Amoxicillin

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test method according to OECD 422. GLP study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate.

Cas Number:

61336-70-7

Molecular formula:

C16H25N3O8S

IUPAC Name:

(2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate.

Constituent 2

Reference substance name:

amoxicillin trihydrate

IUPAC Name:

amoxicillin trihydrate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Amoxicillin trihydrate
- Physical state: White powder
- Stability under test conditions: The test item was found to be stable over a 7 day period (86 %).
- Storage condition of test material: Room temperature, storage in a plastic, glass, or metal container.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Husbandry of laboratory animals of the Experimental Medicine Centre at the Medical University in Białystok.
- Age at study initiation: 12-14 weeks old.
- Weight at study initiation: Males 390.2-410.9 g and females 247.7-248 g
- Fasting period before study: No
- Housing: Same conditions during acclimatisation and experiment, air-conditioned rooms.
- Diet:Altromin 1324 P TPF ad libitum.
- Water: Tap water ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24ºC
- Humidity (%): 35-57 %
- Air changes: 13times/hour
- Photoperiod: 12 hours dark/12 hours light

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% CMC sodium salt

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Suspensions of the test item in vehicle (0.5% Carboxymethylcellulose sodium salt) were prepared once a week (the stability of Amoxicillin in a vehicle in a temperature 4-8ºC during 7-days period was evaluated). During administration, suspensions were kept at room temperature and they were thoroughly mixed.

DIET PREPARATION
- Rate of preparation of diet (frequency): Standard diet was given.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Vehicle control was selected on the basis of solubility of the test item in water.
- Concentration in vehicle: 40mg/mL, 140 mg/mL, 490 mg/mL
- Amount of vehicle (if gavage): 0.5 mL/100 gr
- Lot/batch no. (if required): 1296363
- Other: Name: Carboxymethylcellulose sodium salt, ultra high viscosity, highly purified, purchased from Sigma Aldrich.

Details on mating procedure:

- M/F ratio per cage: 1:1 or 1:2
- Length of cohabitation: As long as required, each morning the females were examined for the presence of sperm or a vaginal plug.
- Proof of pregnancy: Day 0 of pregnancy was defined as the day on witch mating evidence was confirmed (a vaginal plug or sperm was found)
- A replacement of males (unsuccessful pairing was not necessary). The average number of days needed for copulation was 1.5 in the control group, 1.3 in group 1, 1.0 in group 2 and 1.2 in group 3.
- After successful mating each pregnant female was caged: Pregnant females were individually housed with same environmental conditions as explained before. After delivery, females were housed with their offspring.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analytical concentration was measured using a validated HPLC method with DAD detector. The analytical method was validated (Linearity= 1-500 mg/L; Specifity was evaluated with fortitied samples and the matrix. No signal was overlapping. Recovery=92-102.41 %. Precision= Assessed with repetability, RSD= 0.055-0.27 %. LOQ= 1mg/L. LOD= 0.25 mg/L).

The analytical concentrations were measured weekly and ranged as follows:
35.8 – 46.4 mg/mL for the concentration of 40 mg/mL in vehicle (89.5 – 115.9%)
129.0 – 163.9mg/mL for the concentration of 140 mg/mL in vehicle (92.1 – 117.1 %)
442.3 – 574.4 mg/mL for the concentration of 200 mg/mL in vehicle (90.3 – 117.2%).
The results were within the range of 80 -120%.

The test item was stable in vehicle, minimum for one week: Initial concentration = 40 mg/L, after 7 days 34.6-40.2 mg/L.

Duration of treatment / exposure:

Males were dosed for 28 days: 14 days pre-mating and 14 days during mating. Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (51 – 60 days in total). In addition, two satellite groups (10males and 10 females in each group) were used: one treated group (3 SAT) which was given the test item at the highest dose level of 2450 mg/kg b.w./day and one control group (group 0 SAT) receiving the vehicle control. Males from groups 0 SAT and 3 SAT were treated for 28 days (as males from groups 0, 1, 2, and 3). Females from groups 0 SAT and 3 SAT were treated 53 days (on average as long as females from groups 0, 1, 2 and 3). Post treatment, the satellite groups were observed for 14 days in order to evaluate reversibility, persistence, or delayed occurrence of potential toxic changes.

Frequency of treatment:

Once daily, seven days a week.

Details on study schedule:

Males were dosed for 28 days: 14 days pre-mating and 14 days during mating.
Females were dosed throughout the study. This included 2 weeks prior to mating, the variable time to conception, the duration of pregnancy, and at least 13 days after delivery (51 – 60 days in total).

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200 mg/kg-bw/day
Basis:
nominal conc.
Gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
700 mg/kg-bw/day
Basis:
nominal conc.
Gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2450 mg/kg-bw/day
Basis:
nominal conc.
Gavage

No. of animals per sex per dose:

10 males/dose
12 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels for the study were selected on the basis of bibliographic and registrant information.
- Rationale for animal assignment: Animals were randomly assigned to the groups.
- Rationale for selecting satellite groups: In order to evaluate reversibility, persistence, or delayed occurrence of potential toxic changes.
- Post-exposure recovery period in satellite groups: 14 days.

Positive control:

Not required.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Twice a week during the entire experiment.
Body weights of females were measured twice a week before gestation, on days 0, 7, 14, and 20 of gestation, within 24 hours of parturition (day 0 or 1 post-partum), and day 4, 7, 10 and 13 post-partum. Body weights of pups were measured on day 0 (within 24 hours of parturition), 4, 7 and 13 post-partum

FOOD CONSUMPTION: Food consumption was measured, although it is not a feeding study.
Food intake by parental males was measured once a week during the pre-mating period. Food intake by parental females was measured once a week before gestation, on days 0, 7, 14, and 20 of gestation, on day 0 or day 1 post-partum, and on day 4, 7 and 13 post-partum. Food intake by females and males from satellite groups was measured once a week during the entire experiment.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: They were conducted on the day before the introduction of the animals to the experiment and on the day of euthanasia. . In case of the satellite groups, these examinations were additionally conducted after the end of the test item/vehicle administration .
- Dose groups that were examined: All adult animals used in the study were subjected to ophthalmic examinations.

HAEMATOLOGY: Yes (blood samples from the heart)
- Time schedule for collection of blood: After the end of the treatment.
- Anaesthetic used for blood collection: Yes (10 mg xylazine/kg-bw and 100 mg ketamine/kg-bw).
- Animals fasted: Yes, 18 hours
- How many animals: 5males and 5 females from each group.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After the end of the treatment.
- Animals fasted: Yes, 18 hours
- How many animals: 5 males and 5 females from each group.

URINALYSIS: Yes
- Time schedule for collection of urine: During the last day of the experiment.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, 18 hours.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Males were examined at the end of the administration period (1-2 days before euthanasia). Females were examined once during the last week of lactation (1 day before euthanasia).
The behavioural studies on the males and females from satellite groups were performed at the end of the administration period (measurement 1) and then before the end of the additional observation period (measurement 2)
- Dose groups that were examined: Five adult males and five adult females from each group.
- Battery of functions tested: open field observations/ sensiromotor reponses to stimuli / fore and hindlimp grip strenght.
See table 6.

OTHER:
Bone marrow examination:
Bone marrow were collected from the dissected femur after euthanasia. Bone marrow smears were prepared, fixed and stained. The slides were subjected to a qualitative and quantitative evaluation of individual nuclear cells per 1000 tested cells.
The numbers of the following cells were determined: erythrocyte system: proerythroblasts, basophilic erythroblasts, polychromatophilic erythroblasts, and orthochromatic erythroblasts; leukocyte system: myeloblasts, promyelocytes, orthochromatophilic and acidophilic myelocytes, orthochromatophilic and acidophilic metamyelocytes, rod neutrophils and rod eosinophils, filamented neutrophils, filamented eosinophils, and basophils,
different cells: lymphocytes, monocytes, plasmocytes, megakaryocytes, and other cells (cells of reticulum, mast cells, and bare nuclei).

Coagulation examination:
Prothrombin time (PT) was determined using the test strip method.
Partial thromboplastin time (APTT) was determined using a Hemochron apparatus (ITC).

Enzymatic examination:
Blood serum was examined for the activities of the following enzymes
Aspartate aminotransferase (AST) using the IFCC method without P-5-P
Alanine aminotransferase (ALT) using the IFCC method without P-5-P
Alkaline phosphatase (AP) using the IFCC method with p-nitrophenol

The level of thyroid hormone, i.e. total T4 (TT4) in serum were determined once. Blood samples were collected from:
- all dams after termination,
- all adult males after termination,
- two pups per litter on day 4 after birth,
- two pups per litter on day 13 after birth.
Serum obtained from all blood samples were frozen at -20°C ±3°C. Blood samples from 13-day pups and adult males were measured for TT4.

Oestrous cyclicity (parental animals):

Ostreous cycles: Females were screened for normal ostreus cycles in a 2-week pre-treatment period. Ostreus cycles were monitored before treatment starts to select for the study females with regular cyclicity. Females that fail to exhibit typical 4-5-day cycles were not included in the study. Vaginal smears were monitored daily during the pre-treatment period and then from the beginning of the treatment period until evidence of mating. Vaginal smears were examined on the day of necropsy to determine the stage of the ostreus cycle and allow correlation with histopathology of female reproductive organs .

Sperm parameters (parental animals):

Parameters examined in male parental generation:
testis weight, epididymis weight

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, TT4 on day 4 and 13 afterbirth, AGD on day 4 after birth, number of nipple/areolae in male pups on day 13.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals after 14 days after mating.
- Maternal animals: All surviving animals after at least 13 days of delivery.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including external surface of the body, all natural apertures, the cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system. During the necropsy in all parental females the numbers of corpora lutea (separately in the left and the right ovary) and implantation sites (separately in the left and the right horn of the uterus) were determined.

Weights of internal organs
Absolute and relative weights of the testes, epididymides, accessory sex organs (prostate with the seminal vesicles and coagulating glands), thyroid glands and ovaries of all animals were determined. Additionally, absolute and relative weights of the brain, thymus, heart, liver, spleen, kidneys, and adrenal glands of 5 adult males and 5 adult females from each group were determined. Relative weights of internal organs were determined on the basis of absolute weights, with reference to body weights of living animals.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following organs and tissues of animals were examined: testes, epididymides and accessory sex organs (prostate, vesicular and coagulating glands) from all adult males; ovaries from all adult females; thyroid glands from all adult rats and also one male and female day 13 pup from each litter; brain , spinal cord, eyeball with optic nerve, pituitary gland, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, colon, rectum), liver, kidneys, adrenal glands, urinary bladder, spleen, heart, thymus, trachea with esophagus, lungs, mammary gland, lymph nodes and skeletal muscle with peripheral nerve from five adult males and females and additionally uterus with cervix and vagina from five adult females, from control groups 0 and 0 SAT and also in high dose groups 3 and 3 SAT. The histopathological evaluation of those organs was not extended to low dose groups 1 and 2 because there was no treatment- related changes observed in high dose groups. Organs with gross lesions i.e. the liver necrosis focus of the male no. 1 in group 3, the abdominal cavity nodule of the male no. 7 in group 0 and the abdominal cavity nodule of the female no.11 in group 2 were also examined.

Despite of routinely proceeded histopathological evaluation of reproductive system focused on eventual circulatory disorders, inflammation lesions, retrograde or progressive changes, the detailed evaluation of testes and ovaries for the prospective gametogenesis disorders were carried out. In testes spermatogonia, spermatocytes and spermatides were evaluated as well as the accuracy of the particular layers of the sex epithelium structure. The lumen of the seminiferous tubules was examined to exclude the presence of the immature forms of spermatogenetic cells or giant cells. In epididymides the presence of sperm content was evaluated. In the ovaries there were ovarian follicles at various stages of maturation and corpora lutea organisation evaluated.

During the histopathological examination special attention was paid to tissues and organs (gonads, pituitary glands and adrenals in all examined animals, accessory sex organs in males and uterus and vagina in females) which might have been indicators of endocrine disruption influenced by the test item.

Preserved organs and tissues collected from all groups were fixed in a 10% aqueous solution of formalin, embedded in paraffin, and stained using hematoxylin and eosin.The slides were evaluated under a light microscope (100x, 200x, and 600x).

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring was sacrificed at day 4 and 13 of age.
- These animals were subjected to postmortem examinations

GROSS NECROPSY
- Gross necropsy consisted of observations of the external body surface, all natural apertures, and the cranial, thoracic, and abdominal cavities with their content.

Statistics:

The treated groups, i.e. groups 1, 2, and 3 were compared to the control group. The treated satellite group, i.e. 3 SAT was compared to group 0 SAT.
The clinical results were statistically analyzed using the one-way analysis of variance and the Dunnet’s test and Student’s t-test (group 0 SAT, 3 SAT) (p ≤ 0.05).
The clinical-chemical results were statistically analyzed using the one-way analysis of variance and Dunnet’s test (p ≤ 0.05).
The results, i.e. absolute and relative weights of internal organs as well as the numbers of implantation sites and corpora lutea were statistically evaluated using Dunnett’s test (p≤0.05).
The statistical analysis were performed separately for males and females.

Reproductive indices:

Mating index for males
Mating index for females
Fertility index for males
Fertility index for females
Pregnancy index
Length of gestation

Offspring viability indices:

Total number of pups in litter
Number of live births in litter
Number of pups in litter, min.– max.
Percentage of males in litter
Percentage of females in litter

Index of live births
Index of 4-day survival
Index of 13-day survival
Mortality [%]

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Test substance intake: Gavage plus analytical determination

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
During the experiment, no mortality and morbidity of the adult animals was observed.
During the entire experiment, there were no differences in appearance and behaviour between the treated and the control groups. Alopecia on the back was observed in one male from group 1 (change occurred in the second week and remained till the end of experiment). Alopecia on the forelimbs was observed in one female from group 0 (change occurred in the end of gestation period and remained till the end of experiment) and in one females from group 3 (change occurred at the beginning of the lactation period and remained till the end of experiment).

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the study (pre-mating and post-mating periods), there were no statistically significant differences in average body weights of males between the treated and the control groups.
During the pre-mating, gestation period and the lactation periods, there were no statistically significant differences in average body weights of females between the treated and the control groups.
The only exception was a statistically significant increase in the average body weight of females from group 3 on day 4 of the lactation period compared to the control group.
Body weight of one female from group 2 decreased at the end of the lactation period.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS) The study was conducted by gavage. The analytical determination demonstrated that the concentrations were in the range of 80-120 % of the nominal.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
During the pre-treatment and pre-mating period, in all females regular cycles were observed, except one female from control group. This female had normal cycle before treatment, and this deviaton during pre-mating period did not affect fertilization.
During histopathological examination of female reproductive organs the essential findings including arterial involution, the lack of the decidua and the regeneration of the internal elastic lamina were observed. Vaginal smears, that were examined on the day of the necropsy indicated the same stage of the reproductive cycle.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The detailed histopathological examination of testes, epididymides, and ovaries did not reveal any disorders of the process of spermatogenesis and oogenesis in animals exposed at all dose levels. No lesions were observed in the seminal vesicles and coagulating glands. The perivascular infiltration of lymphocytes was observed in a few cases in the prostate in animals exposed at all dose levels. They should not be associated with the test item because similar histopathological changes were noticed in the control group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Twelve females from each group were mated with ten males. All females and males were mated during the scheduled period of mating. The average number of days needed for copulation (the day when males and females were placed together was day 0) was 1.5 in the control group, 1.3 in group 1, 1.0 in group 2 and 1.2 in group 3.
Sperm was found in twelve females from each group. All females from control, 1 and 2 group were pregnant (twelve females delivered offspring). As for group 3, one female (no. 17) was not pregnant (although sperm was found) therefore, only eleven females delivered offspring. Not pregnant female were treated for 53 days (on average, as long as other females from group 0, 1, 2 and 3), next the animal were anesthetized to collect blood for hormonal examination (the sample was frozen for further assessment of TT4), and then euthanized and subjected to post-mortem examinations.
The mating indices for males and females from the control and the treated groups were 100 .
The fertility indices in group 3 were 90.0 for males and 91.7 for females. In the control group and groups 1 and 2 the fertility indices were 100. Hence, the fertility index in group 3 was lower. The pregnancy index from the control and treated groups was 100.
The length of gestation in the treated groups was comparable with the length of gestation in the control group, i.e. 22.3 days in the control group, 22.4 days in groups 1, 22.2 days in group 2, and 22.6 days in group 3.

ORGAN WEIGHTS (PARENTAL ANIMALS)

The results obtained in groups treated with the test item (groups 1, 2, and 3) were compared with the ones obtained in the control group receiving vehicle (group 0).
The analysis of absolute and relative weights of internal organs of the animals from groups 1, 2,
and 3 showed statistically significant changes. There were:
group 1 – increase of absolute and relative weights of kidneys in females
group 2 – increase of relative weights of kidneys in males and females; increase of absolute weights of kidneys in females;
group 3 – decrease in both, absolute and relative weights of thymus in males; increase of relative weights of kidneys in females were noticed;
There were no statistically significant changes in absolute and relative weights of thyroid, testes and accessory sex organs in males from groups 1, 2 and 3. As well as no statistically significant changes in absolute and relative weights of thyroid and ovaries in females from groups 1, 2 and 3 were observed.

These disorders should not be linked causatively with the influence of the test item, since there is no confirmation of histopathological changes, there is no evidence of organ dysfunction.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were several cases of the occurrence of gross changes: in group 0 hypoplasia of the testicle right and the epididymis right and presence of the nodule with a diameter of 10 mm in abdominal cavity defined as a lipoma in 1 male. Also the absence of the left thyroid lobe in 1 female in group 0 was detected, most likely as a birth defect, in 1 female in group 2 the pedunculated nodule with a diameter of 2 mm defined as lipoma in abdominal cavity and in 1 male in group 3 subcapsular necrosis focus with a diameter of 4 mm in the right lobe of the liver and splenomegaly was observed. In the group 0 SAT a nodule (lipoma with a diameter of 4 mm) in 1 female was found. The gross examination of tissues and organs did not reveal any pathological changes associated with the test item.

During the macroscopic examination, the numbers of corpora lutea and implantation sites in the ovaries of the parental females were recorded. On the basis of the results (i.e. the numbers of corpora lutea in the right and the left ovaries determined separately, the numbers of implantation sites in the right and the left uterine horns determined separately, and the statistical analysis), it can be concluded that the test item had no impact on the numbers of corpora lutea and implantation sites as compared to the control group. The one female from group 3 no. 17 was non-pregnant and did not have any of corpora lutea and implantation sites.

Absolute and relative weights of the brain, thymus, heart, liver, spleen, kidneys, and adrenals of 5 adult males and 5 adult females from each group were determined. Statistically significant differences were noticed in case of the thymus, liver, and kidneys. Statistically significant decreased in both absolute and relative weights of the thymus were observed in the groups treated with the test item at the highest dose (group 3 and 3 SAT). A decreased of relative weights of the liver in males in group 3 SAT was also a statistically significant change. As for kidneys increased of relative weights of males in group 2, and increased of absolute weight (group 1 and 2) and relative weight (group 1, 2 and 3) of females were noticed. These disorders should not be linked causatively with the influence of the test item , since there is no confirmation of the present histopathological changes. Additionally, absolute and relative weights of the thyroids, testes, epididymides, accessory sex organs (prostate with the seminal vesicles and coagulating glands) and ovaries of the euthanized animals in the study were statistically calculated. On the basis of the statistical analysis of organs weights of the adult animals, the influence of the test item on weights was not observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
The histopathological examination of organs and tissues of the animals revealed some changes. However, they should not be linked with the test item.
In the liver of 1 male in group 3 subcapsular necrosis focus was observed, but there is small probability that this type of change was caused by the test item. The character and localization of the subcapsular liquefactive necrosis focus suggest that it could have been caused by a small blood clot. There were individual cases of various disorders in the remaining organs such as hyperemia and erythrocytorrhagia in the lungs, hyperemia of the spleen, the infiltration of the lymphocytes in the cecum, colon and rectum, and also proliferation of lymphoid follicle in the colon. In the abdominal cavity a lipoma was noticed. However, it is unlikely that they were caused by the test item because similar histopathological changes were observed in the control group. The detailed histopathological examination of testes, epididymides, and ovaries did not reveal any disorders of the process of spermatogenesis and oogenesis in animals exposed at all dose levels. No lesions were observed in the seminal vesicles and coagulating glands. The perivascular infiltration of lymphocytes was observed in a few cases in the prostate in animals exposed at all dose levels. They should not be associated with the test item because similar histopathological changes were noticed in the control group.

OTHER FINDINGS (PARENTAL ANIMALS)
The ophthalmic examinations did not reveal any pathological changes
The results of the hormonal examinations (TT4) in males from groups 0, 1, 2, and 3 (10 males from each group) are shown in Table 78. No statistically significant changes were observed.

Effect levels (P0)

Dose descriptor:

NOAEL

Effect level:

2 450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed at the highest dose tested.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
There were no statistically significant differences between the treated and the control group.

CLINICAL SIGNS (OFFSPRING)
Some females delivered stillbirths:
two stillborn females (dam no. 11 – one pup and dam no. 19 – one pup) in group 0,
one stillborn male (dam no. 12) in group 1,
four stillborn males (dam no. 13) and one stillborn females (dam no. 13) in group 2,
three stillborn males (dam no. 15 – two pups and dam no. 22 – one pup) and three stillborn
females (dam no. 15) in group 3.
Ten pups were eaten by mothers. Due to cannibalism, the number of pups was reduced in the following groups: four male pups from the control group, one male pup from group 1, four male pups from group 2, and one female pup from group 3.
One pup of dam no. 11 in group 0 was found dead at the day of birth (a few hours after birth) and two pups of dam no. 13 in group 1 was found dead at the 3rd day after birth, hence they were pathomorphologically examined.

The following changes were observed:
temporary hematoma on the neck in one male from group 2;
temporary hematoma on the back in one male from group 0 and one male from group 2;
temporary hematoma on the head in one male from group 0 and one male from group 2;
temporary hematoma on the hindlimb in two male from group 2, and one male from group 3;
temporary scab on the left ear in one male from group 0;
thinning hair in three males and two females from group 0, and four males and two females from group 1;
smaller body in one male and one female from group 0;
smaller and livid body in one male from group 0.

BODY WEIGHT (OFFSPRING)
There were no statistically significant changes in average body weights of the pups per litter between the treated and the control groups

SEXUAL MATURATION (OFFSPRING)
There were no statistically significant changes in AGD measurment in male and female pups between the treated and the control groups.

GROSS PATHOLOGY (OFFSPRING)
There were 571 pups. 10 animals were eaten by mother. Among the remaining 561 animals, 14 of them were stillborn pups (2 females in the control group, 1 male in group 1, 4 males and 1 female in group 2, 3 males and 3 females in group 3), whereas 3 died during observation after the birth
(1 female in group 0, and 2 males in group 1).
The macroscopic examination of the all pups (without pups eaten by mothers) did not reveal any pathological changes or malformations.

OTHER FINDINGS (OFFSPRING)
During the pre-treatment and pre-mating period, in all females regular cycles were observed, except one female from control group. This female had normal cycle before treatment, and this deviaton during pre-mating period did not affect fertilization.
During histopathological examination of female reproductive organs the essential findings including arterial involution, the lack of the decidua and the regeneration of the internal elastic lamina were observed. Vaginal smears, that were examined on the day of the necropsy indicated the same stage of the reproductive cycle.

The results of the hormonal examinations (TT4) in pups 13 days after birth are shown in. No statistically significant changes were found.
There were no statistically significant changes in number of nipples / areolae in male pups on 13th day of life between the treated and the control groups.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 450 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse efects observed at the highest dose tested.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

The indices relating to fertility of the parental animals, including mating indices for males and females, fertility indices for males and females, and pregnancy indices in the treated and the control groups were similar, which suggests that the test item at the dosesof 200, 700, and 2450 mg/kg bw/day did not negatively influence fertility of the parental rats. The test item did not affect the total number of pups in a litter, the numbers of live births, the percentages of males and females in a litter in comparison with the control group. Moreover, no significant changes in anogenital distance (AGD) measurements in male and female pups, and number of nipples/areolae in male pups beetwen treated and control groups were also observed. The indices relating to survival of the offspring of the treated parents show no negative effects on the indices of live births, 4-day and 13-day survival in comparison with the control group. The macroscopic evaluation of the offspring did not reveal any lesions or malformations in liveborn and stillborn pups.

Table 1. List of effects on reproduction/development

OBSERVATIONS	VALUES
Dosage (mg/kg b.w./day)	0	200	700	2450
Pairs started (N)	12	12	12	12
Ostreus cycle (at least mean length and frequency of irregular cycles)	4.0 1/12 (8.3%)	4.0 0/12 (0%)	4.0 0/12 (0%)	4.0 0/12 (0%)
Females showing evidence of copulation (N)	12	12	12	12
Females achieving pregnancy (N)	12	12	12	11
Conceiving days 1 - 5 (N)	12	12	12	11
Conceiving days 6 -…^(1)(N)	0	0	0	0
Pregnancy ≤ 21 days (N)	0	0	0	0
Pregnancy = 22 days (N)	8	7	10	4
Pregnancy ≥ 23 days (N)	4	5	2	7
Dams with live young born (N)	10 + 2*	11 + 1*	11 + 1*	9 + 2*
Dams with live young at day 4 pp (N)	10 + 2**	11 + 1**	10 + 2**	10 + 1**
Corpora lutea/dam (mean)	18.5	17.0	17.8	18.1
Implants/dam (mean)	12.40	13.25	13.40	13.30
Live pups/dam at birth (mean)	11.7	12.3	11.8	11.7
Live pups/dam at day 4 (mean)	11.3	12.0	11.4	11.6
Sex ratio (m/f) at birth (mean)	1.1	1.3	1.4	1.1
Sex ratio (m/f) at day 4 (mean)	1.1	1.2	1.2	1.3
Litter weight at birth (mean) [g]	68.8	75.7	72.4	70.4
Litter weight at day 4 (mean) [g]	111.0	131.4	112.8	118.8
Pup weight at birth (mean) [g]	6.0	6.2	6.1	6.1
Pup weight at the time of AGD measurement (mean males, mean females, PND-4) [g]	♂ 10.3 ♀ 9.7	♂ 10.4 ♀ 10.1	♂ 10.3 ♀ 9.8	♂ 10.7 ♀ 10.3
Pup AGD on the same postnatal day, birth-day 4 (mean males, mean females) [mm]	♂ 5.2 ♀ 3.3	♂ 5.2 ♀ 3.3	♂ 5.2 ♀ 3.3	♂ 5.1 ♀ 3.3
Pup weight at day 4 (mean) [g]	10.0	10.2	10.0	10.5
Pup weight at day 13 (mean) [g]	27.3	27.0	27.0	28.5
Male pup nipple retention at day 13 (mean)	0.10	0.02	0.09	0.01
ABNORMAL PUPS
Dams with 0	5	11	9	10
Dams with 1	6	0	1	1
Dams with ≥2	1	1	2	0

(1)  last day of the mating period

       *     number of dams with at least 1 dead young born
       **    numer of dams with at least 1 dead young at day 4

Table 2. Amoxicillin trihydrate. Mating of parental animals – list of results

Parameter	GROUP
	0	1	2	3
Number of males to mating	10	10	10	10
Number of females to mating	12	12	12	12
Number of males which copulated	10	10	10	10
Number of females which were mated	12	12	12	12
Average number of days to copulation	1.5	1.3	1.0	1.2
Number of fertilized females	12	12	12	11
Number of pregnant females	12	12	12	11
Number of females which delivered	12	12	12	11
Number of not pregnant females	0	0	0	1
Number of females which delivered offspring	12	12	12	11
Number of females which delivered live offspring	12	12	12	11

Table 3. Amoxicillin trihydrate. Indices concerning fertility of parental animals

Parameter	GROUP
	0	1	2	3
Mating index for males	100.0	100.0	100.0	100.0
Mating index for females	100.0	100.0	100.0	100.0
Fertility index for males	100.0	100.0	100.0	90.0
Fertility index for females	100.0	100.0	100.0	91.7
Pregnancy index	100.0	100.0	100.0	100.0
Length of gestation	22.3 ± 0.5	22.4 ± 0.5	22.2 ± 0.4	22.6 ± 0.5

Table 4. Amoxicillin trihydrate. Average number of pups in litter

Parameter	GROUP
	0	1	2	3
Total number of pups in litter	11.8 ± 2.3	12.3 ± 1.6	12.2 ± 2.2	12.3 ± 2.1
Number of live births in litter	11.7 ± 2.2	12.3 ± 1.5	11.8 ± 3.0	11.7 ± 2.4
Number of pups in litter, min.– max.	9 - 15	8 - 14	9 - 15	7 - 15
Percentage of males in litter	48.6	51.4	52.7	50.4
Percentage of females in litter	51.4	48.6	47.3	49.6

Table 5. Amoxicillin trihydrate. Indices connected with survival of offspring

Parameter	GROUP
	0	1 2	2	3
Index of live births	98.6	99.3	96.6	95.6
Index of 4-day survival	97.1	98.0	97.2	99.2
Index of 13-day survival	95.8	97.6	96.6	99.1
Mortality [%]	4.2	2.4	3.4	0.9

Applicant's summary and conclusion

Conclusions:

The NOAEL of the parental toxicity and reproductive toxicity for amoxicillin trihydrate in rats by oral route was determined to be equal or higher than 2450 mg/kg bw/day since no adverse effects were observed at the highest dose tested.

Executive summary:

A combined repeated dose toxicity study with reproduction/developmental toxicity screening was conducted with amoxicillin trihydrate according to OECD Guideline 422 (GLP study). Twuelve to fourteen weeks old Wistar rats were exposed to amoxicillin trihydrate at concentrations of 0 (vehicle control), 200, 700 and 2450 mg/kg-bw/day by oral gavage in 0.5 % carboxymethyl cellulose sodium salt. There were 10 males and 12 females in each group. The test item was administered for 28 days to males and for 51-60 days to females (premating, conception, pregnancy and at least 13 days after delivery). Furthermore 2 satellite groups were included; one control group was given 0.5 % carboxymethyl cellullose sodium salt and a the other group wad administered amoxicillin trihydrate at 2450 mg/kg-bw/day. The satellite groups were not mated. After the exposure, animals from the satellite groups were observed for 14 days to evaluate reversibility, stability or delay in the onset of possible harmful effects. Observations included clinical observations, body weight measurements, food intake, ostreus cycles, mating procedures, behavioural studies, evaluation of reproduction, clinical-chemical examinations, hematological examinations, bone marrow examinations, coagulation examinations, biochemical and enzymatic examinations, urynalisis, hormone testing for TT4, gross examinations, weight of organs, histopathological examinations and evaluation of the immune system. The treatment of male and female rats with amoxicillin trihydrate at dose levels up to 2450 mg/kg-bw/day by repeated oral (gavage) administration revealed no adverse effects on the reproductive performance or fertility of Wistar rats. The no-observed-adverse-effect-level (NOAEL) of amoxicillin trihydrate was determined to be 2450 mg/kg bw/day both for parental toxicity and reproduction toxicity.