Pyrrolidinium, 1-[[(6R,7R)-7-amino-2-carboxy-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-en-3-yl]methyl]-1-methyl-, chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Dose range finding study.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

Only the dose range finding part is reported.

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

yes

Remarks:

Only the dose range finding part is reported.

Qualifier:

equivalent or similar to guideline

Guideline:

other: OPPTS 870.3050

Version / remarks:

2000

Deviations:

yes

Remarks:

Only the dose range finding part is reported.

Principles of method if other than guideline:

The dose range finding part of a subacute toxicity study is reported.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

The test item was suspended in the vehicle (1 % methylcellulose) in concentrations of 20, 60 and 200 mg/mL.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations was performed once during this study. Five aliquots of 5 mL of each formulation to be administered to the animals (20, 60 and 200 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken.
The test item concentrations in the samples varied within the range from 90 % to 92 % in comparison to the nominal values.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Daily once.

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 m + 5 f per group

Control animals:

yes

yes, concurrent vehicle

Positive control:

No.

Observations and examinations performed and frequency:

Examinations performed:
Mortality.
Clinical observations.
Body weight.
Food consumption.
Hematology ablood coagulation.
Clinical chemistry.

Sacrifice and pathology:

Gross pathology and organ weight determinations.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weight gain was reduced during the course of the study resulting in lower mean body weight with respect to the control in male animals at 300 and 1000 mg/kg bw/day from Day 7 up to the end of the study. The difference in body weight with respect to the control group was lower than 10 % therefore this minor change in the body weight development was not considered to be adverse.

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:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not specified

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:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

mortality

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Conclusions:

No toxicologically significant changes in the examined parameters was observed after the 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses.

Executive summary:

No toxicologically significant changes in the examined parameters was observed after the 14-day oral (by gavage) administration of 100, 300 or 1000 mg/kg bw/day doses.

Based on the observations made in this toxicity study the dose levels for a Reproduction/ Developmental Toxicity Screening Test in the Rat (main study) were determined as follows:

Group 1 Vehicle control

Group 2 100 mg/kg bw/day

Group 3 300 mg/kg bw/day

Group 4 1000 mg/kg bw/day

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

not specified

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050

Version / remarks:

2000

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

477-080-2

EC Name:

-

Cas Number:

103121-85-3

Molecular formula:

C13 H19 N3 O3 S . HCl (Hill Formula) C13 H20 N3 O3 S . Cl (CAS Formula)

IUPAC Name:

477-080-2

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Species / Strain: Rat, Hsd.Han Wistar
Source: Toxi-Coop Zrt. 1103 Budapest, Cserkesz u. 90.
The Wistar rat was the system of choice because it has been the preferred and most commonly used species for oral toxicity tests. It is a well-known laboratory model with sufficient historical data.

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: Male animals: 51 – 54 days old at start of the study. Female animals: 49 – 51 days old at start of the study
Body weights: Male animals: 206 – 228 g at start of the study. Female animals: 128 – 146 g at start of the study. The weight variation did not exceed ± 20 per cent of the mean weight.
Number and sex of animals: 20 naïve male and 20 naïve female (nulliparous and non-pregnant animals) rats.
Number of groups: 4 (3 dose levels + 1 control group).
Number of animals/group: 5 animals/ sex/ treatment group.
Animal health: Only healthy animals were used for the study. Healthy status was certified by the breeder.
Acclimatization time: 7 days.

Housing conditions:
Housing: 5 animals of the same sex/ cage
Cage type: Type IV polypropylene/ polycarbonate
Bedding: Certified laboratory wood bedding (Lignocel Hygienic Animal Bedding). 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 central air-condition system.
Environmental conditions are maintained by an air-condition 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 ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water, as for human consumption, ad libitum except overnight food deprivation before the blood sampling.
Animals received tap water from watering bottles.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Details on oral exposure:

The test item and the vehicle were orally administered daily (7 days per week) in graduated doses to three groups of experimental animals for a period of 28 days. Control animals were treated concurrently with the vehicle only. The actual treatment volume was calculated according to the most recent body weight. Animals were not treated on the day of gross pathology.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations was performed twice during this study. Five aliquots of 5 mL of each formulation to be administered to the animals (20, 60 and 200 mg/mL) and five aliquots of 5 mL control substance (vehicle) were taken at both occasions.
The concentration of MPI-ACA in the dosing formulations varied between ranges of 90 and 99 % of nominal concentrations at both analytical occasions.
The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. The recovery of the test item from the vehicle was within the acceptance criteria (relative to nominal concentrations: 98 % at ca. 1 mg/mL and 103 % at ca. 200 mg/mL).
MPI-ACA proved to be stable in a refrigerator (at 5 ± 3 °C) for four days and at room temperature for one day.
A separate analytical report (Study no. 880-100-1637, Section 8, Gaal 2016) provided these data.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5m + 5f

Control animals:

yes

yes, concurrent vehicle

Details on study design:

The dose setting with 0, 100, 300 and 1000 mg/kg bw/day is based on the results of a preliminary dose range finding study with MPI-ACA in rats, see Section 7.5.1, Szakonyine 2017.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

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. Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once weekly thereafter.
In the last exposure week, functional observations were conducted. Sensory reactivity to stimuli of different types, assessment of grip strength and motor activity assessment were examined. General physical condition and behavior of animals were tested. A modified Irwin test were performed.
Body weight and body weight gain: Individual body weights were recorded once during the acclimation. Animals involved in the study were weighed on Day 0 (prior to study start) and once weekly with a precision of 1 g. Individual body weight changes were calculated according to the days of measurements and for the study overall. The animals were also weighed immediately prior to sacrifice (fasted body weight) in order to calculate organ to body weight ratios.
Food consumption: Food consumption was determined with the measurement of non-consumed diet with a precision of 1 g once weekly to coincide with body weight measurements. All animals were fasted overnight prior to blood sampling.
Clinical pathology examinations: Clinical pathology examinations including hematology, blood coagulation and clinical chemistry were conducted in all surviving animals at termination of the treatment (i.e. one day after the last treatment; on Day 28). Animals were food deprived overnight prior to blood collection. Blood samples were harvested from the retro orbital venous plexus under Isofluran anesthesia. Three samples were taken from each animal: one for hematology, one for determination of blood clotting times and the third one to obtain serum samples for clinical chemistry.
Hematology: The following parameters were measured in all animals by SYSMEX XT-2000iV except differential white blood cell count for one control female rat (no. 4321). For this animal, the differential white blood cell count was determined by a light microscope counting blood smears stained with DIA-QUICK PANOPTIC because determination with SYSMEX XT-2000iV was not feasible: WBC White Blood Cell (leukocyte) count, RBC Red Blood Cell (erythrocyte) count, HGB Hemoglobin concentration, HCT Hematocrit (relative volume of erythrocytes), MCV Mean Corpuscular (erythrocyte) Volume, MCH Mean Corpuscular (erythrocyte) Hemoglobin.
Blood coagulation: APTT Activated partial Thromboplastin Time, PT Prothrombin Time.
Clinical chemistry: 17 parameters.

Sacrifice and pathology:

Necropsy: Gross pathology was performed on every experimental animal one day after the last treatment, i.e. 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. All observations were recorded with details of the location, color, shape and size.
Organs/tissues were removed and preserved in 4 % formaldehyde solution, except testes, epididymides, which were preserved in modified Davidson solution and then stored in 4 % formaldehyde solution for histopathological examination.
Organ weight: 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, uterus with fallopian tubes, thymus, spleen, brain and heart. With precision of 0.001 g: Adrenal glands.
Histopathology: Full histological 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). The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Statistics:

Statistical analysis was done with SPSS PC+ software for the following data:
- Body weight
- Food consumption
- Hematology
- Blood coagulation
- Clinical chemistry
- Organ weight

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related clinical signs were not detected at the daily clinical observations. The behavior and physical condition of animals were considered to be normal at each dose level (100, 300 or 1000 mg/kg bw/day) during the course of the 28-day observation period.
Alopecia on the skin of the right side cheek was noted for single female animal (1/5 at 300 mg/kg bw/day) from Day 7 up to and including Day 17. This dermal change (alopecia) occurs spontaneously in untreated experimental rats, which was present only in one mid dose treated animal in the present study. Therefore this was not considered to be toxicologically relevant.

Weekly detailed clinical observations: The animals exhibited normal behavior and physical condition at 100, 300 or 1000 mg/kg bw/day dose levels at the detailed clinical observations during the course of 28-day observation period. The behavior and physical condition of animals were not affected by the test item.

Mortality:

no mortality observed

Description (incidence):

There was no mortality at any of the tested dose levels (100, 300 or 1000 mg/kg bw/day MPI-ACA) or in the control group during the entire observation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The body weight and body weight gain of the male and female animals were no affected in any test item treated groups (100, 300 or 1000 mg/kg bw/day) throughout the entire observation period.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no test item related adverse effect in the mean daily food consumption in male or female animals at 100, 300 or 1000 mg/kg bw/day.

Food efficiency:

not specified

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

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related adverse effects were not identified in the examined hematological or blood coagulation parameters in the male or female animals at 100, 300 or 1000 mg/kg bw/day.
The mean white blood cell count (WBC) was slightly below the control value at 100 mg/kg bw/day in male animals. Slight, but statistically significant differences with respect to the control value were detected in male animals at 300 mg/kg bw/day at the lower mean white blood cell count (WBC), lower mean hemoglobin concentration (HGB) and hematocrit vale (HCT) as well as at the higher mean percentage of monocytes (MONO). The mean white blood cell count (WBC) was slightly but statistically significantly lower than in the control group in male animals at 1000 mg/kg bw/day.
In the female animals, statistically significant difference with respect to the control was observed at the lower mean white blood cell concentration at 100, 300 and 1000 mg/kg bw/day. All other hematological parameters were comparable in the control and test item treated female animals.
There were no differences – statistically or biologically significant – between the control and test item treated groups (100, 300 or 1000 mg/kg bw/day male and female) in the examined blood coagulation parameters (prothrombin time –PT – and activated partial thromboplastin time – APTT).
Alterations in the parameters listed above reached statistical significances with respect to the appropriate controls however there were no dose relevance (MONO, HGB and HCT in male animals, WBC in female animals) and all values remained well within the historical control range. Mean WBC of control group was slightly above the historical control mean both in male and female animals resulting in statistical significances at the lower values in treated animals. Therefore the changes noted in these hematology parameters were considered to be of little or of no toxicologically relevance.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in the male or female animals at 100, 300 or 1000 mg/kg bw/day.
In the male animals at 100 mg/kg bw/day, statistically significant difference with respect to the relevant control was found at the higher mean creatinine (CREA) and at the lower mean concentration of albumin (ALB). In the male animals at 300 mg/kg bw/day, slight but statistically significant differences were observed at lower mean activity of alanine aminotransferase (ALT) and lower mean concentrations of calcium (Ca2+), sodium (Na+) and total protein (TPROT) when compared to their controls. Similarly, statistical significances with respect to the relevant control were detected in male animals at 1000 mg/kg bw/day at the lower mean activity of alanine aminotransferase, at the lower mean concentrations of inorganic phosphorous (Pi), calcium, total protein and albumin: globulin ration (A/G).
All examined clinical chemistry parameters were comparable in the control and test item treated (100, 300 and 1000 mg/kg bw/day) female animals.
Although the mentioned differences between the control and test item treated groups reached statistically significances, values of these parameters (ALT, CREA, Pi, Ca2+, Na+, ALB, TPROT and A/G) were well within the historical control ranges. Some of these changes were only present in the lower dose groups (CREA, ALB) or were not related to doses (ALT, Ca+, TPROT). In the lack of related histopathological changes these findings in the clinical chemistry parameters were not considered to be of toxicological relevance but to be normal, physiological variations and not related to the test item.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Functional observation battery: Functional observation battery did not demonstrate any treatment-related alterations in the behavior or in reactions to different type of stimuli at the end of the treatment period (male and female; control, 100, 300 or 1000 mg/kg bw/day groups; on Day 26).
There were no changes in the physical condition, behavior or in reactions to different types of stimuli in the male or female animals of control and test item treated groups in the examined parameters during the course of the functional observations.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Test item related adverse effects were not detected in the weights of the examined organs of male or female animals administered with 100, 300 or 1000 mg/kg bw/day doses.
Statistical significance was noted for the slightly lower mean heart weight (absolute and relative to body weight) and liver weight relative to body weight in male animals administered with 100 mg/kg bw/day when compared to the appropriate control. In male animals at 300 mg/kg bw/day, the mean heart weight (absolute and relative to body weight) and liver weight relative to body weight were slightly below that of the control group. The mean heart weight (absolute and relative to body weight), liver and testes weight both relative to body weight were slightly but statistically significantly lower than in their control group in male animals at 1000 mg/kg bw/day.
In the female animals, statistical significance with respect to their control was noted for the higher mean weights of several organs (absolute or relative to body or brain weights) in each test item treated groups (100, 300 or 1000 mg/kg bw/day), such as kidneys, thymus, spleen, uterus or adrenal glands. These differences from the control were not related to doses at any organ except uterus. The mean weights of uterus showed slight dose dependence but the differences with respect to the control were not proportional to doses. The changes in the mean absolute and relative organ weights correlated well with the slight differences in the mean body weight and brain weight of test item treated animals comparing to their control. In the lack of supporting clinical chemistry or histopathological findings these organ weight changes were judged to be toxicologically not significant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MPI-ACA did not induce specific macroscopic alterations in the tissues or organs of animals administered with 100, 300 or 1000 mg/kg bw/day.
There were no macroscopic changes in the organs or tissues of male animals in the control, 100, 300 or 1000 mg/kg bw/day groups.
Slight hydrometra was noted for some female animal at 300 mg/kg bw/day (2/5) and at 1000 mg/kg bw/day (2/5). Hydrometra related to sexual cycle (pre-estrous/ estrous) of female animals is a frequent observation in this strain of experimental rats. Histological examinations did not reveal inflammatory or other pathological findings in the uterus at the high dose treated animals therefore hydrometra was not considered to be toxicologically relevant.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Histopathological examination did not reveal specific microscopic alterations related to the test item in the examined organs or tissues of animals in 1000 mg/kg bw/day group (male or female).
Minimal or mild alveolar emphysema was present in the lungs in control (1/5male; 1/5 female) and in 1000 mg/kg bw/day (2/5male) groups. Acute pulmonary emphysema was considered as a consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination.
Mild hyperplasia of bronchus associated lymphoid tissue (BALT), which occurred in control and test item treated male animals (2/5 male and 1/5 female in the control group and 1/5 male in 1000 mg/kg bw/day group) is a physiological phenomenon without inflammatory lesions.
The dilatation of uterine horns in some female animals (2/5 at 1000 mg/kg bw/day) is a slight neuro-hormonal phenomenon in connection with the sexual function – pro-estrus phase – of the inner genital organs.
No morphological evidence of 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 reproductive system or the central, or peripheral nervous system was observed. The structure and the cyto-morphology of the endocrine glands was the same in the control and treated rats.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Effect levels

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:

other: No toxic effects were observed.

Target system / organ toxicity

Key result

Critical effects observed:

no

Organ:

other: No critical organ /tissue/function detected.

Applicant's summary and conclusion

Conclusions:

MPI-ACA did not cause adverse effects in male or female Hsd.Han Wistar rats after the consecutive 28-day oral (by gavage) administration at 100, 300 or 1000mg/kg bw/day doses. The NOEL is 1000 mg/kg bw/day.

Executive summary:

A 28 -days repeated dose oral toxicity study with rats was performed according to the OECD guideline 407. Four groups of Wistar rats consisting of five animals per group and sex were administered orally by gavage once daily at 0 (vehicle only), 100, 300 and 1000 mg/kg body weight. The suitability of the chosen vehicle (recovery and stability) for the test item at the intended concentrations was analytically verified up front. Analytical control of dosing formulations was performed twice during this study. The concentration of the test item in the dosing formulations varied between ranges of 90 and 99 % of nominal concentrations thereby verifying a proper dosing.

Results: MPI-ACA did not cause adverse effects in male or female Hsd.Han Wistar rats after the consecutive 28-day oral (by gavage) administration at 100, 300 or 1000mg/kg bw/day doses.

Based on the observations made in this toxicity study the No Observed Adverse Effect Level (NOAEL) was determined to 1000 mg/kg bw/day for male and female Wistar rats.