[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)

Toxicity to reproduction

Administrative data

Endpoint:

fertility, other

Remarks:

based on test type (migrated information)

Type of information:

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

Adequacy of study:

key study

Study period:

December 20, 1983 – October 28, 1985

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:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: “Animal testing law relating to the effect of pharmaceutical products on reproduction (Notification 529 of Pharmaceuticals and Cosmetics division dated March 31, 1975)”,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Test item : Potassium Clavulanate (CVA-K)
Description : This test compound is easily soluble in water and is a white or pale yellowish-white colored powder.
Received : December 20, 1983
Storage Method : Cold storage, light shielded (Refrigerator)
Test Compound: : Potassium Clavulanate (Hereinafter referred to as CVA-K)

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source:
- Age at study initiation: 140 five-week old Crj: CD (SD) male rats were purchased from Charles River Laboratories Japan Inc., and those which showed no abnormalities in their general condition after a week at the research center were used in the test. Furthermore 140 eleven-week old nulliparous female rats of the same breed were purchased from Charles River Laboratories Japan Inc., 6 weeks after the receipt of the males and they were kept at the research center for 2 weeks.
- Weight at study initiation: (P) Males: 179 ~ 206g g; Females: 243~299g;
- Fasting period before study:
- Housing: A metal mesh cage (320 x 260 x 175mm: Japan Cage K.K.) was divided into two with a divider plate, with each compartment holding one animal,
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): the rats were free to consume solid feed (NMF : Oriental Yeast Co., Ltd.)
- Water (e.g. ad libitum): the rats had free access to tap water (Gotemba City Water).
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
The animals were kept in a room (room 501) set at temperature 22 +/- 2deg C, humidity 55 +/- 10%, ventilation frequency 13 times / hour, and illuminated 12 hours a day (7am – 7pm).

IN-LIFE DATES: From: Day 0 To: End of Study

Administration / exposure

Route of administration:

intravenous

Details on exposure:

Administered fluid volume was made up to 5 ml/kg by adding to 1 vial (5.0g) of the test compound , isotonic sodium chloride solution for injection (Japanese Pharmacopoeia: Otsuka Pharmaceutical Factory, Inc.) at a ratio of 5g/33.3ml, dissolved and formed into 15% w/v solution. Moreover, isotonic sodium chloride solution for injection was used to make the concentration required for each administration group (1.50, 1.00, 0.50 and 0.25% w/v), and these were taken to be the 75.0, 50.0, 25.0, 12.5mg/kg solutions respectively.

Details on mating procedure:

Breeding Method and Confirmation of Mating
Mating began, after the post-administration period of 9 weeks for males, 2 weeks for females. For mating, a male and female from the same administration group were put into the same space at a ratio of 1:1 overnight, and those with copulation plug formation or semen in the vagina were thought to have completed mating and this was taken to be pregnancy day 0. When mating was not confirmed, the mating was re-executed with the same male-female combination. However the mating period was set to be 2 weeks maximum.

Duration of treatment / exposure:

Administration was carried out intravenously, and the test compound of each concentration was administered at a speed of 0.05 ml/sec every day, in males from 9 weeks before mating, during the mating period and from the end of the mating period to the time of autopsy and in females from 2 weeks before mating, during the mating period and from pregnancy day 0 to day 7, once a day in their caudal veins.

Frequency of treatment:

once a day

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
75 mg/kg
Basis:

Doses / concentrations 2

Remarks:

Doses / Concentrations:
50 mg/kg
Basis:

Doses / concentrations 3

Remarks:

Doses / Concentrations:
25 mg/kg
Basis:

Doses / concentrations 4

Remarks:

Doses / Concentrations:
12.5 mg/kg
Basis:

No. of animals per sex per dose:

22 males and 22 females at each concentration

Control animals:

yes, concurrent vehicle

Details on study design:

Control animals:
The control group was administered with isotonic sodium chloride solution for injection (Japanese pharmacopoeia: Otsuka Pharmaceutical Factory, Inc.) in a similar manner. Furthermore the volume of solution administered was decided based on the body weight measured on the administration day, or on a day closest to the administration day, and the test solution adjusted and administered within the time where stability has been confirmed by a test trustee.

Positive control:

None

Examinations

Parental animals: Observations and examinations:

General condition:
The general conditions of the animals were observed every day during the test period. The times of observation during the administration period were before administration, immediately after administration as well as 1 hour post-administration.

Body Weight Measurement:
Measurements were taken twice a week during the administration period prior to mating, and during pregnancy measurements were taken on 0, 4, 7, 11, 14, 17 and 20 days into pregnancy.

Food Consumption Measurement:
During the administration period prior to mating, one day food consumption for the previous day was measured twice a week on the same day as the body weight measurement days, and during pregnancy it was measured on 1, 4, 7, 11, 14, 17 and 20 days into pregnancy.

Observations of the Early Gestation Parent Animal and the Fetus:
All animals confirmed to have mated were killed with carbon dioxide gas on the afternoon of pregnancy day 20, and they were immediately opened up to make observations by eye of the main organs as well as examining the number of live, resorbed and dead fetus and the classifications (resorbed embryo, placental remnant, early macerated fetus, late macerated fetus, dead fetus). After this, the placental weight as well as the weights of the parent animal’s heart, lungs, liver, kidney, spleen and ovaries were measured, then fixed and stored in phosphate buffer 10% formalin.

Oestrous cyclicity (parental animals):

Estrous cycles were observed by collecting vaginal samples in 10 examples from each group during the pre-mating administration period, mating period until confirmation of mating.

Litter observations:

The sex of the live fetus was determined, and after body weights were measured, observations were made for any external abnormalities, including inside the oral cavity. Furthermore, internal abnormalities of each body section were carried out following Nishimura’s method for the chest and the stomach, Wilson’s freehand razor method for the head, and for the investigation of each stomach approximately 1/3 of the live fetuses were fixed in Bouin. The remaining 2/3 of the live fetus was fixed in 70% alcohol, and Alizarin Red S skeletal cleared specimen was formed in accordance with the Dawson method 4) and skeletal abnormalities, variations and progress of ossification were investigated under a stereomicroscope.

Postmortem examinations (parental animals):

Males were killed by Carbon Dioxide gas after the completion of the mating period, the dissection of the main organs were carried out and weights of the heart, lungs, liver, kidneys, spleen, testes and epididymis measured after extraction, and the absolute weights along with relative weights were calculated. Along with the aforementioned measured organs, the seminal vesicle and the prostate were extracted and these were fixed and stored in phosphate buffer 10% formalin. Furthermore, the testes, epididymis, seminal vesicle and the prostate of males confirmed to have mated (female not impregnated) were formed into H.E.-stained specimens via the usual method and histopathological examination executed.

With regards the females, autopsies were carried out at the same time as the early gestation caesarean section and the weights were measured for the heart, lungs, liver, kidneys, spleen and ovaries after extraction, with only absolute weights recorded for the impregnated animals. Non-pregnant animals had their uterus weight measured and the absolute and relative weights of each organ recorded. A part of the uterus and the organs whose weight was measured were fixed and stored in phosphate buffer 10% formalin. Furthermore the ovary and uterus of the non-pregnant animals were formed into H.E.-stained specimens in a similar manner to the males and histopathological examination executed.

Postmortem examinations (offspring):

Observations were made for any external abnormalities, including inside the oral cavity.

Statistics:

Each of the values were processed using a parent animal unit or a group unit, and then for copulation index, fertility index and pregnancy index , chi-squared tests were applied; whilst all other values were tested based on the Kruskal – Wallis test) and the multiple comparison method.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One animal died on day 47 in the 75 mg/kg treatment group, however among the live examples in the 75.0mg/kg administered group and those in the groups administered 50.0mg/kg and below, no notable symptoms were seen.

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:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

These observations were not thought to be dose-dependent and were thought to be incidental changes.

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

In each group, 2-4 were observed in most during the 2 week observation period, and no significant difference was confirmed between the control group and any of the administered groups.

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Details on results (P0)

All examples from all administered groups mated within the mating period, and there were no significant differences confirmed between the control group and the administered groups in the number of days required until copulation. In the mating couplings, there were were 3 examples of non-pregnancy in the control group and 1,2,4 and 3 examples of non-pregnancy in the groups administered 12.5, 25.0, 50.0, 75.0 mg/kg respectively, but there were no significant differences seen between the control group and the administered groups in the pregnancy rate.

Effect levels (P0)

1

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

2

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: In females, other than the observation of reduced food consumption 4 days into their pregnancy in the group with 75.0mg administered, no changes thought to be caused by the test compound were found in the general conditions, body weight or autopsies.

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Effect levels (F1)

Dose descriptor:

NOEL

Generation:

F1

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: With regards the early gestation fetuses, no abnormalities thought to be caused by the test compound administration were seen on their external appearance, skeleton or viscera.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Organ weights:

An increase in the weight of the liver were seen to be dose-dependent, and a significant increase was seen in the absolute weight within the groups administered more than 50.0mg/kg, and a significant increase seen in the relative weight in groups administered more than 25.0 mg/kg. In the testes, a significant increase was seen in the relative weight for the group administered 12.5 mg/kg, in the epididymis, a significant increase was seen in the absolute weight for the group administered 12.5 and 50.0 mg/kg, but these changes were not thought to be dose-dependent.

Discussion:

Using Crj: CD (Sprague Dawley) rats, the effect of administering 12.5, 25.0, 50.0 and 75.0 mg/kg/day in each male rat from 63 days before mating until the day of autopsy following copulation confirmation and in each female rat from 14 days before mating until 7 days pregnant in itscoccygeal vein on their fertility and fetus.

In the post-mating period autopsies, no abnormalities thought to be caused by test compound were observed, but in relation to the organ weights, a dose-dependent increase of the liver was confirmed and a significant difference was seen for the absolute weights in the groups administered more than 50.0 mg/kg, and for the relative weights in the groups administered more than 25 mg/kg.

In females, other than the observation of reduced food consumption 4 days into their pregnancy in the group with 75.0 mg administered, no changes thought to be caused by the test compound were found in the general conditions, body weight or autopsies.

With regards the fertility of the parent rats, there were no effects seen on the females’ estradiol cycles, the mating ability of males and females, fertilization or implantation, and the fetal development post implantation was also normal. With regards the early gestation fetuses, no abnormalities thought to be caused by the test compound administration were seen on their external appearance, skeleton or viscera.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, it is thought that the maximum non-effect does levels of CVA-K for the general toxicological responses, fertility of the parent animals, and the development of the next generation under these test conditions are 25.0mg/kg/day, 75.0mg/kg/day and 75.0mg/kg/day respectively.

Executive summary:

CVA-K (potassium clavulanate) was administered intravenously at 12.5, 25, 50 and 75 mg/kg/day for 9 weeks to male rats and for 14 days to female rats. The animals were then paired and effects of treatment on reproductive performance were studied. Treatment continued during the mating period and for females that had mated, until Day 7 of pregnancy. All pregnant rats were killed and examined by cesarean section on Day 20 of pregnancy and effects of treatment on the fetuses were also examined.

Increased liver weights were found at 50 and 75 mg/kg at autopsy after the mating period. Otherwise, no marked changes attributable to the test compound were noted.

A slight decrease in food consumption was observed at 75 mg/kg in the first stage of the gestation period. Otherwise, no marked changes attributable to the test compound were noted.

Reproductive performance

Copulation index, fertility index and pre-coital interval showed no treatment related changes at any dose level.

Neither were the number of Corpora lutea, implantations or dead and resorbed fetuses affected by treatment.

Fetuses:

No changes attributable to the test compound were observed in any treatment group with respect to fetal body weight and fetal external, visceral or skeletal observations following cesarean section.

In view of the above findings, the maximum non-effect dose levels for general toxicological responses in the parents, for reproductive performance and for the development of the F1 generation are considered to be 25 mg/kg, 75 mg/kg and 75 mg/kg respectively.