[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)

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

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

Adequacy of study:

key study

Study period:

October 1983 - December 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

Limit test:

yes

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
Received : July 26, 1983
Storage Method : Cold storage, light shielded (Refrigerator)

The test compound is Potassium Clavulanate (CVA-K) and was supplied by Beecham Yakuhin K.K. This test compound is easily soluble in water and is a white or pale yellowish-white colored powder. In this test, test compound with lot number: CT12338 (Content: Free Acid 801 µg/mg) was used.

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Details on test animals or test system and environmental conditions:

11-week old Crj: CD (SD) male rats and nulliparous female rats of the same breed 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 for mating. For mating, a 12-week old nulliparous female rat was put into the same space as a male of the same age 1:1 overnight (4pm - 10am), and those with copulation plug formation or semen in the vagina were thought to have copulated and were utilized in the test, with this day taken to be pregnancy day 0. Moreover the body weights on pregnancy day 0 were 210 - 310g.

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, and the rats were free to consume solid feed (NMF : Oriental Yeast Co., Ltd.) and tap water (Gotemba City Water).

However dams delivering naturally were separately and individually collected into plastic Econ Cages (300 x 355 x 175mm : CLEA Japan, Inc.) containing bedding and free access to food and water.

Animal Identification:
Ear tags were used to identify the mating male and female animals during the naturalization period and the offspring from aged 4 weeks, and oil-based ink was used as additional identification aid. Furthermore a table collating the animal numbers and ear tag numbers was created, and the cages were attached with labels showing the test number, administration amount, animal number as well as the ear tag number.

Environmental conditions:
The animals were kept in a room which was automatically kept at a temperature of 22  2C, humidity of 55 +/- 10%, ventilation frequency 13 times / hour, and illuminated 12 hours a day (7am – 7pm).

Administration / exposure

Route of administration:

intravenous

Details on exposure:

Administered fluid volume was made up to 5 ml/kg by adding to 1 vial (0.5g) of the test compound, isotonic sodium chloride solution for injection (Japanese Pharmacopoeia), then dissolving to make 16.7ml and this was taken to be the 150 mg/kg solution (3.0 w/v %). Moreover, isotonic sodium chloride solution for injection was used to make the concentration required for each administration group (1.50, 1.00 and 0.50 w/v %), and these were taken to be the 75.0, 50 and 25 mg/kg solutions respectively. The path of administration was as clinically expected through the vein, and 5 ml/kg of the test solution of each concentration was administered at an administration speed of 0.05 ml/sec. once a day for 11 days, from pregnancy day 7 until pregnancy day 17 into the caudal veins. The control group was administered with isotonic sodium chloride solution for injection (Japanese Pharmacopoeia) in a similar manner. The volumes of solution administered were calculated based on the body weight measured on the day. The test compound solution is adjusted and administered within the period during which its stability is confirmed by the test trustees.

With regards the days of pregnancy, the day when copulation was confirmed was taken to be pregnancy day 0, with regards the days post-delivery, the day of delivery is taken to be 0

Details on analytical verification of doses or concentrations:

The amounts administered were decided with reference to the results from a separately reported preliminary testing (administered amounts: 6.25, 12.5, 25, 50, 100, 150, 200) utilizing pregnant dams. One example of death was confirmed in the group administered 200 mg/kg whilst a slight suppression in weight increase was confirmed in the group administered 150 mg/kg. Therefore 150 mg/kg was suspected to be the volume at which there was a slight onset of toxicity, so 150 mg/kg was set to be the maximum value, and below this, values were set based on a common ratio with 75 mg/kg as the high value, 50 mg/kg as the medium value and 25 mg/kg as the low value. Moreover the administered volume was indicated as the weight of the free acid.

Details on mating procedure:

For mating, a 12-week old nulliparous female rat was put into the same space as a male of the same age 1:1 overnight (4pm - 10am), and those with copulation plug formation or semen in the vagina were thought to have copulated and were utilized in the test, with this day taken to be pregnancy day 0.

Duration of treatment / exposure:

The path of administration was as clinically expected through the vein, and 5 ml/kg of the test solution of each concentration was administered at an administration speed of 0.05 ml/sec. once a day for 11 days, from pregnancy day 7 until pregnancy day 17 into the caudal veins. The control group was administered with isotonic sodium chloride solution for injection (Japanese Pharmacopoeia) in a similar manner.

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 50, 75 and 150 mg/kg
Basis:
other: administered intravenously

No. of animals per sex per dose:

There were 5 treatment groups including the control group, with each group containing 36 - 41 female confirmed to have copulated.

Control animals:

yes

Examinations

Maternal examinations:

The general conditions of the animals were observed every day during the period of administration. The observations were carried out prior to administration, immediately after administration as well as 1 hour after administration. However on holidays it was stopped at the one immediately after administration. Autopsies were carried out on the dead animals, and the development conditions of the fetus were observed.

Body Weight Measurement
Measurements were taken during pregnancy on day 0 and 4 of pregnancy, every day from day 7 to day 20 of pregnancy, and during the nursing period on days 0, 4, 7, 11, 14, 17 and 21 after delivery.

Food Consumption Measurement:
food consumption over the previous 24 hours was measured on days 4, 8, 11, 14, 17 and 20 of pregnancy and 1, 4, 7, 11, 14, 17 and 21 days after delivery.

Dams not killed in pregnancy (11-12 pregnant animal from each group) were made to naturally deliver and the delivery conditions were observed and the pregnancy period and birth rate ( (Number of females giving birth to offspring / Number of pregnant females ) x 100) calculated. The animals which delivered will be left to nurse the offspring for 21 days after delivery, and the nursing conditions were observed. All examples were killed by Carbon Dioxide gas on day 21 after delivery, and the main organs were inspected by eye, and number of implantation sites checked. After this, the weight of the dams’ heart, lungs, liver, kidneys, spleen and ovaries were measured, then fixed and stored in phosphate buffer 10% formalin. The uterus was partially stored in the same manner. The relative weight of each organ was calculated with the weights on day 21 after delivery as the basis.

Ovaries and uterine content:

Dams (20-21 pregnant animals in each treatment group) were killed with Carbon Dioxide gas on the afternoon of pregnancy day 20, opened up immediately and as well as observing the main organs by eye, the ovaries and the uterus were extracted.

Fetal examinations:

Dams (20-21 pregnant animals in each treatment group) were killed with Carbon Dioxide gas on the afternoon of pregnancy day 20, opened up immediately. The number of corpus lutea, number of implantations, number of live fetus, resorbed fetuses and their classifications (resorbed embryo, placental remnant, early macerated fetus, late macerated fetus, dead fetus) were investigated.

The sex of the live fetuses were 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 live fetuses were fixed in 70% alcohol, and Alizarin Red S skeletal cleared specimen was formed in accordance with the Dawson method and skeletal abnormalities, variations and progress of ossification were investigated under a stereomicroscope.

Dams not killed in pregnancy (11-12 pregnant animal from each group) were made to naturally deliver and the delivery conditions were observed and the pregnancy period and birth rate ( (Number of females giving birth to offspring / Number of pregnant females ) x 100) calculated. The number of live offspring and dead offspring were counted, and the existence of external abnormalities, including within the oral cavity, and sex were checked in the live offspring, and the weights were measured, and subsequent to that the weights were measured twice a week until 21 days after birth, and once a week between day 21 to 70.

On day 21 after birth, one male and female animal from each dam were observed for their general condition and functional development based on Irwin profile, locomotor activity based on wheel cage method, neuromuscular activity based on the inclined screen method and the Rotorod method. Furthermore, sight and auditory reflexes were observed using the pupillary and pinna reflex tests. The pupillary reflex test looked at whether or not the pupils contracted when penlight is shone at the animal after it has been adapted to the dark for approximately 30 seconds. Pinna reflex test checked whether the animal’s auricle moves when galton’s whistle (frequency approximately 5200 ~ 6200 Hz) is sounded from a distance of 1m.

Statistics:

After the various values were processed based on the dam units or group units, chi-squared tests were used for the sex ratio, differentiation index, rotarod test, inclined screen method, reflex tests, copulation index and fertility index, and Kruskal-Wallis and multiple comparison methods) used for the testing of other values.

Indices:

The birth rate was calculated as follows:
birth rate ( (Number of females giving birth to offspring / Number of pregnant females ) x 100) calculated

Furthermore, based on the number of live offspring during the nursing growth period, the live birth index ((number of live offspring/number of implantation sites) x 100), 4-day survival index ((number of offspring alive 4 days after birth/number of live offspring) x 100), weaning index ((number of offspring at weaning/number of offspring alive 4 days after birth) x 100) and viability index ((number of offspring alive 70 days after birth / number of offspring after the selections on day 21 after birth) x 100) were calculated.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Visceral and Skeletal Findings in the Fetus:
With regards visceral abnormalities, dilatation of lateral and third ventricle was seen in one example in the group administered with 25 mg/kg, and dilatation of renal pelvis and kinked ureter was seen in one example in the treatment group administered with 75 mg/kg.

Effects on F1:
Skeletal abnormality included one example in the treatment groups administered with 25 and 50 mg/kg (3.0 and 2.2%) of dysplasia of the caudal vertebrae, and one example in the treatment group administered with 75 mg/kg (2.6%) of nodulated ribs, but these rate of emergence showed no significant difference between the control group and the administered treatment groups.

Skeletal variations were seen in 4 examples in the control group (10.8%), in 6~15 examples in the administered treatment groups (13.3% ~ 45.5%), but the rate of emergence showed no significance difference between the control group and the administered treatment groups. The types of variations were 1-2 examples in the 50 and 75mg/kg administered treatment groups (2.2~6.1%) of cervical rib, 2 examples in the control group (5.4%) and 5-7 examples in the 25, 75 and 150 mg/kg administered treatment groups (11.1 ~ 18.4%) of 14th rib, 1 examples in the control group (2.7%) and 2~7 examples in the administered treatment groups (6.1~15.6%) of dysplasia of the metacarpal bones, and 3 examples in the control group (8.1%) and 1 ~ 7 examples in the administered treatment groups (2.2 ~ 18.4%) of extra sternebra.

As the effects seen were few and far between these were not considered to be treatment related.

Effect levels (fetuses)

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

One example of death was confirmed during the administration period in the 150 mg/kg administered group. This example became recumbent immediately after administration on pregnancy day 14 (administration day 7) and died. Other than this no abnormalities in the general conditions were confirmed during the administration period.

Change in Body Weight:

No significant differences were confirmed between the control group and all administered treatment groups for the body weight during the nursing period.

Food consumption:

During pregnancy, a downward trend in the amount of food consumed after the start of administration in the 150 mg/kg administered group. There were no significant differences confirmed between the control group and the administered treatment groups in the food consumed during the nursing period.

Organ weights and macropathological findings:

For macropathological findings, there were dark red spots in all lobes of the lung in one example in the control group, accessory spleen and slight enlargement of the placenta in one example in the 25 mg/kg administered group and dilatation of renal pelvis in one example in the 25 mg/kg administered group and 2 examples in the 50 mg/kg administered group. These rates of occurrence were not thought to show a significant difference relative to the control group.

No significant difference was shown in the organ weights (absolute weight) between the control group and the administered treatment groups.

 Findings at the Caesarean Section:

Significant low values of the live fetus were found in the 150 mg/kg administered group. No significant differences between the control group and the administered treatment groups were confirmed in the number of corpus lutea, number of implantations, dead or resorbed fetuses, live fetuses, sex ratio or placental weights.

External appearance observations showed umbilical hernia in one example in the group administered 150 mg/kg

Visceral and Skeletal Findings in the Fetus:

With regards visceral abnormalities, dilatation of lateral and third ventricle was seen in one example in the group administered with 25 mg/kg, and dilatation of renal pelvis and kinked ureter was seen in one example in the treatment group administered with 75 mg/kg.

No examples of skeletal abnormalities were seen in the control group nor in the administered treatment groups.

Observation of Delivery and Nursing Condition:

No abnormalities were seen with regards the delivery condition in any of the administered treatment groups, and no significant differences between the control group and the administered treatment groups were confirmed in the birth index, gestation length, number of implantation, number of stillborn, number of live born, sex ratio and delivery index. As an external abnormality, absence of tail was seen in one example in the group administered with 150 mg/kg.

Nursing Condition:

There was one example in each of the control group and the group administered with 75 mg/kg where all of the offspring of one dam died in the initial nursing period, but no abnormalities were seen in the other administered treatment groups. In the observation of viability of the offspring, there were no significant differences between the control group and the administered treatment groups in the 4 day survival index, weaning index and viability index on day 70 after birth.

Growth and development of offspring (F1):

Bodyweight:

The average weight of the offspring (F1) in the treatment group with 150 mg/kg administered during nursing and beyond weaning showed a slightly lower value relative to the control group than at the time of birth, but this change was not confirmed to be of significant difference from the control group.

Normal differential and sexual difference:

The differentiation index of the eruption of lower incisor at 11 days after birth and opening of vagina at 30 days after birth for the treatment group administered 75 mg/kg showed a significant low value, but no dose-dependency was confirmed. Furthermore, a significant low value in each administered treatment groups were seen in the separation of eyelid differentiation index on day 14 after birth, but at the observation on day 17 after birth, 100% of the differentiation indices were confirmed in all treatment groups including the control group.

No significant difference between the control group and the treatment administered groups were seen in the differentiation indices of pinna detachment, appearance of abdominal hair and the descent of testis.

 Organ Weights and Macropathological Findings at 4 days old:

No significant difference between the control group and the administered treatment groups was confirmed with regards all organs.

Organ Weights and Macropathological Findings at the Time of Weaning (21 days after birth):

During the autopsy, dilatation of renal pelvis was seen in 2 examples from the control group, 1 example from the treatment group administered with 50 mg/kg, 2 examples from the treatment group administered with 75 mg/kg, but these rates of emergence showed no significant difference compared with the control group.

 Functionality and Behavioral Testing of the Offspring (F₁):

No significant abnormalities were seen in any of the administered treatment groups in the observations of the general condition (Irwin profile), vision tests (pupillary reflect) or hearing tests (Preyer’s reflex). Moreover, no significant difference between the control group and the administered treatment groups were seen in the count in the wheel cage method or the animals falling in the inclined screen method.

Macropathological findings of animals used in behavioral tests:

In males, one example each of dilatation of renal pelvis and flaccid testis were seen in the control group. In females, parietal region loss of hair was seen in 1 example from the treatment group administered with 75 mg/kg.

 Fertility Testing of Offspring (F₁):

Copulation and Fertility Index

In two instances, one couple did not copulate in the group administered 150 mg/kg, but no significant differences were confirmed between the control group and the administered treatment groups with regards the copulation index. Furthermore the fertility index and the number of days until copulation were confirmed to have no significant difference between the control group and the administered treatment groups.

Body Weight Change of Dams (F₁) during Pregnancy until 4 days after delivery:

There was a significantly high value of the body weight during pregnancy and during nursing until 4 days after delivery in the group administered 50 mg/kg, but this change was not thought to be dose dependent.

Delivery and Nursing Condition of Dams (F1):

Delivery abnormalities were confirmed in one example each in the treatment groups administered 25 and 50 mg/kg. One example in the control group and the group administered 25 mg/kg had a delivery which could not be observed, and when autopsied 24 days after the confirmation of copulation, the death of the fetuses inside the uterus was confirmed. Moreover in one example in the group administered 50 mg/kg, delivery was confirmed on day 24 of the pregnancy, but the all of the neonates were cannibalized that day.

No abnormalities were confirmed in the delivery in the treatment groups administered 75 and 150 mg/kg. No significant difference between the control group and the administered treatment groups were confirmed with regards the birth rate, period of pregnancy, number of implantation sites, number of stillborn, number of live births, sex ratio, rate of external abnormalities, birth rate and survival index at 4 days after birth. Moreover no abnormalities were seen in the nursing condition. However a significant reduction of the body weight 4 days after birth of the offspring (F₂) was confirmed in a male in the treatment group administered 75 mg/kg, but no dose-dependency was seen.

Macropathological Findings of the Animals used for Fertility Testing:

Among the males, atrophy of testis was seen in one example from the treatment group administered 150 mg/kg, yellow or yellowish white mass associated with blood-like material on fat was seen in one example from the treatment groups administered 25 and 150 mg/kg and dilatation of renal pelvis was seen in one example from the treatment group administered 50 mg/kg. These rates of emergence showed no significant difference between the control group and the administered treatment groups.

Among the females, dilatation of renal pelvis was seen in one example from the control group.

Applicant's summary and conclusion

Conclusions:

The majority of the effects seen within the study are not treatment related, please see the executive summary for more information. Based on the findings of this study, the maximum no-effect dose levels for general toxicological responses in the dams is 75 mg/kg, for reproductive performance of the dams is 150 mg/kg, and for development of the second generation it is 75 mg/kg.

Executive summary:

Crj: Cv (Sprague Dawley) rats were utilized to see the impact of intraveneous administration of 25, 50, 75 and 150 mg/kg of CVA-k during organogenesis, on the dams, fetuses and offspring.

Among the dams, death was confirmed in one example in treatment group administered with 150 mg/kg immediately after administration on pregnancy day 14. In this example, no abnormalities were seen in the general condition until the administration, and because of the death occurring immediately after the administration it was suspected to be related to the administration speed. In other administered treatment groups, no abnormalities were seen in the general condition and no deaths were confirmed.

During the administration a slight decrease in food consumption and suppression of body weight increase was seen in the treatment group administered with 150 mg/kg but no notable changes were seen in the food consumed during the nursing period. No abnormalities were confirmed by the autopsy observations or the organ weight during weaning and the caesarean section.

No abnormalities were seen in any of the treatment groups with regards the time until pregnancy, pregnancy period, delivery and nursing condition of the dams.

As observations in the caesarean section, a slight reduction in the live fetus body weight was seen in the males and females from the treatment group administered with 150 mg/kg. This change, given that a significant weight increase suppression is seen in the dams of the same treatment group, could also be thought to be a secondary effect based on the change in the body weight of the dams themselves.

However as noted below, this reduction in the fetus body weights was not a change which impacted the fetus morphology or development of the offspring. No significant differences between the control group and the administered treatment groups were seen with regards the number of corpus lutea, number of implantations, number of dead fetuses, number of live fetuses and sex ratio.

There was a low frequency of appearance of visceral, skeletal or external abnormalities as well as skeletal variations, and since no significant difference was seen between the control group and the administered treatment groups, the effects cannot be thought to result from the administration of the test compound. Furthermore the progression of ossification was also not confirmed to be any different from the control group.

The average body weight of the offspring (F₁) in the treatment group administered with 150 mg/kg relative to the control group was slightly lower than at the time of birth, but no significant difference was seen, and in the latter half of the observation period, it was at a level almost the same as the control group. Moreover neither the survival index of the offspring (F₁) nor the macropathological findings at weaning 4 days old showed any abnormalities.

Significant low values of differentiation index were seen in each treatment group for the separation of eyelids on day 14 after birth. However on 17 days after birth, 100% differentiation index were confirmed for all treatment groups, and given that the weight increase of the offspring during this time was smooth, the lowering of the differentiation index was thought to be something temporary.

No changes thought to be caused by the test compound was confirmed with regards the functionality and behavior of the offspring (F₁) , in the functionality test during weaning, open field testing or multiple water T-maze testing. In the conditioned avoidance response testing, a male in the 150 mg/kg administered showed a reduction in the avoidance response ratio in the 2^ndsession, and the lengthening of latency seen in the 2^ndand 3^rdsession, but as testing progressed, the acquirement of the conditioned avoidance response was confirmed, and hence it was thought to be a temporary, incidental change. Furthermore no abnormalities were confirmed with regards fertility.

As discussed above, other than the confirmation of a slight reduction in the fetal body weight where a slight inhibition in weight gain was seen in the dam in the treatment group administered 150 mg/kg, no effect was seen in the dams’ fertility, development of the fetuses, or in the development of offspring (F₁) after birth.

Therefore the maximum no-effect dose levels for general toxicological responses in the dams is 75 mg/kg, for reproductive performance of the dams is 150 mg/kg, and for development of the second generation it is 75 mg/kg.