[2S-[2α,5α,6β(S*)]]-6-[[[[(4-ethyl-2,3-dioxopiperazin-1-yl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Weight of evidence. A fertility study via subcutaneous route, a teratological study via intravenous route and a perinatal and postnatal study via subcutaneous route were performed with the analogue substance piperacillin sodium salt on ICR mice (non-GLP). In all cases, no significant effects were observed in any of the studies compared to the controls, up to 2000 mg/kg bw test item. Based on the study results, the NOAEL for reproductive toxicity of the test item is set at 2000 mg/kg bw. Based on the available information for the read-across approach, the NOAEL for reproductive toxicity of the target substance is set at 2000 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Fertility/Developmental screening.
- Short description of test conditions: the test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy.
- Parameters analysed / observed: Number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched by hand-touch and visual examination, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method).

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks (males), 12 weeks (females)
- Diet: NMF feed ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

subcutaneous

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

males were dosed from 6 weeks after birth and up to pregnancy (60+ days), females were dosed from 2 weeks before mating up to 6 days after pregnancy.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

(control)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a preliminary toxicity study, it was observed that a dose of 3000 mg/kg bw resulted in ulcers at the administration site. Therefore, 2000 mg/kg was selected as the top dose, 1000 mg/kg as the intermediate dose and 500 mg/kg as the low dose.
- Route of administration: because of the difficulty of delivering long-term intravenous administration to mice, subcutaneous injections were chosen.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (not a feeding study)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

Sperm parameters (parental animals):

Parameters examined in [P] male parental generations: not examined.

Litter observations:

PARAMETERS EXAMINED
The number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method).

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animalsas soon as possible after the last litter was produced.
- Maternal animals: All surviving animals at the 18th day of pregnancy.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
The heart, lungs, liver, kidneys and spleen were weighed. Microscopic examination not specified.

Offspring viability indices:

Death rate of maternal rats, death rate of fetuses, occurrence rate of morphological abnormality, body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses, number of ossification

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, suppression of body weight was observed from weeks 2 to 7, but there was no significant difference compared with the control group. See Fig.1-4 ('Illustration'). There were no differences between the control group and the treated groups.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Dose descriptor:

NOAEL

Generation:

F1

Remarks on result:

not determinable because of methodological limitations

Critical effects observed:

not specified

Key result

Reproductive effects observed:

no

Table 2. Effect of subcutaneous administration of T-1220 in mouse fetuses and pregnant mice on fertility study.

Dose    (mg/kg)		control	500	1000	2000
No. or mating mice		27	30	27	30
No. of mice with vaginal plug (%)		26 (96.3)	28 (93.3)	25 (92.6)	30 (100)
No. of pregnant mice (%)		24 (92.3)	27 (96.4)	23 (92.0)	27 (90. 0)
No. of corpora lutea (mean ± S. E.)		264 (11.0 ± 0.41)	308 (11.4 ± 0.36)	274 (11.9 ± 0.35)	309 (11.4 ± 0.22)
No. of implants (mean ± S. E.)		254 (10. 6 ± 0.43)	287 (10. 6 ± 0.40)	255 (11.1 ± 0.30)	288 (10. 7 ± 0.26)
No. of pre implantation loss (%)		10 (3.8)	21(6.8)	19 (6.9)	21 (6.8)
No. of post implantation loss (%)		17 (6.7)	28 (9.8)	13 (5.1)	26 (9.0)
Total implantation loss (%)		27 (10.2)	49 (15.9)	32 (11.7)	47 (15.2)
Alive fetuses (mean±S. E.)		237 (9. 9 ± 0.43)	259 (9. 6 ± 0.44)	242(10. 5 ± 0.33)	262 (9. 7 ± 0.36)
Mean body weight of fetuses (g±S.E.)		1.29 ± 0.021	1.35 ± 0.028	1.33 ± 0.016	1.35 ± 0.017
Sex	Male	122	124	122	144
	Female	115	135	120	118
Malformed fetuses (%)		9 (3.8)	6 (2.3)	7 (2.9)	14 (5.3)
Open eyelid		8	1	2	12
Club foot		1	3	5	1
Kinky tail		—	2	—	1
Mean organ weight of pregnant mice (g±S.E.)
Heart		0.18 ± 0. 006	0.18 ± 0.005	0.20 ± 0.006	0.19 ± 0.005
Lung		0.20 ± 0.006	0.22 ± 0.012	0.21 ± 0.005	0.21 ± 0.004
Liver		2. 48 ± 0.071	2. 28 ± 0. 048	2. 37 ± 0.057	2. 45 ± 0.046
Kidney	left	0.19 ± 0. 006	0.21 ± 0.006	0.20 ± 0.005	0.21 ± 0.003
	right	0.21 ± 0.006	0.21 ± 0.007	0.22 ± 0.006	0. 22 ± 0.003
Spleen		0.13 ± 0.008	0.14 ± 0.008	0.13 ± 0.007	0.13 ± 0.007

Table 3. Effect of subcutaneous administration of T-1220 on skeletal development and visceral malformation in mouse fetuses.

Dose    (mg/kg)	control	500	1，000	2,000
No. of fetuses examined m skeletal development	102	108	110	104
No. of ossified proximal phalanges of right forelimb, mean ± S. E.	3. 9 ± 0.04	4.0 ± 0.02	4.0 ± 0.01	4.0 ± 0.02
No. of ossified caudal vertebrae, mean ± S. E.	7. 5 ± 0.40	8.0 ± 0.34	8. 0 ± 0. 30	8. 5 ± 0.25
Retarded ossification of sternebrae (%)	2 (2.0)	3 (2.8)	1(0.9)	—
Extra sternebrae (%)	1(1.0)	—	—	1(1.0)
14th rib (%)	20(19.6)	31(28.7)	28(25.5)	23(22.1)
Bifurcation or split of 2nd cervical vertebrae (%)	13(12. 8)	13(12.0)	15(13. 6)	12(11.5)
No. of fetuses examined in visceral malformations	92	89	100	92
Malformed fetuses	1(1.1)	4 (4.5)	1(1.0)	2 (2.2)
Dilatation of lateral ventricles	1	3	—	2
Dilatation of third ventricle Cerebral hernia	—	1	1	—

 

 

Conclusions:

Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Executive summary:

A study was conducted to assess the potential toxicity to reproduction of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). The test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy. The number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method). Under test conditions, no abnormality of reproductive function was observed in male or female mice. External, internal and skeletal abnormalities were similar in all treated groups and control group. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Perinatal and postnatal study.
- Short description of test conditions: the test item was administered subcutaneously to female mice at the doses of 500, 1000 and 2000 mg/kg/day from the 15th day of getation to the 21st day after delivery. All pups were allowed to grow up to 6 weeks, the mothers were sacrificed at weaning. At 6 weeks, 20 m/f mice per group were kept, the remaining offspring were sacrificed and autopsied. When they reached 12 weeks of age, the surviving animals were mated to determine reproductive ability.
- Parameters analysed / observed: Delivery rates, weaning rates, survival rates, external malformation, behaviour, differentiation after birth, growth, and reproductive function of F1. Macroscopical observation and organ weights of F1.

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 14 weeks (males)
- Diet: NMF feed ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

subcutaneous

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

P0 females were dosed from the 15th day of gestation up to 21 days after delivery.
F1 animals were not treated with test item.

Frequency of treatment:

daily

Details on study schedule:

- F1 parental animals not mated until 6 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 6 weeks of age.
- Age at mating of the mated animals in the study: 12 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

(control)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

20 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a preliminary toxicity study, it was observed that a dose of 3000 mg/kg bw resulted in ulcers at the administration site. Therefore, 2000 mg/kg was selected as the top dose, 1000 mg/kg as the intermediate dose and 500 mg/kg as the low dose.
- Route of administration: because of the difficulty of delivering long-term intravenous administration to mice, subcutaneous injections were chosen.

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes (weekly)

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (not a feeding study)
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

NECROPSY
- The mother was sacrificed at weaning and macroscopic observation was performed; the fetal birth rate was calculated from the number of implantation traces.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no, the animals dosed during the study were allowed to litter normally and rear their progency to the stage of weaning without standardisation. After weaning, only 20 m/f animals per group were randomly kept, the rest were sacrificed.

PARAMETERS EXAMINED
The following parameters were examined in [F1] offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, differentiation after birth, growth and reproductive function.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities, organ weights and ratios.

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: no.

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: no.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals as soon as possible after the last litter was produced.
- Maternal animals: All surviving animals after the last litter was weaned.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS: not examined.

Postmortem examinations (offspring):

GROSS NECROPSY
- The F1 offspring not selected as parental animals were sacrificed at 6 weeks of age. These animals were subjected to postmortem examinations. (macroscopic examination).
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGTHS
The heart, lung, liver, kidneys and spleen were weighed. Histopathology not performed.

Offspring viability indices:

Death rate of maternal rats, death rate of fetuses, occurrence rate of morphological abnormality, body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses, number of ossification

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No differents were observed during the gestation period, but significant weight gain was observed in the treated groups every week after parturition.

Food consumption and compound intake (if feeding study):

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Behaviour (functional findings):

not specified

Histopathological findings: non-neoplastic:

not specified

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Enlargement of the renal pelvis was observed only in one case in the control group and two cases in the 1000 mg/kg group.

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

- No difference was observed between the control group and the offspring of the treated groups in mating rate, pregnancy rate, fetal survival rate or average body weight.
- External malformations (F2): There were cases of open eyelid and club foot legs, both in animals of the control and the treated groups, for example: 2 cases of clubfoot in the control, 1 case at 500 mg/kg, 1 at 1000 mg/kg and 4 at 2000 mg/kg. However, no significant differences were observed between the treated groups and the controls.

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 2. Effect of subcutaneous administration of T-1220 on delivery and postnatal development of F1 mice on perinatal and postnatal study. 

Dose    (mg/kg)		control	500	1000	2000
No. of litters		20	18	23	19
No. oi implants		233	208	263	206
No. of new born young		207	179	223	186
Delivering rate (%)		88.8	86.1	84.8	90.3
Weaning rate		87.0	89.9	94.6	95.2
Survival rate (%)		85.0	86.6	94.6	93.0
No. and mean body weight of offspring (g ± S.E.)
at birth	Male	110    1.52 ± 0.027	92    1.56 ± 0.031	119    1.58 ± 0.027	102     1.56 ± 0.031
	Female	97      1.48 ± 0.031	87 1.47 ± 0.026	104    1.49 ± 0.031	84      1.50 ± 0.034
1 st week	Male	107     3. 45 ± 0.103	86 3. 71 ± 0.132	116     3.58 ± 0.146	99      3.65 ± 0.118
	Female	88     3.31 ± 0.117	80 3. 61 ± 0.135	98      3.49 ± 0.158	80      3.58 ± 0.108
2 nd week	Male	106   5.40 ± 0.135	86 5. 98 ± 0.222	115     5.88 ± 0.257	98      5.97 ± 0.159
	Female	83     5.22 ± 0.176	79 5. 73 ± 0.194	97       5.81 ± 0.256	89      5.96 ± 0.148
3 rd week	Male	103   8.48 ± 0.381	85 9. 08 ± 0.600	114     8.89 ± 0.701	98     9.45 ± 0.531
	Female	82     8.45 ± 0.399	78 8. 78 ± 0.560	97      8.80 ± 0.691	79      9.12 ± 0.518
4 th week	Male	101    13.50 ± 0.555	84 15.00 ± 0.706	114    14.83 ± 0.702	98    15.46 ± 0. 557
	Female	79      12.79 ± 0.528	77 13.98 ± 0.603	97      13.97 ± 0.583	79    14. 22 ± 0.377
5 th week	Male	100    20.02 ± 0.663	83    21.56 ± 0.902	114     21.29±0.715	97    22.99 ± 0.536
	Female	78     18.35 ± 0.647	73    19.35 ± 0.644	97      19.45 ±0.588	79    20.20 ± 0.536
6 th week	Male	99     24.08 ± 0.447	83    25.61 ± 0.718	114     26.00 ± 0.661	96   26.91 ± 0.514
	Female	77    21.92 ± 0.508	72    22.36 ± 0.466	97      22.77 ± 0.536	77   23.63 ± 0.448

Table 3. Organ-body weight ratio and visceral malformation of Fi mice at 6 weeks administered T-1220 subcutaneously on perinatal and postnatal study.

Dose    (mg/kg)	control	500	1000	2000
No. of fetuses examined	56	59	82	64
Heart	0.66 ± 0.032	0.67 ± 0.021	0.67 ± 0.016	0.70 ± 0.022
Lung	0.81 ± 0.030	0.82 ± 0.027	0.77 ± 0.015	0.78 ± 0.016
Liver	6.28 ± 0.134	5. 95 ± 0.101	6.12 ± 0.133	6.06 ± 0.119
Kidney left	0.81 ± 0.036	0.82 ± 0.016	0.79 ± 0.020	0.81 ± 0.022
right	0.85 ± 0.031	0.87 ± 0.022	0.83 ± 0.024	0.86 ± 0.028
Spleen	0.62 ± 0.048	0.67 ± 0.050	0.62 ± 0.020	0.64 ± 0.027
Visceral malformation
Pyelectasia (%)	1(1.8)	—	2 (2.4)	—

 

Table 4. Effect of T-1220 on fertility in Fi mice of perinatal and postnatal study.

Dose    (mg/kg)		control	500	1000	2000
No. of mated mice		20	20	20	20
No. of mice with vaginal plug (%)		19(95.0)	17(85.0)	19(95.0)	20 (100)
No. of pregnant mice (%)		17(89.5)	17 (100)	19 (100)	19(95.0)
Survival rate of tetuses (%)		93.8	97.2	91.0	96.3
Mean body weight of fetuses (g ± S.E.)		1.30 ± 0.016	1.40 ± 0.021	1.36 ± 0.017	1.36 ± 0.015
Sex	Male	84	92	90	115
	Female	81	80	92	93
Malformed fetuses (%)		3 (1.8)	1(0.6)	2 (1.1)	6 (2.9)
Open eyelid		1	—	1	2
Club foot		2	1	1	4

 

Conclusions:

Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Executive summary:

A study to assess the perinatal and postnatal toxicity of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). 14-week-old male and female mice were mated, and the pregnant females were administered the test item subcutaneously at the doses of 500, 1,000 and 2,000 mg/kg/day from the 15th day of gestation to the 21st day after delivery. All pups were allowed to grow up to 6 weeks, the mothers were sacrificed at weaning. At 6 weeks, 20 m/f mice per group were kept, the remaining offspring were sacrificed and autopsied. When they reached 12 weeks of age, the surviving animals were mated to determine reproductive ability. In the pregnancy period, which was normal in all groups, no significant differences of body weight were observed versus the control, but after delivery, a remarkable increase in body weight gain was observed in all treated groups, altough no other effects were observed. The delivery rates, the weaning rates and the survival rates of all treated groups had no significant difference from those of the control, respectively. External malformation, behavior, differentiation after birth and growth of newborn were also normal in all groups. No abnormality of reproduction of F1 mice was observed. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Effect on fertility: via oral route

Endpoint conclusion:

no study available

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

Weight of evidence:

-A fertility study was conducted to assess the potential toxicity to reproduction of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP).The test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and till the performance of copulation; it was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy. The number of corpus luteum, number implants, number of deaths and number of live fetuses were recorded. Regarding the living fetuses, the exterior abnormalities, the gender and the body weights were recorded. Half of the living fetuses were stained with Alizarin red and their skeletons were examined for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method). Under test conditions, no abnormality of reproductive function was observed. External, internal and skeletal abnormalities were similar in all treated groups and control group. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

- A study to assess the perinatal and postnatal toxicity of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). 14-week-old male and female mice were mated, and the pregnant females were administered the test item subcutaneously at the doses of 500, 1000 and 2000 mg/kg/day from the 15th day of gestation to the 21st day after delivery. All pups were allowed to grow up to 6 weeks, the mothers were sacrificed at weaning. At 6 weeks, 20 mice per group were kept, the remaining offspring were sacrificed and autopsied. When they reached 12 weeks of age, the surviving animals were mated to determine reproductive ability. In the pregnancy period, which was normal in all groups, no significant differences of body weight were observed versus the control, but after delivery, a remarkable increase in body weight gain was observed in all treated groups, although no other effects were observed. The delivery rates, the weaning rates and the survival rates of all treated groups had no significant difference from those of the control, respectively. External malformation, behaviour, differentiation after birth and growth of new-born were also normal in all groups. No abnormality of reproduction of F1 mice was observed. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Based on the available information for the read-across approach, the NOAEL for reproductive toxicity of the target substance is set at 2000 mg/kg bw.

Effects on developmental toxicity

Description of key information

Weight of evidence. A fertility study via subcutaneous route, a teratological study via intravenous route and a perinatal and postnatal study via subcutaneous route were performed with the analogue substance piperacillin sodium salt on ICR mice (non-GLP). In all cases, no significant effects were observed in any of the studies compared to the controls, up to 2000 mg/kg bw test item. Based on the study results, the NOAEL for reproductive toxicity of the test item is set at 2000 mg/kg bw. Based on the available information for the read-across approach, the NOAEL for developmental toxicity of the target substance is set at 2000 mg/kg bw.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: Fertility/Developmental screening.
- Short description of test conditions: the test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy.
- Parameters analysed / observed: Number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched by hand-touch and visual examination, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and
their internal organs were examined (Wilson method).

GLP compliance:

no

Limit test:

no

Species:

mouse

Strain:

ICR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks (males), 12 weeks (females)
- Diet: NMF feed ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

subcutaneous

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

- males were dosed from 6 weeks after birth and up to pregnancy (60+ days),
- females were dosed from 2 weeks before mating up to 6 days after pregnancy.

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 males and 30 females per group

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: In a preliminary toxicity study, it was observed that a dose of 3000 mg/kg bw resulted in ulcers at the administration site. Therefore, 2000 mg/kg was selected as the top dose, 1000 mg/kg as the intermediate dose and 500 mg/kg as the low dose.
- Route of administration: because of the difficulty of delivering long-term intravenous administration to mice, subcutaneous injections were chosen.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (not a feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #18
- Organs examined: heart, lung, liver, kidneys, spleen.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter. The exterior abnormalities were searched, the gender was recorded, and the body weights were measured.
- Soft tissue examinations: Yes: half per litter. Half of the living fetuses were fixed with Bouin's solution and their internal organs were examined (Wilson method).
- Skeletal examinations: Yes: half per litter. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities.
- Head examinations: No data

Indices:

Death rate of maternal rats, death rate of fetuses, occurrence rate of morphological abnormality, body weight, food consumption, corpora lutea count, implantation count, number of fetuses and weight of fetuses, number of ossifications.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In the 1000 mg/kg group, suppression of body weight was observed from weeks 2 to 7, but there was no significant difference compared with the control group. See Fig.1-4 ('Illustration'). There were no differences between the control group and the treated groups.

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were cases of open eyelid and club foot legs, both in animals of the control and the treated groups: In the control group, there were 8 cases of open eyelid and 1 clubfoot leg in the control group; 1 open eyelid, 3 clubfoot legs and 2 kinky tails in the 500 mg/kg group; 2 open eyelids and 5 clubfoot legs in the 1000 mg/kg group; 12 open eyelid, 1 clubfoot and 1 kinky tail at 2000 mg/kg. However, the expression frequency of these external malformations was not significant, no difference related to the administration of test item was observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An excess of thoracic nucleus was detected in one case in the control and in one case in the 2000 mg/kg group. Formation of the 14th rib was observed in all groups: 20 cases in the control, 31 at 500 mg/kg, 28 at 1000 mg/kg and 23 at 2000 mg/kg. The second cervical vertebra was separated or branched in 13 cases in the control group, 13 at 500 mg/kg, 15 at 1000 mg/kg and 12 at 2000 mg/kg. In each case, no differences were observed between the incidence in the control group and treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Three cases of enlargement of the lateral ventricle and one case of dilatation of the third ventricle were observed at 500 mg/kg, but no significant difference with the control group was observed.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Table 2. Effect of subcutaneous administration of T-1220 in mouse fetuses and pregnant mice on fertility study.

Dose    (mg/Kg)		control	500	1000	2000
No. or mating mice		27	30	27	30
No. of mice with vaginal plug (%)		26 (96.3)	28 (93.3)	25 (92.6)	30 (100)
No. of pregnant mice (%)		24 (92.3)	27 (96.4)	23 (92.0)	27 (90. 0)
No. of corpora lutea (mean±S. E.)		264 (11.0±0.41)	308 (11.4±0.36)	274 (11.9±0.35)	309 (11.4 ± 0.22)
No. of implants (mean±b. E.)		254 (10. 6±0.43)	287 (10. 6±0.40)	255 (11.1±0.30)	288 (10. 7 ± 0.26)
No. of pre implantation loss (%)		10 (3.8)	21(6.8)	19 (6.9)	21 (6.8)
No. of post implantation loss (%)		17 (6.7)	28 (9.8)	13 (5.1)	26 (9.0)
Total implantation loss (%)		27 (10.2)	49 (15.9)	32 (11.7)	47 (15.2)
Alive fetuses (mean±S. E.)		237 (9. 9±0.43)	259 (9. 6 ±0.44)	242(10. 5 ± 0.33)	262 (9. 7 ± 0.36)
Mean body weight of fetuses (g±S.E.)		1.29 ± 0.021	1.35 ± 0.028	1.33 ± 0.016	1.35±0.017
Sex	Male	122	124	122	144
	Female	115	135	120	118
Malformed fetuses (%)		9 (3.8)	6 (2.3)	7 (2.9)	14 (5.3)
Open eyelid		8	1	2	12
Club foot		1	3	5	1
Kinky tail		—	2	—	1
Mean organ weight of pregnant mice (g±S.E.)
Heart		0.18 ± 0.006	0.18 ± 0.005	0.20 ± 0.006	0.19 ± 0.005
Lung		0.20 ± 0.006	0.22 ± 0.012	0.21 ± 0.005	0.21 ± 0.004
Liver		2.48 ± 0.071	2. 28 ± 0.048	2. 37 ± 0.057	2.45 ± 0.046
Kidney	left	0.19 ± 0.006	0.21 ± 0.006	0.20 ± 0.005	0.21 ± 0.003
	right	0.21 ± 0.006	0.21 ± 0.007	0.22 ± 0.006	0.22 ± 0.003
Spleen		0.13 ± 0.008	0.14 ± 0.008	0.13 ± 0.007	0.13 ± 0.007

Table 3. Effect of subcutaneous administration of T-1220 on skeletal development and visceral malformation in mouse fetuses on fertility study.

Dose    (mg/kg)	control	500	1，000	2,000
No. of fetuses examined m skeletal development	102	108	110	104
No. of ossified proximal phalanges of right forelimb, mean ± S. E.	3.9 ± 0.04	4.0 ± 0.02	4.0 ± 0.01	4.0 ± 0.02
No. of ossified caudal vertebrae, mean ± S. E.	7.5 ± 0.40	8.0 ± 0.34	8.0 ± 0.30	8.5 ± 0.25
Retarded ossification of sternebrae (%)	2 (2.0)	3 (2.8)	1(0.9)	—
Extra sternebrae (%)	1(1.0)	—	—	1(1.0)
14th rib (%)	20(19.6)	31(28.7)	28(25.5)	23(22.1)
Bifurcation or split of 2nd cervical vertebrae (%)	13(12. 8)	13(12.0)	15(13. 6)	12(11.5)
No. of fetuses examined in visceral malformations	92	89	100	92
Malformed fetuses	1(1.1)	4 (4.5)	1(1.0)	2 (2.2)
Dilatation of lateral ventricles	1	3	—	2
Dilatation of third ventricle Cerebral hernia	—	1	1	—

Conclusions:

Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Executive summary:

A study was conducted to assess the embryotoxic, fetotoxic, and teratogenic potential of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). The test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and then was continued to administer till the performance of copulation after mating, and was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy. The number of corpus luteum, number implants, number of deaths and number of live fetuses. Regarding the living fetuses, the exterior abnormalities were searched, the gender was recorded, and the body weights were measured. Half of the living fetuses were stained with Alizarin red and their skeletons were examined with a magnifying glass to check for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method). Under test conditions, no abnormality of reproductive function was observed in male or female mice. External, internal and skeletal abnormalities were similar in all treated groups and control group. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: The study evaluates the period of organogenesis (days 5-15 in the rodent).
- Short description of test conditions: the test item was administered intravenously at the doses of 500, 1,000 and 2,000 mg/kg/day to pregnant mice from the 6th to the 15th day of gestation. 20/30 dams were sacrificed on day 18, 10/30 females per group were allowed to spontaneously deliver and within 12 hours of birth, the number of babies, their viability and death, weight etc. was measured, and the mothers were allowed to nurse themselves until pups were 6 weeks old.
- Parameters analysed / observed: Number of implants, fetal mortality, external, skeletal and visceral malformations, body weight and sex of each fetus by cesarean section were examined on the 18th day of gestation.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

ICR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Age at study initiation: 14 weeks
- Diet: NMF feed ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

intravenous

Analytical verification of doses or concentrations:

not specified

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused: 14-week old male and female mice were crossed, and females that got pregnant were used for the study; no further details.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy.

Duration of treatment / exposure:

10 days, from the 6th day of gestation to 15th day of pregnancy.

Frequency of treatment:

Daily

Duration of test:

about 9 weeks

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

(control)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

30 females per dose.

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: Based on a preliminary toxicity study, where 3/8 pregnant females died in the 3000 mg/kg bw dose group and 0/8 died on the 2000 mg/kg bw, 2000 mg/kg bw was selected as the top dose. 1000 mg/kg bw was selected as the intermediate dose and 500 mg/kg bw was selected as the low dose.

Maternal examinations:

CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 days for pregnant females; weekly for surviving pups.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes (not a feeding study)

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 18 (only 20 females per group; 10 per group were sacrificed after delivery)
- Organs examined: heart, lungs, liver, kidneys, spleen.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number of malformed fetuses.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Figure 6 (see 'Illustration') shows the changesbody weight. There was no difference between the control group and the treated groups for maternal body weight.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Figure 5 (see 'Illustration') shows the changes in food intake. There was no difference between the control group and the treated groups for the increase in food intake.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No abnormalities were found in the weight of the maternal organs.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No abnormalities were found in the macroscopic findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

not specified

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were cases of open eyelid and club foot legs, both in animals of the control and the treated groups, no difference related to the administration of test item was observed.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External and visceral malformations were not seen in all treated groups and control group, except that the 14th rib formation was observed in group of 1000 mg/kg bw/day. An excess of thoracic nucleus was detected in one case in the 1000 mg/kg group, one case in asymmetric thymic nucleus in the control group, two in the 500 mg/kg group, one in the 1000 mg/kg group, and 3 cases in the 2000 mg/kg group; 12 cases of separation or split in the second cervical vertebrae were observed in the control group, 10 at 500 mg/kg, 8 at 1000 mg/kg group, and 12 at 2000 mg/kg; but in each case, no differences were observed between the incidence in the control group and treated groups.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Dilatation of the pelvis of the kidney was observed in 5 cases in the control group, 1 case at 500 mg/kg, 1 case at 1000 mg/kg and 5 cases at 2000 mg/kg bw. Two cases of enlargement of the lateral ventricle were observed at 500 mg/kg, but no significant difference with the control group was observed.

Other effects:

no effects observed

Description (incidence and severity):

F1: Growth and behaviour of newborns of the treated group had no significant difference from control in the period from birth to the 6th week after birth. F1 mice, at 12 weeks old, had normal reproductive function.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

effects observed, non-treatment-related

Localisation:

visceral/soft tissue: urinary

Key result

Developmental effects observed:

no

Treatment related:

no

Table 2.Effect of intravenous administration of T-1220 in mouse fetuses and pregnant mice on teratological study.

Dose    (mg/kg)		control	500	1000	2000
No. of litters		20	20	20	20
Mean body weight gain of pregnant mouse (g ± S.E.)
Day 0-6 of pregnancy		1.67 ± 0.242	1.26 ± 0.183	1.59 ± 0.178	1.94 ± 0.276
Day 0-15 of pregnancy		12. 55 ± 0.431	12.09 ± 0.569	12.78 ± 0.492	12. 84 ± 0. 526
Day 0-18 of pregnancy		20. 00 ± 0.727	21.13 ± 0.773	22.19 ± 0.830	22. 65 ± 0.813
No. of implants (mean ± S. E.)		237 (11.9 ± 0.37)	224 (11.2 ± 0.39)	236 (11.8 ± 0.60)	236 (11.8 ± 0.53)
Alive fetuses (mean± S. E.)		223 (11.2 ± 0.48)	208 (10. 4 ± 0.50)	218 (10. 9 ± 0.66)	222 (11.1 ± 0.57)
Dead fetuses {%)		14 (5.9)	16 (7.1)	18 (7.6)	14 (5.9)
Mean body weight of fetuses (g ± S.E.)		1.27 ± 0.027	1.25 ± 0.020	1.29 ± 0.024	1.27 ± 0 014
Sex	Male	124	91	107	107
	Female	99	117	111	115
Malformed fetuses {%)		8 (3.6)	5 (2.4)	3 (1.4)	9 (4.1)
Open eyelid		6	3	2	6
Club foot		2	2	1	3
Mean organ weight of pregnant mice (g ± S.E.)
Heart		0.18 ± 0.008	0.18 ±0.007	0.19 ± 0 007	0.18± 0.007
Lung		0.22 ±0.010	0.22 ± 0.008	0.23 ± 0.010	0. 23 ± 0.011
Liver		2.40 ± 0.071	2. 46 ± 0.056	2. 46 ± 0.037	2. 57 ± 0.061
Kidney	left	0.19± 0.006	0. 20 ± 0.005	0. 22 ± 0.005	0.21 ± 0.005
	right	0.21 ± 0.006	0.21 ± 0.006	0.23 ± 0.006	0.23 ± 0.006
Spleen		0.15 ±0.008	0.13 ±0.004	0.15 ±0.013	0.15 ±0.009

Table 3. Effect of intravenous administration of T-1220 on skeletal development and visceral malformation in mouse fetuses on teratological study.

Dose    (mg/kg)	control	500	1000	2000
No. of fetuses examined in skeletal development	117	109	115	117
No. of ossified proximal phalanges of right forelimb, mean ± S.E.	4. 0 ± 0. 04	4.0 ± 0.01	4.0 ± 0	4.0 ± 0.01
No. of ossified caudal vertebrae, mean ± S. E.	7.4 ± 0.35	7. 3 ± 0.32	7. 5 ± 0.28	7. 4± 0.26
Retarded ossification of sternebrae (%)	1 (0.9)	—	1 (0.9)	1(0.9)
Extra sternebrae (%)	—	—	1 (0.9)	—
Asymmetry of sternebrae (%)	1 (0.9)	2 (1.8)	1 (0.9)	3 (2.6)
14th rib (%)	24 (20. 5)	30 (27. 5)	44 (38. 3)*	30 (26.1)
Bifulcation or split of 2nd cervical vertebrae (%)	12 (10.3)	10 (9.2)	8 (7.0)	12 (10.4)
No. of fetuses examined in visceral malformations	106	99	103	105
Malformed fetuses (%)	1(0.9)	3 (3.0)	1(1.1)	4 (3.7)
Pyelectasia	1	1	1	4
Pyelectasia + dilatation of lateral ventricles	—	2	—	—

Table 4. Effect of intravenous administration of T-1220 on delivery and posnatal development of F1 mice on teratological study.

Dose    (mg/kg)		control	500	1000	2000
No. or litters		10	10	10	10
No. oi implants		117	105	107	116
No. of new born young		107	96	99	100
Delivering rate (%)		91.5	91.4	92.5	86.2
Weaning rate (%)*		84.1	91.7	88.9	95.0
Survival rate (%)**		83.0	91.7	85.9	93.0
No. and mean body weight of offsprings (g ± S.E.)
at birth	Male	58    1.55 ± 0.029	44   1.61 ± 0.039	57   1.58 ± 0.028	54   1.66 ± 0.043
	Female	49    1.47 ± 0.029	52   1.50 ± 0.041	42   1.47 ± 0.028	46   1.53 ± 0.034
1 st week	Male	53    3. 30 ± 0.214	40   3. 69 ± 0.341	55   3. 39 ± 0.133	53   3. 59 ± 0.163
	Female	42    3. 32 ± 0.218	50   3. 57 ± 0. 317	34   3. 27 ± 0.137	44   3. 40 ± 0.144
2 nd week	Male	52    5. 74 ± 0.180	40  6. 08 ± 0.473	55   5.64 ± 0.191	53   6.17 ± 0.319
	Female	42    5. 68 ± 0.211	48   5.95 ± 0.511	34   5. 52 ± 0.240	44   5. 87 ± 0.268
3 rd week	Male	51    7.63 ± 0.358	40   9.16 ± 0.691	55   7. 55 ± 0.365	52   8. 72 ± 0.468
	Female	42    7. 65 ± 0.350	48   8.87 ± 0.553	34  7.55 0 ± 0.313	44   8. 45 ± 0.464
4 th week	Male	49   12. 64 ± 0.676	40  16.18 ± 1.185	54  11.95 ± 0.697	52   14.74 ± 0.941
	Female	41  12.34 ± 0.616	48  14. 26 ± 1.040	34  11.42 ± 0.562	43  13. 63 ± 0.532
5 th week	Male	48   19. 26 ± 0.952	40  22. 26 ± 1.269	53  17.22 ± 1.005	52  21.41 ± 0.754
	Female	40   17. 68 ± 0.814	48  19.13 ± 1.286	32  15.73 ± 0.784	43  18. 81 ± 0.628
6 th week	Male	48   24. 77 ± 0.865	40  26. 20 ± 1.002	53  23. 60 ± 0.930	50  26. 74 ± 0.590
	Female	40   21.94 ± 0.666	48  22. 07 ± 1.147	32  20. 41 ± 0.873	43  23. 38 ± 0.586

Table 5. Organ-body weight ratio and visceral malformation of Fi mice at 6 weeks administered T-1220 intravenously on teratological study

Dose    (mg/kg)		control	500	1000	2000
No. of fetuses examined		58	57	41	58
Heart		0. 58±0.013	0.61 ±0.026	0.63 ±0.027	0.60 ± 0.015
Lung		0.78±0.018	0.78 ± 0.023	0.83 ±0.039	0.73±0.014
Liver		6. 63 ± 0.169	6.66 ± 0.145	6.47 ± 0.070	6.54 ± 0.105
Kidney	left	0.77 ±0.041	0.71 ±0.024	0.78 ±0.027	0.75±0.019
	right	0.82 ±0.043	0.80 ± 0.025	0.80 ± 0.031	0.80 ± 0.015
Spleen		0.61 ±0.038	0.64 ±0.037	0.66 ± 0.038	0. 52±0. 030
Visceral malformation		5 (8.6)	1(1.8)	1(2.4)	5 (8.6)
Pyelectasia (%)

 

Table 6. Effect of T-1220 on fertility in F1 mice of teratological study

Dose    (mg/kg)		control	500	1000	2000
No. of mated mice		20	20	20	20
No. or mice with vaginal plug (%)		20 (100)	20 (100)	20 (100)	20 (100)
No. of pregnant mice (%)		20 (100)	20 (100)	20 (100)	20 (100)
Survival rate of fetuses (%)		93.6	93.6	94.6	94.8
Mean body weight of fetuses (g ± S.E.)		1.25 ± 0.020	1.33±0.015	1.29 ± 0.021	1.30±0.018
Sex	Male	104	111	105	99
	Female	100	92	87	103
Malformed fetuses (%)		1(0.5)	7 (3.5)	2 (1.0)	4 (2.0)
Open eyelid		—	—	—	1
Club foot		1	7	1	1
Cleft palate		一	—	1	1
Exencephaly		—	—	—	1

 

Conclusions:

Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

Executive summary:

A study was conducted to assess the embryotoxic, fetotoxic, and teratogenic potential of the intravenous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). 14-week old mice were mated, and 30 pregnant female mice per group were used for the study. Based on the results of a preliminary toxicity group, the animals were administered doses of 0 (control), 500, 1000 and 2000 mg/kg bw test item intravenously for 10 days, from day 6 to day 15 of gestation. On day 18, 20 females per group were sacrificed and uterine content was examined. Maternal examinations included clinical observations, body weights, food consumption, gross necropsy, and organ weights. Number of implants, fetal mortality, external, skeletal and visceral malformations, body weight and sex of each fetus were examined. The remaining 10 females per group were allowed to spontaneously deliver and within 12 hours of birth, the number of babies, their viability and death, weight etc. was measured, and the mothers were allowed to nurse themselves until pups were 6 weeks old. External and visceral malformations were not seen in any treated groups or the control group, except that 14th rib formation was observed in group of 1,000mg/kg/day, but it was not considered to be dose-related. Growth and behaviour of newborns of the treated group had no significant difference from control in the period from birth to the 6th week after birth. F1 mice, had normal reproductive function at 12 weeks of age. Based on the test results, the test item, up to 2000 mg/kg did not exhibit any maternal toxicity, fetotoxicity, embryotoxicity, or teratogenicity in mice. Therefore, the NOAEL of the analogue substance in mice was set at 2000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no study available

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

Weight of evidence:

- A fertility study was conducted to assess the potential toxicity to reproduction of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP).The test item was administered subcutaneously to male mice at the doses of 500, 1000 and 2000 mg/kg/day for 60 days from 6 weeks after birth prior to mating and till the performance of copulation; it was also injected to female mice at the same doses from 2 weeks before mating to 6 days after pregnancy. The number of corpus luteum, number implants, number of deaths and number of live fetuses were recorded. Regarding the living fetuses, the exterior abnormalities, the gender and the body weights were recorded. Half of the living fetuses were stained with Alizarin red and their skeletons were examined for abnormalities. The remaining half was fixed with Bouin's solution and their internal organs were examined (Wilson method). Under test conditions, no abnormality of reproductive function was observed. External, internal and skeletal abnormalities were similar in all treated groups and control group. Based on the test results, the NOAEL for the analogue substance was set at 2000 mg/kg bw/d.

- A teratological study was conducted to assess the embryotoxic, fetotoxic, and teratogenic potential of the subcutaneous administration of the analogue substance sodium salt of piperacillin in ICR mice (non-GLP study). 14-week old mice were mated, and 30 pregnant female mice per group were used for the study. The animals were administered doses of 500, 1000 or 2000 mg/kg bw test item intravenously for 10 days, from day 6 to day 15 of gestation. On day 18, 20 females per group were sacrificed and uterine content was examined. Number of implants, fetal mortality, external, skeletal and visceral malformations, body weight and sex of each fetus were examined. The remaining 10 females per group were allowed to spontaneously deliver and within 12 hours of birth, the number of babies, their viability and death, weight etc. was measured, and the mothers were allowed to nurse until pups were 6 weeks old. External and visceral malformations were not seen in any treated groups or the control group, except that 14th rib formation was observed in group of 1,000 mg/kg/day, but it was not considered to be dose-related. Growth and behaviour of newborns of the treated group had no significant difference from controls. Based on the test results, the NOAEL of the analogue substance in mice was set at 2000 mg/kg bw/d.

Based on the available information for the read-across approach, the NOAEL for developmental toxicity of the target substance is set at 2000 mg/kg bw.

Justification for classification or non-classification

Based on the available information, the substance is not classified for toxicity to reproduction in accordance with CLP Regulation (EC) No. 1272/2008.