Amoxicillin

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

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 oral 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.

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 (Validated method: 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; Stability in vehicle, initial concentration = 40 mg/L, after 7 days 34.6-40.2 mg/L).

The analytical concentrations were verified weekly and they ranged from:
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%.

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.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200 mg/kg-bw (Group 1)
Basis:
other: Nominal by gavage

Doses / concentrations 2

Remarks:

Doses / Concentrations:
700 mg/kg-bw (Group 2)
Basis:
other: Nominal by gavage

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2450 mg/kg-bw (Group 3)
Basis:
other: Nominal by gavage

No. of animals per sex per dose:

10 males/dose
12 females/dose
10 animals/sexe/dose in the satellite groups

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

Observations and examinations performed and frequency:

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 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.
- Parameters checked in table 2-6 were examined.

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.
- Parameters checked in table No 7 were examined.

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.
-The general urine examination involved the determination of urine colour and volume, specific gravity (density), pH, proteins, ketones, bilirubin (qualitative test), blood, urobilinogen, leukocytes, and glucose.
Urine sediment was examined microscopically using a 40x objective . Appropriate scale values were assigned to individual elements of urine sediment in a vision field (the squamous and columnar epithelium, leukocytes, erythrocytes, bacteria, and crystals).

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.

OTHER:
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 .

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.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All adult animals were euthanized and pathologically.

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: Yes
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; 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.

Other examinations:

Evaluation of the immune system
The preliminary evaluation of the immune system was performed using the following results of the clinical-chemical and post mortem examinations:
- blood morphology with a picture of peripheral blood and bone-marrow,
- parameters of clinical chemistry, i.e. albumin concentration (as an acute phase negative protein),
total protein, and albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin,
- activity of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase,
- absolute and relative weights of the thymus and spleen,
- histopathologic evaluation of the thymus, spleen, and lymph nodes.

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.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

Some effects were observed, however since such observations were not reproducible in the satellite groups (highest dose and control) are considered irrelevant.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Although histologically there were no indications of dysfunction.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY
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 WEIGHT GAIN
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 (highest dose) on day 4 of the lactation period compared to the control group.
Body weight of one female from group 2 (mid-dose) decreased at the end of the lactation period.
There were no statistically significant differences in average body weights of males and females between the treated and the control satellite groups during the study period

FOOD CONSUMPTION
During the pre-mating period, food intake by males and also by females from the treated and the control groups was similar.
During the gestation period, there were statistically significant differences in food intake by females from group 1, 2 and 3 compared to the control group. A statistically significant increase in food intake by females from group 1 and 2 was observed on days 7, 14 and 20 of gestation compared to the control group. A statistically significant increase in food intake by females from group 3 was observed on days 14 and 20 of gestation compared to the control group.
During the lactation period, there were statistically significant differences in food intake by females from group 1, 2 and 3 compared to the control group. A statistically significant increase in food intake by females from group 1 was observed on days 4, 7 and 13 of lactation period compared to the control group. A statistically significant increase in food intake by females from group 2 was observed on day 4 of lactation period compared to the control group. A statistically significant increase in food intake by females from group 3 was observed on days 4 and 13 of lactation period compared to the control group.
Food intake by the treated and the control satellite groups was similar.

OPHTHALMOSCOPIC EXAMINATION
The ophthalmic examinations did not reveal any pathological changes.

HAEMATOLOGY
There was no statistically significant differences in hematological examinations of periferal blood between the treated and the control groups.
The results of the leukocytogram did not show any significant differences between the treated and the control animals.
The results of the bone marrow erythrocyte did not show any statistically significant difference between the treated and the control groups.
No statistically significant changes in the bone marrow leukocyte system between the treated and the control groups were observed.
No statistically significant changes in the bone marrow different cells between the treated and the control groups were observed.
Coagulation examination:
No statistically significant differences in PT and APTT between the treated and the control groups were observed

CLINICAL CHEMISTRY
No statistically significant changes in biochemical examinations between the treated and the control groups were observed.
The only exceptions were:
- Decreased chlorides concentration in males from group 2 and 3,
The decrease in chloride concentration was small, by about 2% compared to the control group and did not increase with increasing dose. Because of that, chloride concentrations analysis was extended to other 5 males from groups 0, 1, 2, and 3. Statistical analysis of all results (n=10) did not reveal statistically significant changes. For this reason statistically significant change which was observed during statistical analysis of results from 5 males from each group may be considered as not treatment-related.
- Decreased creatinine concentration in females from group 2. This change appeared only in females from this group, not in females from the third group. There was no dose-response-relationship, so the effect was considered not to be test item-related.

No statistically significant changes were found in enzymatic examinations except the decreased activity of alkaline phosphatase in females from group 1, 2 and 3.
Due to the lack of different changes in paremeters determing the liver function (total bilirubin, Asp At, Al At), determination of alkaline phosphatase activity was extended to other females from groups 0, 1, 2, and 3.
Since statistical analysis of alkaline phosphatase activity in 12 females per group showed statistically significant change only in females from second group, this change was absent in other groups (group 1 and 3), it may be considered as not treatment related.
In the satellite groups there were no differences in the activities of individual enzymes between the treated and the control group.

URINALYSIS
General urinalysis
There were several statistically significant changes stated:
- the decreased volume of urine in males from group 2 and 3,
- an increase in specific gravity in males from group 2
- increased pH in males from group 1
- decreased pH in females from group 1 and 3
No statistically significant changes were observed in the satellite groups.
Urine sediment examinations
No statistically significant changes were found. The only exception was the reduced quantity of bacteria in males from group 1, 2, and 3 and females from group 1 and 3.
There were not any statistically significant differences between the satellite group and the control group.

An increase in specific gravity in males from group 2 may be considered as fortuitous change because there is no dose-effect observed. Increased pH in males from group 1 and decreased pH in females from group 1 and 3 can also be fortuitous because these changes are opposing. The reduced quantity of bacteria in urine sediment examination in males from group 1, 2, and 3 and females from group 1 and 3 were observed . This change is beneficial and it results from the mechanism of action of the test item.
The decreased volume of urine in males from group 2 and 3 were observed. This change was considered as not treatment-related effect due to the following reasons:
- it was not observed in treated females and animals from satellite groups,
- there were no changes in kidney parameters and parameters assessing water balance in males from group 2 and 3,
- there was no changes in number of urinations that were measured during open field observations


NEUROBEHAVIOUR
Open field observations
In case of males and females from groups 1, 2, 3 and control group, no involuntary clonic and tonic movements, changes in gait, or stereotypical behaviour were observed.
A slight decrease in arousal was noticed in one male and one female from group 0, one male and one female from group 1. A slight increase in arousal was noticed in two females from group 0, one male from group 2, three males and one female from group 3. A strong increase in arousal was noticed in one female from group 3.
There were no statistically significant differences in the number of fecal boluses left by males and females between the treated groups, i.e. 1, 2, and 3, and the control group. There were no statistically significant differences in the number of urine pools left by males and females between the treated and the control groups.
There were no statistically significant differences in horizontal and vertical locomotor activities of males between groups 1 and 2 compared to the control group. A statistically significant increase of horizontal and vertical locomotor activity was noticed in males from group 3 compared to the control group.
There were no statistically significant differences in horizontal and vertical locomotor activities of females between the treated and the control groups.
Evaluation of sensorimotor responses to stimuli
There were no treatment-related effects on responses to visual, tactile, auditory, and pain stimuli.
As for the latency of pain responses of males and females, there were no differences between the treated and the control groups
Measurement of the fore- and hindlimb grip strength
There were no statistically significant differences in the forelimb grip strength of males between the treated and the control groups. The hindlimb grip strength of males from group 3 was statistically significantly greater compared to the control animals.
There were no statistically significant differences in the fore- and hindlimb grip strength of females between the treated and the control groups.

ORGAN WEIGHTS
The results obtained in groups treated with the test item 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.

The observed 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.

Satellite groups
The results obtained in the group treated with the test item (group 3 SAT) were compared with the ones obtained in the control group receiving a vehicle.
There were: decrease of absolute and relative weights of the thymus of males, decrease of relative weights of the liver of males;
There were no statistically significant changes in absolute and relative weights of the other organs in the satellite groups.
There were no statistically significant changes in absolute and relative weights of thyroid, testes and accessory sex organs in males from group 3 SAT. As well as no statistically significant changes in absolute and relative weights of thyroid and ovaries in females from group 3 SAT were observed.

GROSS PATHOLOGY
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.

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: NON-NEOPLASTIC
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
No statistically significant changes were observed in hormonal examinations of TT4 nor in parental animals, satellite groups or offspring.

The clinical-chemical parameters which can be used to evaluate the immune system are as follows: hematological, albumin, total protein, and albumin/globulin ratio, creatinine, urea nitrogen, cholesterol, total bilirubin, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase. These parameters did not change statistically significantly.

Effect levels

Dose descriptor:

NOAEL

Effect level:

2 450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

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

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Amoxicillin trihydrate. Clinical signs

Clinical signs	Group / sex / number of animals / computer numbers of animals
	0		1		2		3
	males	females	males	females	males	females	males	females
Test animals	10	12	10	12	10	12	10	12
Animals with clinical signs	0	1	1	0	0	1	0	1
Animals with ophthalmic changes	0	0	0	0	0	0	0	0
Death of animals	-	-	-	-	-	-	-	-
BODY WEIGHT
Body weight loss	-	-	-	-	-	1 [21]	-	-
SKIN AND COAT
Alopecia on the back	-	-	1 [7]	-	-	-	-	-
Alopecia on the forelimbs	-	1 [22]	-	-	-	-	-	1 [13]

[ ] computer numbers of animals

Table 2 . Amoxicillin trihydrate. Results of hematological and coagulological examination –statistically analyzed

Parameter		GROUP
		0 n=5	1 n=5	2 n=5	3 n=5
HEMOGLOBIN                   g/L	Males	155.20 ± 5.07	162.60 ± 6.66	157.20 ± 2.17	157.60 ± 3.51
	Females	151.60 ± 8.20	152.20 ± 4.44	153.40 ± 9.66	154.40 ± 5.22
HEMATOCRIT1/1	Males	0.48 ± 0.01	0.50 ± 0.02	0.48 ± 0.01	0.48 ± 0.01
	Females	0.46 ± 0.02	0.46 ± 0.02	0.47 ± 0.03	0.47 ± 0.02
ERYTHROCYTES       x 10^12/L	Males	8.85 ± 0.13	9.24 ± 0.34	9.05 ± 0.11	8.83 ± 0.31
	Females	7.69 ± 0.22	7.61 ± 0.20	7.96 ± 0.32	8.08 ± 0.26
MCV fL	Males	54.00 ± 1.22	54.00 ± 1.22	53.20 ± 1.10	54.20 ± 1.10
	Females	60.00 ± 1.00	59.80 ± 1.92	59.20 ± 1.79	58.60 ± 1.14
MCHpg	Males	17.54 ± 0.57	17.62 ± 0.47	17.40 ± 0.34	17.84 ± 0.43
	Females	19.74 ± 0.58	19.98 ± 0.38	19.26 ± 0.55	19.10 ± 0.44
MCHCg/L	Males	324.00 ± 8.19	326.00 ± 3.08	325.80 ± 2.28	329.20 ± 2.39
	Females	329.40 ± 6.19	333.40 ± 6.43	325.60 ± 5.59	327.20 ± 5.89
RETICULOCYTES             1/1	Males	0.019 ± 0.004	0.021 ± 0.005	0.019 ± 0.005	0.018 ± 0.004
	Females	0.026 ± 0.009	0.021 ± 0.002	0.018 ± 0.006	0.018 ± 0.003
THROMBOCYTES       x 10^9/L	Males	593.40 ± 57.06	638.80 ± 81.51	625.60 ± 74.55	598.00 ± 41.86
	Females	647.60 ± 50.92	694.60 ±103.95	643.60 ± 93.40	535.00 ±171.48
LEUKOCYTES             x 10^9/L	Males	3.78 ± 0.45	3.48 ± 0.99	3.54 ± 1.00	3.42 ± 0.37
	Females	4.10 ± 0.94	5.76 ± 2.73	3.50 ± 1.38	4.98 ± 1.07
PTs	Males	13.66 ± 0.71	14.88 ± 1.04	14.88 ± 1.19	14.52 ± 1.13
	Females	10.68 ± 0.31	10.22 ± 0.40	10.42 ± 0.74	10.12 ± 0.38
APTTs	Males	48.60 ± 4.62	47.40 ± 3.71	57.60 ± 19.93	48.80 ± 2.95
	Females	48.80 ± 7.82	51.40 ± 1.82	48.40 ± 8.11	42.60 ± 6.99

 

Table 3. Amoxicillin trihydrate. Results of hematological examination –

Percentage content of leukocytes (leukocytogram), (statistically analyzed)

 

 

Parameter		GROUP
		0 n=5	1 n=5	2 n=5	3 n=5
NEUTROCYTES                1/1	Males	0.21 ± 0.07	0.24 ± 0.04	0.20 ± 0.05	0.24 ± 0.11
	Females	0.43 ± 0.13	0.47 ± 0.15	0.49 ± 0.16	0.42 ± 0.11
EOSINOCYTES                 1/1	Males	0.01 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	Females	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
LYMPHOCYTES                1/1	Males	0.78 ± 0.07	0.75 ± 0.04	0.78 ± 0.07	0.76 ± 0.11
	Females	0.57 ± 0.13	0.52 ± 0.14	0.50 ± 0.16	0.57 ± 0.10
MONOCYTES                    1/1	Males	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00
	Females	0.00 ± 0.00	0.00 ± 0.00	0.00 ± 0.00	0.01 ± 0.00
OTHER CELLS                  1/1	Males	0.00 ± 0.00	0.01 ± 0.01	0.01 ± 0.01	0.00 ± 0.00
	Females	0.00 ± 0.00	0.01 ± 0.01	0.00 ± 0.00	0.01 ± 0.01

 

Table 4. Amoxicillin trihydrate. Results of hematological studies – bone marrow examination

Erythrocyte system – (statistically analyzed)

Parameter		GROUP
		0 n=5	1 n=5	2 n=5	3 n=5
PROERYTHROBLASTS 1/1	Males	0.020 ± 0.004	0.018 ± 0.002	0.018 ± 0.005	0.018 ± 0.005
	Females	0.018 ± 0.002	0.018 ± 0.006	0.021 ± 0.006	0.020 ± 0.003
BASOPHILIC ERYTHROBLASTS 1/1	Males	0.023 ± 0.003	0.021 ± 0.003	0.020 ± 0.003	0.024 ± 0.003
	Females	0.026 ± 0.005	0.024 ± 0.006	0.024 ± 0.004	0.026 ± 0.005
POLYCHROMATOPHILIC ERYTHROBLASTS             1/1	Males	0.138 ± 0.016	0.148 ± 0.014	0.145 ± 0.011	0.141 ± 0.017
	Females	0.157 ± 0.010	0.150 ± 0.014	0.152 ± 0.012	0.153 ± 0.008
ORTHOCHROMATIC ERYTHROBLASTS 1/1	Males	0.218 ± 0.019	0.214 ± 0.013	0.217 ± 0.013	0.218 ± 0.018
	Females	0.229 ± 0.016	0.230 ± 0.022	0.229 ± 0.012	0.223 ± 0.016
TOTAL 1/1	Males	0.400 ± 0.009	0.401 ± 0.011	0.399 ± 0.013	0.402 ± 0.027
	Females	0.429 ± 0.013	0.423 ± 0.006	0.426 ± 0.022	0.422 ± 0.015

 

Applicant's summary and conclusion

Conclusions:

The NOAEL for the subacute repeated dose toxicity of 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 at dose levels up to 2450 mg/kg-bw/day by repeated oral (gavage) administration revealed no consistent and dose-related effects. The no-observed-adverse-effect-level (NOAEL) of the test item was determined to be 2450 mg/kg b.w./day for parental toxicity.