Phenoxymethylpenicillin potassium

Toxicity to reproduction

Administrative data

Endpoint:

extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the extended one-generation reproductive toxicity study does not need to be conducted because there are no results from available repeated dose toxicity studies that indicate adverse effects on reproductive organs or tissues, or reveal other concerns in relation with reproductive toxicity

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
No adverse effects were observed in reproductive organs or tissues when the substance was tested in sub-acute, sub-chronic or chronic animal studies. See the cross-referenced studies.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline available

Principles of method if other than guideline:

Two-years study design. Groups of 50 rats and 50 mice of each sex received penicillin VK in corn oil by gavage 5 days for 103-104 weeks. Rats received 0, 500, or 1000 mg/kg penicillin VK. Mice 0, 500, or 1000 mg/kg penicillin VK. The control groups received corn oil alone. At the conclusion of the 2-year dosing period, animals were kept for 1 to 2 weeks of observation without dosing and killed by carbon dioxide inhalation. Necropsy was performed on all animals. ods (Cox, 1972). Differences in survival were analyzed by life-table methods (Cox, 1972).

GLP compliance:

not specified

Specific details on test material used for the study:

USP grade lots C9014 and H1688

Species:

other: Rodents : Male and female F344/N rats and B6CF3 mice obtained from Charles River Breeding Laboratories (portage, MI)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Rodents were placed on study at 7-8 weeks of age. Animals were assigned to groups using a table of random numbers and were housed by sex, five
per cage, in polycarbonate cagescovered with fiber filters and were provided with heat-treated hardwood chips as bedding (Ancare Corp., L.I., NY). Tap water and NIH 07 feed (Zeigler Bros, Gardners, PA) were provided ad libitum. The animals were maintained in a room that was kept at 66-81°F [19 - 27 °C] with 10- 12 air changes per hour, and a 12-hr fluorescent light cycle. All animals were checked daily for clinical signs and moribund animals were necropsied. Animal body weights were taken once a week during the first 13 weeks of study and thereafter every 4 weeks.

Route of administration:

oral: feed

Vehicle:

corn oil

Details on oral exposure:

Rodents were placed on study at 7-8 weeks of age. Animals were assigned to groups using a table of random numbers and were housed by sex, five
per cage, in polycarbonate cagescovered with fiber filters and were provided with heat-treated hardwood chips as bedding (Ancare Corp., L.I., NY). Tap water and NIH 07 feed (Zeigler Bros, Gardners, PA) were provided ad libitum. The animals were maintained in a room that was kept at 66-81°F [19 - 27 °C] with 10- 12 air changes per hour, and a 12-hr fluorescent light cycle. All animals were checked daily for clinical signs and moribund animals were necropsied. Animal body weights were taken once a week during the first 13 weeks of study and thereafter every 4 weeks.

Analytical verification of doses or concentrations:

yes

Remarks:

Dose solutions were checked throughout the course of the study and were within ± 10% of the targeted concentrations (Dunnick, 1987, 1988).

Duration of treatment / exposure:

103-104 weeks

Frequency of treatment:

Daily once. 5 days weekly

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Groups of 50 rats and 50 mice of each sex received Penicillin VK in corn oil.

Control animals:

yes, concurrent no treatment

Positive control:

NO

Observations and examinations performed and frequency:

All animals were checked daily for clinical signs and moribund animals were necropsied. Animal body weights were taken once a week during the first 13 weeks of study and thereafter every 4 weeks.

Sacrifice and pathology:

Necropsy was performed on all animals. Tissues were preserved in 10% neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The following tissues were examined microscopically: grossly observed tissue masses,mandibular, or mesenteric lymph nodes; salivary glands;
bone including marrow;
thyroid gland, parathyroids;
small intestine;
large intestine;
liver;
prostate/testes/epididymis or ovaries/uterus;
lungs with mainstem bronchi;
skin;
heart;
esophagus;
stomach;
brain;
thymus;
trachea;
pancreas;
spleen;
kidneys;
adrenal glands;
urinary bladder;
pituitary gland;
eyes (if gross lesions were evident);
and mammary glands.

Statistics:

Differences in survival were analyzed by life-table methods (Cox, 1972). For the analysis of tumor incidence data, survival-adjusted procedures were used to assessdose-response trends and to make pairwise comparisons between dosed groups and controls (Haseman, 1984). Fisher exact tests and Cochran rmitage trend tests were also utilized to assesstumor incidence data. Results are considered as significant where the p value is ~0.05.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs were observed sporadically and included diarrhea in rats and male
mice dosed with penicillin.

Mortality:

no mortality observed

Description (incidence):

Survival in all groups of rats was 50% or greater until Week 92. Survival of
mice dosed with penicillin VK was comparable to vehicle controls.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weights of male and female rats receiving penicillin VK were similar to the corresponding vehicle control groups. Mean body weights of male mice receiving penicillin VK were comparable to controls, but mean body weights of dosed female mice were 4- 16% lower than those of the vehicle controls from Week 28 to the end of the study.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Nonneoplastic lesions of the glandular stomach were seen in dosed male
and female mice, and lesions of the gallbladder were seen in male mice. The incidence of hepatocellular adenomas was decreased in high-dose male mice (14/50, 15/49,4/49).

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

No compound-related increases in neoplasms were seen in male or female rats or mice.

Key result

Dose descriptor:

LOAEL

Effect level:

> 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

500 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

There is no evidence of carcinogenicity in rats or mice after penicillin VK dosing. Short-term genetic toxicity test results with ampicillin trihydrate and penicillin V and VK indicate that these drugs are not genotoxic (Dunnick, 1987, 1988). The most common side effects reported in humans after penicillin tratment are hypersensitivity (anaphylactoid reactions). Gastrointestinal toxicity was seen in mice after penicillin V Potassium amministration and this target organ toxicity is similar to what has been observed in humans.

Executive summary:

Toxicology and carcinogenesis studies of ampicillin trihydrate and penicillin VK, two widely used β-lactam antibiotics, were performed in F344/N rats and B6C3F, mice. In these studies ampicillin trihydrate was administered for 2 years to rats at doses of 0, 750, or 1500 mg/kg and to mice at doses of 0, 1500, or 3000 mg/kg, and penicillin VK was administered to rats and mice at doses of 0, 500, or 1000 mg/kg. Both drugs were administered
by oral gavage in corn oil. Toxic lesions of the stomach were seen in rats and mice after ampicillin trihydrate administration and in mice after penicillin VK administration. There was no evidence for
carcinogenic activity in female rats or male and female mice after ampicillin trihydrate administration or in rats and mice after penicillin VK administration.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12/12/1979 - 21/03/1980

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

Thirteen-week studies were conducted to evaluate the cumulative toxic effects of repeated administration of penicillin VK and to determine the concentrations to be used in the 2-year studies.
Five-week-old male and female F344/N rats and 4- to 6-week-old male and female B6C3F1 mice were obtained from Charles River Breeding Laboratories and were observed for 15 days before the studies began. Rats and mice were housed five per cage in polycarbonate cages. NIH 07 Rat and Mouse Ration pellets and water (half deionized/half tap) were available ad libitum.
Groups of 10 rats of each sex were administered 0, 180, 370,750, 1,500, or 3,000 mg/kg penicillin VK in corn oil by gavage, 5 days per week for 13 weeks. The 3,000 mg/kg groups of rats were given two doses of 1,500 mg/kg, 5 hours apart. Groups of 10 mice of each sex received 0, 250, 500, 1,000, 2,000, or 3,000 mg/kg on the same schedule.
Animals were checked two times per day; moribund animals were killed. Individual animal weights were recorded weekly. At the end of the
13-week studies, survivors were killed. A necropsy was performed on all animals except those excessively autolyzed or cannibalized.

GLP compliance:

yes

Species:

other: rodents

Strain:

other: F344/N rats and B6C3F1 mice

Details on species / strain selection:

Five-week-old male and female F344/N rats and 4- to 6-week-old male and female B6C3F1 mice were obtained from Charles River Breeding Laboratories and were observed for 15 days before the studies began.

Sex:

male/female

Route of administration:

oral: gavage

Details on route of administration:

The chemical (94% or 98% pure, USP grade) was administered orally (by gavage in corn oil) because oral administration is the primary route used to treat infections in humans.

Vehicle:

corn oil

Details on oral exposure:

Groups of 10 rats of each sex were administered 0, 180, 370,750, 1,500, or 3,000 mg/kg penicillin VK in corn oil by gavage, 5 days per week for 13
weeks. The 3,000 mg/kg groups of rats were given two doses of 1,500 mg/kg, 5 hours apart. Groups of 10 mice of each sex received 0, 250, 500, 1,000, 2,000, or 3,000 mg/kg on the same schedule.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability studies of penicillin VK mixed with NIH 07 Rat and Mouse Ration indicated that penicillin VK at a concentration of 1,000 ppm was unstable when stored for 2 weeks at temperatures ranging from 5 °C to 45 °C. Recovery of penicillin VK after 2 weeks' storage, sealed and protected from air and light, was 85% a t 5 °C, 40% at 25"C, and 7%at 45 °C. Because of the instability of penicillin VK mixed in rodent feed, corn oil was investigated as a possible vehicle for gavage studies. Penicillin VK and corn oil were mixed to give the desired concentrations. Stability studies of a 10 mg/ml dose mixture stored for 2 weeks at room temperature or 6 °C were performed by extraction with 0.01 M sodium dihydrogen phosphate:methanol (1:4) and analysis by high-performance liquid chromatography on a Varian MicroPak Cl8 column with an aqueous 0.01 M sodium dihydrogen phosphate:methanol (52:48) mobile phase a t a flow rate of 1ml/minUte and ultraviolet detection at 254 nm. The concentration of penicillin VK was calculated from an acetanilide internal standard. Penicillin VK (10 mg/ml) in corn oil was found to
be stable when stored at room temperature or 5"C for 14days. In the 13-week dose mixtures were stored a t 4 °C for no longer than 2 weeks. Formulations of penicillin VK in corn oil were periodically selected at random at the study laboratory, extracted with the same extraction solvent listed above, and analyzed in duplicate by ultraviolet spectroscopy (269 nm) to estimate the accuracy with which formulations were prepared over the course of the studies. Dose mixtures were analyzed once during the 13-week studies; the results ranged from 94% to 107% of the target concentrations. Because 40/41 dose mixtures analyzed were within +10% of the tar get concentrations, the dose mixtures were estimated to have been within specifications 98%of the time throughout the 2-year studies. Referee analyses were periodically performed by the analytical chemistry laboratory. The penicillin VK concentrations in one referee sample were outside of specifications.

Duration of treatment / exposure:

13 weeks

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

rat and mice

Dose / conc.:

180 mg/kg bw/day (actual dose received)

Remarks:

rats

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

mice

Dose / conc.:

370 mg/kg bw/day (actual dose received)

Remarks:

rats

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

mice

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

rats

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

mice

Dose / conc.:

1 500 mg/kg bw/day (actual dose received)

Remarks:

rats

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

mice

Dose / conc.:

3 000 mg/kg bw/day (actual dose received)

Remarks:

rats and mice: given two doses of 1,500 mg/kg, 5 hours apart.

No. of animals per sex per dose:

10 males and 10 females of each species

Control animals:

yes, concurrent vehicle

Positive control:

No

Observations and examinations performed and frequency:

All animals were observed two times per day, and clinical signs were recorded at least once per month. Body weights by cage were recorded once per week for the first 12 weeks of the studies and a t least once per month thereafter.
Mean body weights were calculated for each group. Animals found moribund and those surviving to the end of the studies were humanely killed.

Sacrifice and pathology:

A necropsy was performed on all animals including those found dead, unless they were excessively autolyzed or cannibalized, missexed, or found missing. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and is not necessarily equal to the number of animals that were placed on study.
During necropsy, all organs and tissues were examined for grossly visible lesions. Tissues were preserved in 10%neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin.
Necropsy performed on all animals. Histologic exam performed on vehicle
control and 3,000 mg/kg groups and on animals that died before the end of the
studies; tissues examined include:
adrenal glands,
brain,
esophagus,
eyes (if grossly abnormal),
gross lesions,
heart,
kidneys,
large intestine,
liver,
lungs and mainstem bronchi,
mammary gland,
pancreas,
parathyroids,
pharynx (if grossly abnormal),
pituitary gland, prostatel teste depididymis or ovaries/uterus,
salivary glands,
skin,
small intestine,
spinal cord (if neurologic signs present),
spleen,
sternebrae or femur or vertebrae including marrow, ù
stomach,
thymus,
thyroid gland,
tissue masses,
trachea,
and urinary bladder

Statistics:

Data Recording: Data on this experiment were recorded in the Carcinogenesis Bioassay Data System (Linhart et al., 1974). The data elements include descriptive information on the chemicals, animals, experimental design, survival, body weight, and individual pathologic results, as recommended by the International Union Against Cancer (Berenblum, 1969).

Survival Analyses: The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is presented in the form of graphs. Animals were censored from the survival analyses a t the time they were found to be missing or dead from other than natural causes; animals dying from natural causes were not censored. Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone’s (1975) life table test for a doserelated trend. When significant survival differences were detected, additional analyses using these procedures were carried out to determine
the time point at which significant differences in the survival curves were first detected. All reported P values for the survival analysis are two-sided.
Calculation of Incidence: The incidence of neoplastic or nonneoplastic lesions is given as the ratio of the number of animals bearing such lesions at a specific anatomic site to the number of animals in which that site was examined. In most instances, the denominators include only those animals for which the site was examined histologically. However, when macroscopic examination was required to detect lesions (e.g., skin or mammary tumors) prior to histologic sampling, or when lesions could have appeared a t multiple sites (e.g., lymphomas), t h denominators consist of the number of animals on which a necropsy was performed.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Rats: Diarrhea was observed for males that received 750 mg/kg or more and for females that received 1,500 or 3,000 mg/kg. Minimal to mild mucous cell metaplasia of the glandular stomach was observed in 719 males and 818 females that received 1,500mg/kg and in 416 males and 818 females that received 3,000 mg/kg. This change occurred primarily adjacent to the junction of
the glandular stomach and forestomach and consisted of a replacement of parietal and chief cells by a mucous cell type.

Mice: Inflammation, mucous cell metaplasia of the glandular stomach, and papillary hyperplasia or hyperkeratosis of the forestomach were seen at increased incidences in dosed groups. The degree of severity was dose dependent. Eosinophilic cytoplasmic change, characterized by the accumulation of eosinophilic material in the cytoplasm of glandular epithelial cells near the junction of the glandular stomach and forestomach,occurred in dosed mice.

Mortality:

no mortality observed

Description (incidence):

Mice: all deaths were considered to be due to gavage error.
Rats:

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Rats: Final mean body weights of rats that received 3,000 mg/kg were 11%lower than those of the vehicle controls for males and 6% lower for females.

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Key result

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg bw/day (actual dose received)

System:

gastrointestinal tract

Organ:

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

In the 13-week studies, male and female rats received doses of 180-3,000 mg/kg and male and female mice received doses of 250-3,000 mg/kg. No compound-related deaths were seen in rats or mice. Final mean body weights of rats that received 3,000 mg/kg were 11% lower than those of the vehicle controls for males and 6% lower for females. For mice, mean body weights were comparable. Diarrhea occurred in male rats at doses of 750mg/kg and above and in female rats at doses of 1,500and 3,000 mg/kg. Mucous cell metaplasia of the glandular stomach was observed in male and female rats receiving 1,500 and 3,000mg/kg. Lesions of the glandular stomach (inflammation, mucous cell
metaplasia, and eosinophilic cytoplasmic change) and the forestomach (papillary hyperplasia and hyperkeratosis) were seen in all groups of dosed mice. The severity of lesions at 1,000mg/kg or below was considered minimal. Based on these results, doses selected for rats and mice in the 2-year studies were 0, 500, or 1,000mg/kg

Executive summary:

In the 13-week studies, penicillin VK was administered at doses up to 3,000 mg/kg. The gas trointestinal tract was the site primarily affected, with glandular stomach and/or forestomach lesions observed in dosed rats and mice. No dose-related deaths occurred; the extent and severity of gastrointestinal lesions and body weight data were the primary factors used to select doses for the 2-year studies. In both the 13-week and 2-year studies of penicillin VK in F344/N rats and B6C3F1 mice, the gastrointestinal tract was a primary site for toxicity. In the ampicillin trihydrate 2-year studies, gastrointestinal toxicity also was seen in F344/N rats and B6C3F1 mice (NTP, 1987). In rats administered ampicillin trihydrate, diarrhea and hyperkeratosis and acanthosis of the
forestomach were seen for dosed animals. Mice administered ampicillin trihydrate had doserelated forestomach lesions (not glandular stomach lesions as seen in the penicillin VK study), including inflammation, hyperkeratosis, acanthosis, and ulcers. D-Lactam antibiotics have been shown to cause gastrointestinal toxicity in humans (Kitano et al., 1984; Braver, 1983;
Norrby, 1986)and in rodents (Murakami, 1971; Berg, 1981). Oral penicillin administration altered the population of enteric bacteria in mice, decreased the total anaerobe population, and allowed an overgrowth of gram-negative enteric bacilli in the ceca (Berg, 1981) Although penicillin is an antimicrobial agent, Salmonella can be used to assay its mutagenic activity because an end point other than cell death is monitored; the mutagenic activity of
penicillin was measured at doses that did not produce excessive toxicity. The presence of the plasmid pKMlOl in strains TA98 and TA100 confers resistance to penicillin but not total immunity. Therefore, the doses of penicillin VK tested in these two strains are almost 100-fold higher than the highest nontoxic dose tested in strainsTA1535 and TA1537; in all cases, no evidence for mutagenicity was observed before toxic concentrations were reached. Penicillin VK, other penicillins, and penicillin analogs are consistently nonmutagenic when tested in bacteria.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 November 2019 - 05 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

data waiving: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 October 2008

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Name:Pen V Potassium
Batch No.: B519322
Chemical name: Phenoxymethylpenicillin Potassium
Structural formula:C16H17KN2O5S
Active ingredient:≥ 99 %
Appearance:White, solid powder
Expiry date: April 30, 2024
Storage condition: At room temperature (15-25 oC), protected from light
Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials should be applied to assure personnel health and safety.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species / Strain:Han:WIST rat of Wistar originSource:Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Hygienic level: SPF (Specific pathogen-free) at arrival and kept in good conventional environment during the study.
Age of animals at start of the study :
Male animals: 40 – 44 days
Female animals: 39 – 42 days
Body weights at start of the study:
170 – 188 g for male animals
114 – 130 g for female animals
The weight variation did not exceed ± 20 per cent of the mean weight.
Number and sex of animals: 40 rats (20 male and 20 female - nulliparous and non-pregnant animals) Number of groups: 4 (3 dose levels + 1 control group)
Number of animals/groups: 10 (5 male; 5 female)
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder.
Acclimatization time:7 days

Reason for selection of species
The rat is a commonly used species for toxicological studies in accordance with international recommendations. The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests and is a well-known laboratory model with sufficient historical data.

Housing conditions
Animal room no.:18/1
Housing: 5 animals of the same sex/ cage
Cage type: Type IV polypropylene/ polycarbonate Bedding: Certified laboratory wood bedding (SAFE 3/4-S FASERN produced by J. Rettenmaier & Söhne GmbH+Co.KG; D-73494 Rosenberg Holzmühle 1 Germany). see Appendix 13). The cages and bedding were changed twice a week.
Illumination:Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C Relative humidity:30 - 70 %
Ventilation: Above 10 air-exchanges/ hour by a central air-condition system.
Environmental conditions were maintained by an air-conditioning system.
Temperature and relative humidity were verified and recorded daily during the study.

Animals received ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniffSpezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum except overnight food deprivation before the blood sampling. The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Animals received tap water from watering bottles. Water quality control analysis and microbiological assessment are performed once every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Váci út 174. Budapest, H-1138 Hungary). The quality control results are available at Toxi-Coop Zrt.’s archives

Route of administration:

oral: gavage

Details on route of administration:

The test item was administered orally via gavage. The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item. Animals were randomly assigned to test groups. All animals were sorted according to body weight by computer and grouped according to weight ranges. There were an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping was controlled by SPSS/PC+ computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.

Vehicle:

water

Remarks:

Animals in Group 1 only received the vehicle.

Details on oral exposure:

The dose setting with 0, 100, 300 and 1000 mg/kg bw/day has been chosen on the basis of the results of the 14-day oral (by gavage) study with Pen V Potassium in rats (Toxi-Coop study no. 880-400-4596) and in agreement with the Sponsor. Pen V Potassium did not cause adverse systemic effects – clinical signs, changes in body weight, food consumption, hematology and blood coagulation, clinical chemistry, organ pathology or organ weights – in male or female Han:WIST rats after the consecutive 14- day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day. Doses were selected with the aim of inducing toxic effects but no mortality or suffering at the highest dose and a NOAEL at the lowest dose. In addition, the study was intended to allow the dose setting for the planned OECD 421/422 study.

Duration of treatment / exposure:

The experimental period involved 7 days of acclimatization, 28 days of treatment and observation period and necropsy on the following day (study Day 28). The day of first treatment was considered as Day 0 of examination

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Remarks:

Group 1: Dosing volum: 5mL/kg bw Male 5 + Female 5

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Group 2: Dosing volum: 5mL/kg bw Male 5 + Female 5

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Remarks:

Group 3: Dosing volum: 5mL/kg bw Male 5 + Female 5

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Group 4: Dosing volum: 5mL/kg bw Male 5 + Female 5

No. of animals per sex per dose:

Male 5 + Female 5

Control animals:

yes, concurrent vehicle

Details on study design:

Selection of animals
Forty (40) healthy rats (twenty males and twenty females) were used in the study. Animals were selected for this study on the basis of adequate body weight, a body weight within± 20% of the mean within a sex and free from clinical signs of disease or injury. Selected rats were distributed by randomization according to stratification by body weight so that there was no statistically significant difference among group body weight means within a sex.

Administration of test item
The test item was orally administered by gavage daily (7 days per week) in graduated doses to three groups of experimental animals for a period of 28 days. A constant treatment volume of 5 mL/kg body weight was administered in all groups. Control animals were treated concurrently with the vehicle only. The actual treatment volume was calculated according to the most recent body weight. A treatment volume of 5 mL/kg body weight was applied. Animals were not treated on the day of gross pathology. Animals were not treated on the day of necropsy.

Mortality
Animals were inspected for signs of morbidity and mortality twice daily ( at the beginning and end of each working day).

Clinical observations
General clinical observations were made cage-side once a day, after treatment at approximately the same time.On the day prior to the first treatment with the test item and approximately once weekly thereafter, detailed observations were conducted while handling the animal on days that the animals were weighed and food consumption measurements were taken. Potential signs noted included but were not limited to: changes in skin, fur, eyes, and mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g., lacrimation, piloerection, pupil size, and unusual respiratory pattern). Likewise, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotype activities (e.g., excessive grooming, repetitive circling), or bizarre behavior (e.g., self-mutilation, walking backwards) were considered. All observations were recorded.In the last exposure week, functional observations were conducted. Sensory reactivity to stimuli of different types, assessment of grip strength and motor activity assessment wereexamined. General physical condition and behavior of animals were tested. A modified Irwin test were performed. (Irwin, S. Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968; Toxi-Coop SOP no. ATK 001).

Body weight and body weight gain
Individual body weights were recorded once during acclimatization. The body weight of animals involved in the study were determined on Day 0 (prior to study start) and twice weekly with an accuracy of 1 g. The animals were weighed prior to sacrifice in order to calculate organ to body weight ratios. Individual body weight changes were calculated according to the days of measurements and for the study overall.

Food consumption measurement
Food consumption was determined with the measurement of given and non-consumed diet with an accuracy of 1 g once weekly to coincide with body weight measurements (Days 0, 7, 14, 21 and 27). Food consumption were evaluated and reported by weekly interval for each group.

Observations and examinations performed and frequency:

Clinical pathology
Hematological, blood coagulation and clinical biochemistry examinations were conducted at termination of the treatment (i.e. one day after the last treatment: on Day 28). Animals were food deprived for approximately 16 hours prior to blood collection. Blood samples were harvested from the retro orbital venous plexus under Isofluran CP® anesthesia. Three samples were taken from each animal: one for determination of blood clotting times, one for hematology and the third one to obtain serum samples for clinical chemistry.

Hematology
Blood samples for hematology measurements were collected in tubes containing K3EDTA (MiniCollect® EDTA tubes, spray-dried, 0.5 mL, Greiner Bio-One International AG) and tubes were filled up to the final volume marked on the tubes. Blood were analyzed after the sampling.

Blood coagulation
Blood samples for determination of blood clotting times (APTT and PT) were collected in tubes containing 9NC Coagulation sodium citrate 3.2 % (MiniCollect® 1 mL; manufactured by Greiner Bio-One International AG, Kremsmünster, Austria). Tubes were filled up to the final volume marked on the tubes. Blood were centrifuged at 2500 rpm for 15 minutes within 20 – 30 minutes after the sampling. Supernatant plasma samples were measured.

Clinical chemistry
Blood samples collected for clinical chemistry measurements were drawn in tubes without anticoagulant (VACUETTE® Serum Tube, 2.5 mL, Greiner Bio-One International AG). At least 1.0 mL blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm for 15 minutes. Serum samples were stored at 2-8°C and measured.

Sacrifice and pathology:

Necropsy
Gross pathology was performed on every animal one day after the last treatment, on Day 28. Animals were anesthetized with Isofluran CP® and were exsanguinated from the abdominal aorta after verification of deep narcosis.The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size. The following organs/tissues were removed and preserved in 4 % formaldehyde solution, except testes and epididymides, which were preserved in modified Davidson solution and then stored in 4 % formaldehyde solution.

Thyroid and parathyroid were preserved together with larynx but larynx was not processed histologically. Organs and tissues were excised, trimmed of any adherent tissue, as appropriate, weighed, and preserved as described above.

The following organs were weighed and recorded. Paired organs were weighed together. With precision of 0.01g: Liver, kidneys, testes, epididymides, seminal vesicles with coagulating gland and prostate as a whole, thymus, spleen, brain and heart. With precision of 0.001g: Adrenal glands, ovaries .

Histopathology Histopathological examinations were performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively). In addition, gross lesions noted in animals in low and mid dose test groups at the time of terminal sacrifice were also examined as follows: -kidneys in 1/5 male and 1/5 female at 100 mg/kg bw/day; 3/5 male at 300 mg/kg bw/day; -diaphragm including liver in 1/5 male at 300 mg/kg bw/day; -uterus in 1/5 female at 100 mg/kg bw/day; Examination of parathyroids were performed if it was feasible (i.e. section plane of thyroids crosses parathyroids).The fixed tissues were trimmed, processed (dehydrated), embedded in paraffin, sectioned with a microtome (at a thickness of 2-4 μm, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

There was no mortality in the control, 100, 300 or 1000 mg/kg bw/day groups during the 28-day observation period (male and female). There were no test item related clinical signs. The behavior and physical condition of allanimals (male and female) were normal in the control, 100, 300 and 1000 mg/kg bw/day during the course of 28-day administration. Slightly softer than normal stools were observed in the bedding material in the cages of male and female animals at 300 mg/kg bw/day between Days 3-7 and 13-15 at 1000 mg/kg bw/day from Day 2 up to and including Day 17. This minor change in consistence of the faces was considered to have little or no toxicological relevance as there were no related changes in the body weight or food consumption data.

Weekly detailed clinical observations The behavior and physical condition of animals were not affected by the test item.The animals exhibited normal behavior and physical condition at 100, 300 or 1000 mg/kg bw/day dose levels at the detailed weekly clinical observations during the course of the entire observation period (male and female).

Functional observation battery did not demonstrate any treatment-related difference with respect to the controls in the physical state, behavior or in reactions to different type of stimuli of animals at 100, 300 or 1000 mg/kg bw/day group (male and female, 5/5, each).

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Test item related adverse effect on the body weight development was not detected 100, 300 or 1000 mg/kg bw/day in male or female animals.The mean body weight was similar – there were no statistical significances with respect to their control – in male and female animals in the control, 100, 300 and 1000 mg/kg bw/day treated groups during the entire observation period.Statistically significant difference with respect to the control was observed at the higher mean body weight gain of male animals at 300 mg/kg bw/day and at the lower mean body weight gain in male animals at 1000 mg/kg bw/day between Days 14 and 18. In the female animals, statistical significance was detected comparing to the control at the lower mean body weight gain at 100 and 300 mg/kg bw/day between Day 7-11 and at the higher mean body weight gain at 300 mg/kg bw/day between Days 21 and 25. These minor changes in body weight gain resulted in only slight differences in the mean body weight (not statistically or biologically significant). Therefore, these were considered to have little or no toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Test item influence on the mean daily food consumption was not detected in male or female animals during the study. The mean daily food consumption was comparable in the control and 100, 300 and 1000 mg/kg bw/day groups (male and female) during the 28-day observation period.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Description (incidence and severity):

There were no adverse test item related alterations in the examined hematological or blood coagulation parameters in male or female animals at 100, 300 or 1000 mg/kg bw/day compared to their controls. All examined hematological and blood coagulation parameters were comparable in the male animals in the control and 100 mg/kg bw/day dose treated groups. In the female animals at 100 mg/kg bw/day, lower mean percentage of neutrophil granulocytes (NEU), higher mean percentage of lymphocytes (LYM), lower mean hemoglobin concentration (HGB) and shorter mean activated prothrombin time (APTT) were observed when compared to the control. In the male animals, s tatistical significance with respect to the control was detected at the slightly lower mean percentage of monocytes (MONO) at 300 mg/kg bw/day. There were no statistically or biologically significant differences between the control and 300 mg/kg bw/day groups in the examined hematological and blood coagulation parameters in the female animals. At 1000 mg/kg bw/day, slightly higher mean hematocrit (HCT) and slightly shorter mean prothrombin time (PT) was observed in male animals when compared to their control. The percentage of the reticulocytes was elevated in one male animal (no. 6538; 1/5) in high dose group and this was considered as an individual finding. In the female animals administered with 1000 mg/kg bw/day, lower mean corpuscular hemoglobin concentration (MCHC), higher mean platelet count (PLT) and higher mean percentage of reticulocytes (RET) were detected with respect to their control. The differences from control in these parameters were with low degree and most of values met well the historical control (MONO, HCT, PT, NEU, LYM, HGB, MCHC). Some individual value of NEU, LYM, PLT and RET were out of the historical control. Taking into account the low incidence of these findings and lack of dose relevance, these minor changes were judged to be of little or no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Clinical chemistry evaluation did not reveal pathological changes in the investigated parameters at 100, 300 or 1000 mg/kg bw/day (male and female). Statistical significance with respect to the control was observed at the slightly higher mean activity of alanine aminotransferase (ALT) in male animals at 100 mg/kg bw/day and at the lower mean concentration of inorganic phosphorous (Pi) in male animals at 100 and 300 mg/kg bw/day. In the female animals, lower mean concentration of total bilirubin (TBIL) was observed at 1000 mg/kg bw/day. All other examined parameters were comparable with their control in male and female animals in the test groups. The changes in mentioned clinical chemistry parameters were considered to be of little or no toxicological significance as values corresponded well with the historical controls and there was no dose relevance (ALT, Pi).

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Test item related changes were not detected in the weights (absolute or relative to body and brain weights) of the investigated organs.The weights of the examined organs (absolute and relative to body weight) were comparable in male and female animals of the control group and test groups at 100, 300 and 1000 mg/kg bw/day. Statistically significant differences with respect to the control were detected at the higher mean kidneys weight relative brain weight in male animals at 100 mg/kg bw/day and at the lower mean heart weight relative to brain weight in female animals at 100, 300 and 1000 mg/kg bw/day, Although the differences from the control reached statistical significances in the weights of mentioned organs, these findings were considered to be toxicologically not relevant in the lack of dose relevance and related changes in clinical chemistry parameters or organpathology.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Specific macroscopic alterations were not detected in the organs or tissues of male or female animals in the control, 100, 300 or 1000 mg/kg bw/day at the necropsy. All macroscopic findings were judged to be individual or species-specific changes and not related to the test item. Hemorrhage in the lungs (1/5 male in the control group) and pyelectasia was observed in male animals as follows: 1/5 in the control group (both sides), 1/5 at 100 mg/kg bw/day(both sides), 3/5 at 300 mg/kg bw/day (right or both sided) and 2/5 at 1000 mg/kg bw/day (right side). Pea-sized Hernia diaphragmaticaincluding a part of the liver was noted for single male animal at 300 mg/kg bw/day (1/5). In female animals, hydrometra and renal pyelectasia were observed in several cases as follows:Hydrometra: 1/5 control (slight); 2/5 at 100 mg/kg bw/day (slight or moderate); 2/5 at 300 mg/kg bw/day (slight) and 2/5 at 1000 mg/kg bw/day (slight or moderate);Pyelectasia: 1/5 in control group (right side); 1/5 at 100 mg/kg bw/day (both sides); 2/5 at 1000 mg/kg bw/day (right side); In one female animal at 100 mg/kg bw/day, a mustard seed sized soft formation was seen in the left uterine horn (1/5). Hemorr hage in the lungs was probably due to the exsanguination procedure in one control animal.Pyelectasia is a common finding in experimental rats of this strain with similar age. There were no inflammatory or other pathological signs accompanying this renal change, therefore it was considered to be toxicologically not relevant in this study. Hydrometra, related to the female sexual cycle, is also a frequent observation in experimental rats. In the lack of related inflammatory or other pathological signs, it wasjudged to be toxicologically not relevant and not test item related as no dose response was noted. Hernia diaphragmatica and mustard seed sized soft formation in the uterine horn are also frequent findings in this strain of experimental rats. Both of these findings occurred in single animals in the mid or low dose groups.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Pen V Potassium did not cause any toxic or other test item related lesions detectable by histological examination of the investigated organs. Minimal alveolar emphysema (1/5) and minimal acute hemorrhage (2/5) in the lungs occurred sporadically in control male animals. This finding could be in connection with the hypoxia, dyspnea and circulation disturbance, developed during the exsanguinations. Mild hyperplasia of BALT (Bronchus Associated Lymphoid Tissue) without inflammatory lesions is a physiological immune-morphological phenomenon and it was seen in single male animal at 1000 mg/kg bw/day (1/5).Pyelectasia was detected in one or both sided kidneys as follows: -1/5 control (both sides), 1/1 at 100 mg/kg bw/day (both sides), 3/3 at 300 mg/kg bw/day (2/3 one side and 1/3 both sides) and 2/5 at 1000 mg/kg bw/day (one side, both) in male animals;-1/5 in control group (one side), 1/1 at 100 mg/kg bw/day (both sides), 2/5 at 1000 mg/kg bw/day (one side) in female animals; Pyelectasia is a common finding in Wistar rats and has no toxicological significance without degenerative, inflammatory or other histopathological lesions. Diaphragmatic hernia along with moderate focal fibrosis in the Glisson’s capsule of the liver due to mechanical irritation (1/1 male at 300 mg/kg bw/day) and focal endometrial proliferation ( 1/1 female at 100 mg/kg bw/day) were considered as individual diseases occurring also in not treated experimental rats.The dilatation of uterine horns in some female animals (1/5 control and 2/5 at 1000 mg/kg bw/day) is a slight neuro-hormonal phenomenon in connection with the sexual function – proestrus phase – of the inner genital organs. No morphological evidence of test item related acute or subacute injury (degeneration, inflammation, necrosis etc.) of the gastrointestinal tract, liver, pancreas, cardiovascular system, urinary system, lymphoid system, hematopoietic system, the skeleton, the muscular system, the male and female reproductive system or the central, or peripheral nervous system, the eyes, the lachrymal glands and the integumentary system was observed. The structure and the cytomorphology of the endocrine glands were the same in the control and treated rats.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

mortality

organ weights and organ / body weight ratios

other: serum/plasma biochemistry (migrated information)

Critical effects observed:

not specified

Conclusions:

Under the conditions of the present study, Pen V Potassium did not cause adverse systemic effects – cli nical signs, changes in body weight, food consumption, hematologyand blood coagulation, clinical chemistry organ pathology or organ weights – in male or female Han:WIST rats after the consecutive 28-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day . ased on these observations, the No Observed Adverse Effect Level (NOAEL) was determined as 1000 mg/kg bw/day in male and female Han:WIST rats.

Executive summary:

The objective of this study was to obtain first information on the toxic potential of Pen V Potassium in male and female rats likely to arise from repeated e xposure to the test item over a 28-day repeat-dose test period. Estimation of the no-observed-adverse-effect level (NOAEL) was also sought for each sex. The dose setting with 0, 100, 300 and 1000 mg/kg bw/day was made on the basis of the results of the 14-day oral (by gavage) study with Pen V Potassium in rats and in agreement with the Sponsor. Doses of 0 (vehicle only), 100, 300 and 1000 mg/kg bw/day were orally administered (by gavage) to four groups of Han:WIST rats consisting of five animals per group and sex at a dosing volume of 5 mL/kg in concentrations of 20, 60 and 200 mg/mL. A group of vehicle (distilled water) treated animals (n= 5/sex) served as a control.The suitability of the chosen vehicle for the test i tem was analytically verified. A sufficient stability and homogeneity in the chosen vehicle were verified over the range of relevant concentrations at the appropriate frequency of preparation. Measured concentrations of formulations applied in the study varied in the acceptable range (between 97 % and 109 % of the nominal concentrations), thereby confirming proper dosing. General clinical observations were performed daily after the treatment. Detailed weekly clinical observation and terminal functional observations were also performed. Body weights were recorded twice weekly. The food consumption was determined weekly to coincide with body weight measurements during the study. Clinical pathology (hematology, blood coagulation and clinical chemistry) and gross pathology examinations were conducted one day after the last treatment (on Day 28). Selected organs were weighed.Histopathological examinations were performed on the preserved organs and tissues of the animals from both the control and high (1000 mg/kg bw/day) dose groups (Groups 1 and 4, respectively). In addition, organs with gross lesions of animals in the low (100 mg/kg bw/day) and mid (300 mg/kg bw/day) dose groups were also processed and evaluated histologically.The results of this study were summarized as follows:Results:Mortality No mortality occurred (control, 100, 300 or 1000 mg/kg bw/day) during the 28-day observation period. Clinical observationsThere were no clinical signs in animals of control, 100, 300 or 1000 mg/kg bw/day groups during the course of 28-day observation period. Slightly softer than normal stool was detected transiently in the bedding material of male and female animals at 300 and 1000 mg/kg bw/day. This finding was considered to have little or no toxicological relevance as there were no related changes in the body weight, food consumption or water balance. Body weight and body weight gain The body weight development of the male and female animals was not affected in any testitem treated groups (100, 300 or 1000 mg/kg bw/day) throughout the entire observation period. Clinical pathology There were no test item related changes in the examined hematological, blood coagulation or clinical chemistry parameters in male or female animals administered with 100, 300 or 1000 mg/kg bw/day dose of the test item.NecropsySpecific macroscopic alterations related to the test item were not detected in the organs or tissues of male or female animals at 100, 300 or 1000 mg/kg bw/day at the necropsy. Organ weight Test item related adverse effects were not found on the weight of the examined organs in male or female animals (100, 300 and 1000 mg/kg bw/day). Histopathology Histopathological investigation did not reveal test item related alterations in the organs or tissues of male or female animals 1000 mg/kg bw/day. Conclusion Under the conditions of the present study, Pen V Potassium did not cause adverse systemic effects – clinical signs, changes in body weight, food consumption, hematologyand blood coagulation, clinical chemistry organ pathology or organ weights – in male or female Han:WIST rats after the consecutive 28-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day. Based on these observations, the No Observed Adverse Effect Level (NOAEL) was determined as 1000 mg/kg bw/day in male and female Han:WIST rats.

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:

not specified