[2R-(2α,3Z,5α)]-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, compound with tert-butylamine (1:1)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Treatment period was between May 14th and November 18th 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Materials and methods

Principles of method if other than guideline:

The study has been designed to meet the requirements of the Food and Drug Administration and the study conduct will comply with the requirements of "Good Laboratory Practices" published in the Federal Register, December 22, 1978.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium clavulanate

IUPAC Name:

Potassium clavulanate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Six hundred (600) air tight light resistant vials of BRL 14l51K (manufactured December, 1978) were received at Bio-Research Laboratories on February 9, 1979. Each vial was labelled with the code number BRL 14151K, the batch number CT 9267, the expiry date December 31, 1979 and the weight 6.3 g. The test article was an off-white powder which was identified on each vial as containing 78.8% pure free acid of BRL 1415lK. For the purposes of dosimetry, solutions of BRL 14151K were prepared assuming a 78.8% purity and all subsequent doses were expressed in terms of mg of pure free acid.

All supplies of BRL 1415lK were kept refrigerated at a temperature of 5°C prior to and during the course of the experiment.

Test animals

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 26 male and 26 female purebred, registered Beagle dogs (five to six months of age, body weight range males: 6 - 9.5 kg females: 4.75 to 9 kg) were received from White Eagle Laboratories, Doylestown, Pennsylvania on March 23, 1979. The supplier had immunized all dogs against rabies, canine distemper, infectious canine hepatitis and leptospirosis.

Males and females were identified by a permanent ear tattoo and were randomly assigned to the different experimental groups. The dogs were housed individually in conventional stainless steel cages which were equipped with t gallon water bottles fitted with gravity sippers. Fresh water was available ad libitum and 450 g of a standard dog food (50:50 mixture of Romar 90 and Purina Dog Chow) was given once daily during a one hour feeding period. The animals were conditioned to the laboratory setting during a four week acclimation period.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Solutions of BRL 14151K in distilled water were prepared fresh daily 'and were administered by oral gavage within 30 minutes of preparation. The dosing volume was set uniformly across all groups at 1 ml/kg and the dose concentrations were such that daily dose levels of 5, 10, 20 and 50 mg/kg pure free acid of BRL 14151K were administered to animals in Groups II, III, IV and V respectively.

VEHICLE
The vehicle controls received a daily dose of water at a volume of 1 ml/kg.

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

Treatment was initiated for Replicates A, B, C, D and E on May 14, May 15, May 16, May 17 and May 18, 1979 respectively.
The test and control articles were administered once daily, seven days a week for a period of six months. Dose volumes were adjusted weekly in order to take into account changes in body weight.

Frequency of treatment:

Solutions of BRL14151K in distilled water were prepared fresh daily 'and were administered by oral gavage within 30 minutes of preparation.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 5, 10, 20 and 50 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group 1 - vehicle control (7 males and 7 females)
Group 2 - 5 mg/kg/day (7 males and 7 females)
Group 3 - 10 mg/kg/day (7 males and 7 females)
Group 4 - 20 mg/kg/day (7 males and 7 females)
Group 5 - 50 mg/kg/day (7 males and 7 females)

Control animals:

yes, concurrent vehicle

Details on study design:

- Post-exposure recovery period:
After the completion of the six month dosing period, three male and three female animals from Group I (dogs No. 105 to 107 and 155 to 157) and Group V (dogs No. 505 to 507 and 555 to 557) were allowed a thirty day period off treatment before necropsy in order to assess the reversibility of any treatment related effects.

Examinations

Observations and examinations performed and frequency:

Clinical observations: Were performed daily
Mortality was observed daily

Fecal occult blood determinations were carried out on all animals for three consecutive days pretreatment, after 1, 3 and 6 months of test article administration and at the end of the regression period.

Individual body weights were determined weekly during the pretreatment, treatment and regression periods.

Food Consumption
A standard diet (450 g of a 50:50 mixture of Purina Dog Chow and Romar 90) was given to each dog daily during a one-hourfeeding period.

Ophthalmoscopy:
Once prior to the initiation of treatment, after one, three and six months of drug administration and again after a one month regression period. the eyes of each dog were subjected to a complete funduscopic and biomicroscopic examination by a certified ophthalmologist

Other examinations:

Hematology:
Blood samples were collected via the jugular vein from all animals twice pretreatment, after one, three and six months of treatment and again at the end of the regression period. Each hemogram consisted of: RBC count, packed cell volume, hemoglobin level, total and differential WBC count, prothrombin time, activated partial thromboplastin time, platelet count, reticulocyte count and a calculation of Wintrobe's constants (MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation

Mortality:

mortality observed, treatment-related

Description (incidence):

salivation

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

it is concluded that daily treatment with BRL 14151K for six consecutive months had no effect on food consumption in Beagle dogs.

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

In summary, daily treatment with BRL 14151K at doses of 5, 10, 20 and 50 mg/kg had no effect on fluid consumption in male or female Beagle dogs.

Ophthalmological findings:

effects observed, treatment-related

Description (incidence and severity):

Effects were noted however none of these findings can be attributed to treatment with BRL 14151K.

Haematological findings:

no effects observed

Description (incidence and severity):

Control and treated animals were homogeneous with respect to values obtained for hematological parameters evaluated during the course of the study.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No non reversible treatment related effects were observed

Urinalysis findings:

no effects observed

Description (incidence and severity):

Control and treated animals were homogeneous with respect to values obtained for urinary parameters evaluated during the course of the study.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

organ weight assessmentrevealed no effect of treatment with BRL l4l5lK.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Reddening of the gastric mucosa was seen in treated dogs at necropsy, but this was also seen in control males and females and therefore cannot be attributed to treatment with BRL 14151K.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histopathological studies revealed a reversible hepatic hydropic vacuolization in 4/8 dogs, after six months of treatment at a dose level of 50 mg/kg/day. These changes were not observed in the regression group and considered to be reversible.

Histopathological findings: neoplastic:

no effects observed

Details on results:

There were no deaths during the course of this study and the only treatment related effect consisted of a slightly higher incidence of salivation and emesis after daily treatment with BRL 14151K at dose levels of 50 mg/kg.

It may be seen that the greatest incidence of salivation during and/or after daily treatment was observed in Group V males and females (BRL 14151K, 50 mg/kg/day). Salivation occurred less frequently in animals from other treated groups and was not observed at all in the vehicle controls.

Emesis was observed in animals from all groups both before and after daily treatment. The incidence of emesis in Group V males and females tended to be slightly higher than control levels throughout the six month treatment period whereas the incidence of emesis in animals from Groups II to IV inclusive was not different from that observed in controls.

Emesis generally occurred within 1.5 hours of treatment. The vomitus was described as consisting of a clear white to yellowish foamy liquid containing partially digested food.

Incidental clinical signs noted during the treatment period consisted of sporadic occurrences of diarrhea in dogs from all groups. cuts and abrasions to the feet and legs due to caging and a variety of skin conditions. The ventral surface of the pinna and the external ear canal of dogs in several groups were found to be reddened, dry and inflamed.

In summary, daily administration of BRL 14151K caused a slightly elevated incidence of salivation and emesis after daily treatment in comparison to controls. These differences were most pronounced in groups receiving a daily dose of 50 mg/kg pure free acid. No other treatment related effect was apparent.

A very low incidence of positive reaction for fecal occult blood was reported in both males and females. None of the positive reactions recorded appeared to be consistently related to one group of animals and therefore could not be attributed to treatment with BRL 14151k.

Bodyweight measurements:
During the 30 day regression period body weights of control and high dose males and females remained stable or slightly increased indicating that body weights were not adversely affected by the gavage procedure or by BRL 14151K administration. In summary, body weight measurements revealed no adverse effect of treatment with BRL 14151K at doses of 5, 10, 20 and 50 mg/kg/day.

In summary, body weight measurements revealed no adverse effect of treatment with BRL 14151K at doses of 5, 10, 20 and 50 mg/kg/day.

Food consumption:
The food consumption of individual dogs was determined daily during the acclimatization, treatment and regression periods by subtracting the amount of food remaining at the end of each feeding period from the initial 450 g given. It was concluded that daily treatment with BRL 14151K for six consecutive months had no effect on food consumption in Beagle dogs.

Fluid consumption:
Daily fluid consumption values were averaged on a weekly basis for each dog. In general, males tended to consume more water daily than females and had a larger range of fluid consumption. No differences between the five experimental groups were present and isolated increases or decreases in fluid consumption in males or females during the pretreatment, treatment or regression periods usually could be attributed to one or two animals.

In summary, daily treatment with BRL 14151K at doses of 5, 10, 20 and 50 mg/kg had no effect on fluid consumption in male or female Beagle dogs.

ophthalmological findings:
Abnormalities observed consisted of a congenital iris adhesion in male No. 103 and the presence of a corneal sub-epithelial infiltrate in male No. 503 pretreatment. Females no. 352 and 553 had persistent edematous membrana nicitans and a bilateral sub-epithelial haziness was observed pretreatment in the cornea of female No. 454. None of these findings can be attributed to treatment with BRL 14151K.

Hematology:
For each parameter examined, a considerable range of normal variability is possible. Both pretreatment data and values obtained for the control animals on all assessment occasions indicate the possible range of variation.

Considering group mean data for both males and females over the six month treatment period, it was apparent that treated and control groups were comparable for all parameters evaluated. Group mean regression data in males and fema~es showed a slightly increased lymphocyte count and a slightly decreased segmented neutrophil count in comparison to the six month data. These differences, however, may be attributed to a few individual animals.

In summary, six months of treatment with BRL 14151K at doses of 5, 10, 20 and 50 mg/kg had no effect on the hematological parameters.

Urinalyses:
On each sample
collected, an assessment of the following qualitative or semi-quantitative parameters was conducterl: Appearance (colour, odour and transparency), pH, glucose, bile pigments, ketone bodies, protein and occult blood.

A microscopic examination of the urinary sediment was carried out following centrifugation.

Occult blood, ketones, protein and bilirubin were detected sporadically in the urine of males and females from all groups pretreatment, after one, three and six months of treatment and at the end of the regression period. Similarly, an evaluation of urinary sediment revealed the presence of white cells and epithelial cells in the urine of animals from all groups. These findings are normal for Beagle dogs and in no way reflect effects of treatment with BRL 14151K.

No difference between groups was discernible in terms of the complement of crystals present and therefore one may conclude that treatment with BRL 14151K had no effect on this parameter. In summary, treatment with BRL 14151K at doses of up to 50 mg/kg/day had no adverse effect on the urinary parameters examined in this study.

Blood Biochemical Analyses:
The individual data revealed isolated values falling outside of normal ranges. In view of the large number of parameters examined, the identification of a few parameters exceeding normal ranges is to be expected.

In summary, six months of daily administration of BRL 14151K at dose levels of 5, 10, 20 and 50 mgjkg was without effect on the blood biochemical parameters evaluated in this experiment.

Gross Pathological Findings:
At the conclusion of six months of treatment and at the end of the regression period, the appropriate dogs were killed routinely by intravenous administration of sodium pentobarbital and a gross pathological examination of each animal was performed.
Mild reddening or petechiation of the mucosal surface throughout the gastrointestinal tract and particularly at the ileocecal junction, was observed with equivalent frequency in treated and control groups both after six months of treatment and after the one month regression period.

When only the glandular portion of the stomach. was considered, there was a tendency for the focal reddening of the mucosa to be more prevalent in treated than control animals. The exceptions to this, however, were male and female controls sacrificed after one month regression. The generalized and non-specific nature of the changes observed suggest that they were not caused by treatment with BRL 14151K.

Similarly, no systematic change associated with treatment could be identified in the livers or kidneys of male or female dogs in this study. Numerous incidental lesions were identified with equivalent frequency in treated and control animals. The low incidence of these lesions coupled with the fact that control and treated animals were equally affected indicate that they cannot be attributed to treatment with BRL 14151K.

Organ Weights:
The majority of organs were weighed within 20 minutes of the onset of the necropsy procedures and all measurements were complete within 45 minutes. In summary, organ weight determinations carried out after six months of treatment and at the end of the regression period, failed to reveal any effect of treatment with 8RL 14151K.

Histopathology:
Oral administration of BRL 14151K at the high dose level (50 mg/kg/day) produced mild hydropic vacuolization in the livers of some dogs.
Hydropic vacuolization was characterized by the presence of clear vacuoles within the hepatocytic cytoplasm. These vacuoles stained negatively with fat and glycogen stain. The nuclei were well preserved and no overt hepatic necrosis was revealed. As is indicated in the table, one of four males (No. 502) and three of four females (Nos. 551, 553 and 554) of the high dose group showed this change. The change was reversible as it was not demonstrable in the regression animals. Animals in other treated and untreated groups did not reveal this change.

In this study, oral administration of BRL 14151K for a period of six months at a dose level of 50 mg/kg/day produced a reversible hepatic hydropic vacuolization in four of eight dogs. No other effects of treatment with BRL 14151K were apparent at any of the other dose levels.

Effect levels

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The potential toxicity of orally administered BRL 14151K was investigated in the Beagle dog during a six month treatment period. There were no deaths during the course of this study. Effects were seen over the course of the study however the majority of these effects were seen in both control male and female control animals. No non reversible effects were seen throughout the study therefore the No Observed Adverse Effect Level has been set to 50 mg/kg which was the highest dose group selected.

Executive summary:

The potential toxicity of orally administered BRL 14151K was investigated in the Beagle dog during a six month treatment period. Five groups of dogs were treated daily either with distilled water or with an aqueous solution of BRL 14151K. The doses evaluated in the four treatment groups were 5, l0, 20 and 50 mg pure free acid of BRL 14151K per kg of body weight. At the end of the six month treatment period, three males and three females from each of the high dose and vehicle control groups were maintained for 30 days off treatment before they were killed in order to evaluate the regression of treatment related effects.

There were no deaths during the course of this study.

Emesis was associated with treatment with BRL 14151K and the occasional presence of blood streaks was noted. Similarly, salivation was associated with treatment with BRL 14151K, high doses exhibiting salivation at an earlier study date than other treated groups. In males, the number of animals affected in the high dose was higher than in the lower doses. Females exhibited salivation only at the higher doses with the number of animals affected the same as the male high dose group.

Tests performed to detect fecal occult blood failed to demonstrate a dose-related effect although positive results were found during the course of the study in all treated and control groups for both males and females.

Body weight and food and fluid consumption measurements revealed no adverse effect of treatment with BRL 14151K in this study.

The biomicroscopic and funduscopic examinations conducted during the experiment demonstrated no finding related to treatment with the test article.

Control and treated animals were homogeneous with respect to values obtained for hematological and urinary parameters evaluated during the course of the study. The only noteworthy finding was the presence of abnormal cytoplasmic granulations in neutrophils which were observed more frequently in animals from the high dose group (BRL 14151K, 50 mg/kg/day.

In general, the blood biochemical data were unremarkable and revealed no effect of treatment with BRL 14151K.

Electrocardiographic studies revealed no effect of BRL 14151K on cardiac function during the six months of treatment.

Reddening of the gastric mucosa was seen in treated dogs at necropsy, but, since at regression, control males and females displayed similar findings, this change could not be attributed to treatment with BRL 14151K.

Histopathological studies revealed a reversible hepatic hydropic vacuolization in four of eight.dogs after six months of treatment with BRL 14151K at a dose level of 50 mg/kg/day. This was characterized by the presence of clear vacuoles within the hepatocytic cytoplasm in high dose males and females. These changes were not observed in dogs of the regression group and therefore were considered to be reversible. Gastric changes noted at necropsy were not judged to be treatment related since microscopic alterations were seen equally in both control and treated groups. Bone marrow examinations and organ weight assessmentrevealed no effect of treatment with BRL 14151K.