincubation test according to Haworth et al., Environ. Mutagen. 5 (Suppl.1), 3-142, (1983) and Zeiger E. and Drake J.W., IARC Scientific Publ. No. 27, 303-313, (1980)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Name of test material (as cited in study report): Peniccilin V Potassium (CAS: 132-98-9)

Target gene:

his

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Species / strain / cell type:

S. typhimurium TA 1535

Species / strain / cell type:

S. typhimurium TA 1537

Metabolic activation:

with and without

Metabolic activation system:

1254 Aroclor induced rat and hamster liver

Test concentrations with justification for top dose:

0; 0.10; 0.33; 1.0; 3.3; 10 mg/plat

Vehicle / solvent:

water

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

other: There were 5 concentrations, 1 solvent control and 1 positive control with and without rat and hamster S-9 metabolic activation were performed. 4 strains (TA98, TA100, TA1535, TA1537) were tested.

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other:

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

Evaluation criteria:

A dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold, was evaluated as a "mutagenic response". When no increase in the number of revertants was elicited by the chemical, this was evaluated a "negative response" and when there was an absence of a clear-cut dose-related increase in revertants, when the dose-related increase in the number of revertants were not reproducible or when the response was of insufficient magnitude to support a determination of mutagenicity, this was evaluated a "questionable response".

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Penicillin V Potassium [CAS Nr. 132-98-9] was negative in these tests and the highest ineffective dose tested in any S. typhimurium strain was 10 mg/plate.

Conclusions:

Penicillin V Potassium [CAS Nr. 132-98-9] was negative in these tests and the highest ineffective dose tested in any S. typhimurium strain was 10 mg/plate.

Executive summary:

Penicillin V Potassium [CAS Nr. 132-98-9] was tested in Mortelsmans, K. et al. "Salmonella mutagenicity tests: II. Results from the testing of 270 chemicals" [Environ. Mutagen., Vol. 8 Suppl. 7, 1986 , S. 1-119]: the publication includes data of Salmonella mutagenicity results on 270 coded chemicals, encompassing 329 tests performed by three laboratories under contract to the National Toxicology Program (NTP). The preincubation modification of the Salmonella/mammalian microsome assay was used to test chemicals in up to five Salmonella strains in the presence and absence of rat and hamster liver S-9. With a few exceptions, inter- and intralaboratory reproducibility was good.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

No indications were obtained to justify a classification of the test item.