[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)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Link to relevant study records

Reference

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.

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

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

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

Remarks:

Doses / Concentrations:
75 mg/kg
Basis:

Remarks:

Doses / Concentrations:
50 mg/kg
Basis:

Remarks:

Doses / Concentrations:
25 mg/kg
Basis:

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

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.

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: 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

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.

Dose descriptor:

NOEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

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.

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

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.

Reproductive effects observed:

not specified

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.

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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

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 ofCorpora 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.

Short description of key information:
Based on the results of this study, it is thought that the maximum non-effect does levels of CVA-K (potassium clavulanate) 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.

Justification for selection of Effect on fertility via oral route:
The study was conducted on the analogue material potassium clavulanate. The analogue material is considered to be similar enough to the substance of interest for results to be used for the purpose of Health and Hazard assessment.
The study has been assigned a Klimisch reliability rating of 2 (reliable with restriction). Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Effects on developmental toxicity

Description of key information

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. 

Link to relevant study records

Reference

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.

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

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).

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

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

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.

Details on maternal toxic effects:

Maternal toxic effects:no effects

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.

Dose descriptor:

NOEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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.

Effect on developmental toxicity: via oral route

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Two studies have been conducted. Both studies were conducted on the analogue material potassium clavulanate. The analogue material is considered to be similar enough to the substance of interest for results to be used for the purpose of Health and Hazard assessment. Furthermore, both studies were conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results and as a result were assigned the Klimisch rating of 2 (reliable with restrictions).

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Yamauchi 1985: Teratology study

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.

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 ormultiple 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.

Furukawa 1985: Organogenisis study

The CVA-K (potassium clavulanate) fetal organogenesis test was conducted, using New Zealand White rabbits. 37.5, 75 and 150mg/kg were administered intravenously from the 6^thto 18^thdays of pregnancy and effects on mother bodiesand fetus were analyzed.

The effects of CVA-K administration were not observed in general symptoms, food and water intake, weight transition and organ findings during Caesarian section in mother bodies in the groups with 37.5, 75mg/kg administrations.

After onset of administration in the group with 150mg/kg administration, reductions in food and water intake and increases in diarrhea and liver weights were observed and one case developed death during the administration period. Each one of these was considered to be due to CVA-K administration.

 There were no effects of CVA-K observed in corpora lutea, implantation number and ratio, surviving embryo number, dead embryo number, fetal survival rate, sex ratio, fetal and placenta weight and number of premature offspring in fetal observation. In addition, the effect of CVA-K administration was not observed in the exterior, internal organs and skeletal examinationof surviving fetuses.

According to the results mentioned above, it is considered that the NOEL regarding general toxicological effects on CVA-K rabbit mother bodies is 75mg/kg and the NOEL regarding pregnancy maintenance of mother bodies is 150mg/kg. Moreover, for this study, CVA-K is considered not to possess lethal action and teratogenic use against fetus. Therefore, the NOEL concerning CVA-K rabbit fetuses is concluded to be 150mg/kg.

Justification for selection of Effect on developmental toxicity: via oral route:
Both teratology studies in rat and rabbit are acceptable however the NOEL for fetal development was lower in the rat study than the rabbit and therefore the rat study was used as the Key study as it represents the worst case scenario.
The study has been assigned a Klimisch reliability rating of 2 (reliable with restriction). Study conducted in accordance with generally accepted scientific principles, possibly with incomplete or methodological deficiencies, which do not affect the quality of relevant results.

Justification for classification or non-classification

Although all the reproduction and developmental studies were carried out on an analogue material, the analogue is considered to be sufficiently similar to the substance of interest (please see attached data matrix and justification in Section 13 for additional details) for it to be used for the purposes of health and environment risk assessments.

All studies were performed according to recognised guidelines and are therefore considered to be reliable.

Assessment of reproduction showed no effects at the maximum stated dose of 75 mg/kg/day whilst developmental effects where seen were thought to be incidental and therefore not attributable to the test material. Based on these results, the case for classification is thought to be unjustified.

Although the fertility study cannot be compared with a multigenerational study due to the minimal information on neonatal development, the neonatal evaluation conducted in the teratology study supports the case for no classification. 

In conclusion the substance is considered to be not classified for reproductive toxicity according to current classification and labelling guidelines.