[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

Repeated dose toxicity: other routes

Administrative data

Endpoint:

short-term repeated dose toxicity: other route

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

acute toxicity: other routes

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 available

Principles of method if other than guideline:

- Principle of test: method for assessing acute toxicity that involves the identification of a dose level that causes evidence of non-lethal toxicity.
- Short description of test conditions: The test item was administered i.v. to groups of 7 Wistar SPF rats per sex (8 or 16 weeks old) at 12 doses ranging from 1.81 to 3.54 g/kg bw test item.
- Parameters analysed / observed: Mortalities and clinical signs were monitored during 7 days post-dosing after which animals were necropsied and subjected to a gross examination.

GLP compliance:

not specified

Limit test:

yes

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dilution with water.

Species:

rat

Strain:

Wistar

Remarks:

SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Nippon CLEA Co., Ltd.
- Age at study initiation: 8 and 16 weeks old. The LD50 were compared in the stages of growth.
- Weight at study initiation: 8 weeks: (8 weeks old: males = 220-290 g, females = 160-195 g), 16 weeks (body weight: males 400 to 450 g).
- Diet: ad libitum
- Water: tap water ad libitum
- Acclimation period: one week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 60 ± 5%

Route of administration:

intravenous

Vehicle:

water

Doses:

12 doses from 1810 mg/kg bw to 3540 mg/kg-bw

No. of animals per sex per dose:

7

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 7 days.
- Frequency of observations and weighing: daily observations.
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, macroscopic observations.

Statistics:

95% confidence limits were calculated by Litchfield-Wilcoxon’s method.

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

2 710 mg/kg bw

Based on:

test mat.

95% CL:

>= 2 360 - <= 3 120

Remarks on result:

other: 8 weeks old

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

2 790 mg/kg bw

Based on:

test mat.

95% CL:

>= 2 210 - <= 3 500

Remarks on result:

other: 16 weeks old

Key result

Sex:

male

Dose descriptor:

LD50

Effect level:

2 260 mg/kg bw

Based on:

test mat.

95% CL:

>= 2 100 - <= 2 430

Remarks on result:

other: 16 weeks old

Mortality:

No mortality was observed.

Clinical signs:

At 2000 mg/kg bw, only a decrease in hyperactive locomotor activity was observed.
At 2660 mg/kg bw, transient effects that included lack of control of limb movement and unresponsiveness to stimulae for about 30 min post-administration. The surviving animals recovered about 2h after administration. 4 males and 3 females in this group showed ankylosing convulsions for 15-30 min, and died.

Gross pathology:

At necropsy of the dead animals of the 2660 mg/kg bw group, pulmonary haemorrhage and mild subdural haemorrhage of the brain were found in most cases. These were not observed at necropsy of the surviving animals of the same group.

Table 1. Death rate in acute toxicity tests of T-1220. Rat, i.v.

Age	Dose (g/kg)	Male									Female
		Time in days								Death rate	Time in days								Death rate
		0	1	2	3	4	5	6	7		0	1	2	3	4	5	6	7
8 w	3.54	7								7/7	7								7/7
	3.22	6	0	0	0	0	0	0	0	6/7	7	0	0	0	0	0	0	0	7/7
	2.93	4	0	0	0	0	0	0	0	4/7	4	0	0	0	0	0	0	0	4/7
	2.66	4	0	0	0	0	0	0	0	4/7	3	0	0	0	0	0	0	0	3/7
	2.42	1	0	0	0	0	0	0	0	1/7	3	0	0	0	0	0	0	0	3/7
	2.20	1	0	0	0	0	0	0	0	1/7	0	0	0	0	0	0	0	0	0/7
	2.00	0	0	0	0	0	0	0	0	0/7	0	0	0	0	0	0	0	0	0/7
16 w	2.66	7	0	0	0	0	0	0	0	7/7
	2.42	5	0	0	0	0	0	0	0	5/7
	2.20	3	0	0	0	0	0	0	0	3/7
	2.00	1	0	0	0	0	0	0	0	1/7
	1.81	0	0	0	0	0	0	0	0	0/7

 

Table 2. Summary of results.  

Animal	Route	Age	LD50 (g/kg)
			Male	Female
Rat	i.v.	8 wk	2.71 (2.36 – 3.12)	2.79 (2.21 – 3.50)
		16 wk	2.26 (2.10 – 2.43)

*(): 95% confidence limits, calculated by Litchfield-Wilcoxon’s method.

Conclusions:

The intravenous LD50 of the test item in rats was > 2000 mg/kg-bw.

Executive summary:

An acute intravenous toxicity study was conducted in order to determine the toxicological properties of the sodium salt of piperacillin with a method similar to OECD guideline 401 (non-GLP). The test item was administered i.v. to groups of 7 Wistar SPF rats per sex (8 or 16 weeks old) at 12 doses ranging from 1.81 to 3.54 g/kg bw test item. Mortalities and clinical signs were monitored during 7 days post-dosing after which animals were necropsied and subjected to a gross examination. All animals died at the highest dose tested, and toxic effects were observed until 2.66 g/kg bw. At 2.00 g/kg bw no mortalities occured, the clinical signs were limited to mild transient locomotor supression, no gross abnormalities were observed at necropsy. The intravenous LD50 was found to be > 2000 mg/kg bw in rats.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1977

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: Subacute study via intravenous route in rats, for the determination of the No-Observed Adverse Effect Level (NOAEL).
- Short description of test conditions: The test substance is intravenously administered daily in graduated doses to several groups of experimental animals, one dose level per group for a period of 30 days. During the period of administration the animals are observed closely, each day for signs of toxicity. Animals that die or are euthanised during the test are necropsied and at the conclusion of the test surviving animals are euthanised and necropsied.
- Parameters analysed / observed: clinical observations, body weight, food consumption, haematology, clinical chemistry, urianalysis, gross necropsy, histopathology.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-Thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, 6-[[[[(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-, monosodium salt, [2S-[2α,5α,6β(S*)]]-

EC Number:

261-868-6

EC Name:

4-Thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid, 6-[[[[(4-ethyl-2,3-dioxo-1-piperazinyl)carbonyl]amino]phenylacetyl]amino]-3,3-dimethyl-7-oxo-, monosodium salt, [2S-[2α,5α,6β(S*)]]-

Cas Number:

59703-84-3

Molecular formula:

C23H26N5O7S.Na

IUPAC Name:

sodium;(2S,5R,6R)-6-[(2S)-2-[4-ethyl-2,3-dioxopiperazin-1-yl)carbonylamino]-2-phenylacetamido]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylate

Test material form:

solid

Test animals

Species:

rat

Strain:

Wistar

Remarks:

SPF

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 6 weeks
- Diet: standard feed (Oriental MF) ad libitum.
- Water: tap water ad libitum.
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 60 ± 5

Administration / exposure

Route of administration:

intravenous

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

30 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: A preliminary study was performed by administering doses of 500, 1000 and 2000 mg/kg bw test item to 5 male rats per group. No deaths or relevant effects were observed. Therefore, the maximum dose for the main study was 2000 mg/kg bw.
- Rationale for selecting satellite groups: a group of 5 males and 5 females was used as a recovery group.
- Post-exposure recovery period in satellite groups: 1 month.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:

BODY WEIGHT: Yes
- Time schedule for examinations: every 2 days.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X100 calculated as time-weighted averages from the consumption and body weight gain data: No
WATER CONSUMPTION: Yes (weekly)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 7 per sex per group
- Parameters examined: Red blood cell count, white blood cell count (Coulter counter model D), hematocrit (capillary method), hemoglobin (cyanometric hemoglobin method), leukocyte percentage.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during euthanasia procedure.
- Animals fasted: No data
- How many animals: 7 per sex per group
- Parameters examined: GOT, GPT (Reitman-Frankel method), ALP (Kind-King method), Ch-E (Takahashi-Shibata method), Cholesterol (Zurkowski method), BUN (Urease-Indophenol method), total Bilirubin (Evelyn-Malloy method), LDH (Babson modified), all measured by Olympus ACA automatic analyzer; total protein (refractive index method), A/G ratio (cellulose acetate membrane electrophoresis method), blood glucose (Reflectance meter), Na, K, BSP examination (3 male and 3 female in each group, using cardiac blood collection 30 min after BSP 2.5 mg/100 g injection)

URINALYSIS: Yes
- Time schedule for collection of urine: Not specified.
- Parameters examined: Urine volume (ml / 17 hr), sugar, protein, macotonia, blood cells, pH (Ames Labstix), virinolein (Ames Ictostix)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
- After 1 month administration, 7 male and 7 female rats each were exsanguinated from the carotid artery and autopsied.

HISTOPATHOLOGY: Yes (see table)
- After macroscopic observation, heart, * lung *, liver *, kidney *, spleen *, adrenal gland *, submandibular gland *, thyroid *, thymus *, ovary *, testis *, brain, pituitary gland, mesenteric lymph nodes, stomach, duodenum, jejunum, ileum, cecum, colon, bone marrow, bladder, femoral skeletal muscle were excised and histologically examined by formalin fixation and HE staining. For the organs marked with *, the weight was measured.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the administration period, loose stools were observed for about 2 weeks from the start of administration in the 2000 mg/kg administration group, but the symptoms returned to normal after 3 weeks of administration. Similar symptoms were also observed in the ABPC control group.

Mortality:

mortality observed, treatment-related

Description (incidence):

In the high dose group (2000 mg/kg bw), 5/15 males and 7/15 females died during the 30-day administration period. In females, the first death started right after administration, showing piloerection, and movement suppression immediately after administration, then dyspnea until death. The necropsy findings showed massive hemorrhage in the lungs in all the dead animals.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weight gain was slightly depressed in both male and female rats at the 2000 mg/kg group (P <0.01).

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the 2000 mg/kg administration group, a mild decrease in GOT and an increase in LDH were observed, but both were mild and change in the physiological range. The BSP test was not carried out because there were many deaths in the T-1220 2000 mg / kg administration group, but no abnormality was found in the BSP value performed in 3 males and 3 females in the other group. In the recovery test, no change beyond the physiological range was observed.

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In the males of the T-1220 administration group, a mild decrease in heart weight and heart weight ratio was observed, but it was not proportional to the dose and in females this change was not observed. In females at the 2000 mg/kg dose group, a slight increase in the right kidney weight ratio and a decrease in the spleen weight ratio were observed.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slight degree of enlargement of the caecum cavity was observed at all doses, but it was not deemed relevant. In addition, necrosis of the tail of the administration site was observed at 2000 mg/kg test item, but this was a change due to a local disorder caused by leakage of the specimen.

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 2 Mortality in rats treated intravenously with T-1220 for 1 month.

Experimental group	Sex	Time (Day)
		5	10	15	20	25	30	Death rate
Control (saline)	M	0/15	0/15	0/15	0/15	0/15	0/15	0/15
	F	0/15	0/15	0/15	0/15	0/15	0/15	0/15
T-1220 500 mg/kg	M	0/15	0/15	0/15	0/15	0/15	0/15	0/15
	F	0/15	0/15	0/15	0/15	0/15	0/15	0/15
T-1220 1000 mg/kg	M	0/15	0/15	0/15	0/15	0/15	0/15	0/15
	F	0/15	0/15	0/15	0/15	0/15	0/15	0/15
T-1220 2000 mg/kg	M	0/15	1/15	1/15	3/15	3/15	5/15	5/15
	F	1/15	2/15	2/15	5/15	6/15	7/15	7/15
ABPC 2000 mg/kg	M	0/15	0/15	0/15	0/15	0/15	0/15	0/15
	F	0/15	0/15	0/15	0/15	0/15	0/15	0/15

 

Table 3 Food and water intake in rats treated intravenously with T-1220 for 1 month

Food intake (g/day)			Week
	Dose		0	1	2	3	4
Male	Control	(saline)	19. 2	22.6	22.0	22.0	22. 8
	T-1220	500 mg/kg	17. 4	21.0	22.4	23.2	20.8
	T-1220	1000 mg/kg	18.0	22.4	22.4	24.4	24.0
	T-1220	2000 mg/kg	19.0	22.0	22. 0	21.0	23.0
	ABPC	2000 mg/kg	18.4	21.8	21.0	23. 0	22.0
Female	Control	(saline)	15.8	16.8	17.8	18. 4	17.6
	T-1220	500 mg/kg	18. 4	16.6	18.4	17.4	14.6
	T-1220	1000 mg/kg	17.8	17. 0	18. 0	18.8	17.4
	T-1220	2000 mg/kg	16.4	18. 4	16. 3	17.3	16.0
	ABPC	2000 mg/kg	14. 4	15.8	15. 6	15.2	15. 6

Mean ( n = 5)

 

Water intake (ml/day)			Week
	Dose		0	1	2	3	4
Male	Control	(saline)	25.0	27.5	30.0	33. 0	43.0
	T-1220	500 mg/kg	29.0	29.0	30. 0	32.0	28.0
	T-1220	1000 mg/kg	26.0	35.0	37.0	39.0	40.0
	T-1220	2000 mg/kg	31.0	38.0	37.5	38.8	41.3
	ABPC	2000 mg/kg	25.0	34.0	33.0	32.0	27.0
Female	Control	(saline)	28.0	31.0	31.0	28.0	32. 0
	T-1220	500 mg/kg	26.0	25.0	26.0	31.0	34.0
	T-1220	1000 mg/kg	30.0	28.0	32.0	32.0	31.0
	T-1220	2000 mg/kg	28.8	27.0	35.2	33.0	32.0
	ABPC	2000 mg/kg	24.0	27.0	35.0	31.0	35.0

Mean ( n = 5)

Table 4. Urinalysis in rats treated intravenously with T-1220 for 1 month.

Dose/day	Sex	Control	T-1220			ABPC
Item		(saline)	500 mg/kg	1000 mg/kg	2000 mg/kg	2000 mg/kg
Volume (ml/17 hr)	M	7. 9±2. 05	3.4 ± 0. 76	6.2±0.80	4.9±0.61	7.5±1.53
	F	2. 7±0.29	4.1 ± 0.50	4.2 ± 0. 42	4.7 ± 0. 67	11.5 ± 1.77**
Occult blood	M	—(5)	—(5)	—(5)	—(5)	—(3)	+ (2)
	F	—(5)	—(5)	—(5)	—(5)	—(4)	+ (1)
Ketones	M F	—(5)	—(5)	—(5)	—(5)	—(5)
Glucose	M	—(5)	—(5)	—(5)	—(5)	—(5)
	F	—(5)	—(5)	—(5)	—(5)	—(4)	+(5)
pH	M	6.5 - 7. 5	6.5 - 8.0	7.0 - 8. 0	6.0 - 7.5	7.5 - 9.0
	F	7.0 - 8. 5	6.5 - 7.0	7.5 - 8. 5	6.5 - 8. 5	8.5 - 9.0
Bilirubin	M	—(5)	—(5)	—(5)	—(5)	—(5)
	F	—(5)	—(5)	—(5)	—(5)	—(5)
Protein	M	+(5)	+(5)	+(5)	+(5)	+(5)
	F	+(5)	+(5)	+(5)	+(5)	+(5)

 

Table 5. Hematological findings.

	Dose/day	Sex	Control	T-1220			ABPC
Item			(saline)	500 mg/kg	1000 mg/kg	2000 mg/kg	2000 mg/kg
R. B. C. (x10^4/mm3)		M	807 ± 14.2	801 ± 25.1	834 ± 14.9	841 ± 9.8	807±9.2
		F	828 ± 8.5	826±5. 4	844±18.4	853 ± 11.9	811 ± 12. 5
W. B. C. (x 10^2/mm3)		M F	114 ± 10.5 98 ± 9.2	95 ± 5. 2 85 ± 7. 5	91 ± 3. 4 96±7. 4	98±11.8 119±6. 8	68 ± 6.5** 84 ± 1.5
Hemoglobin (g/dl)		M	15.3±0. 3	16.0±0.2	16.0 ± 0.1	16.0 ± 0.3	15.3 ± 0.3
		F	15.9 ± 0. 2	15.5 ± 0.2	15.1 ± 0. 2	14.7 ± 0.3*	14.9 ± 0. 2*
Hematocrit (%)		M	49.6 ± 1.5	51.1 ± 1.3	51.6 ± 0. 7	52.4 ± 1.0	52.1 ± 1.3
		F	54.9 ± 0. 3	51.2 ± 1.0**	50. 6±1.1	50.3 ± 1.0**	50.7±0.8**
	Neutro.	M	30.0±4. 6	19.1±5. 8	16.4 ± 3.3*	17.3±3.1*	15.0 ± 1.2**
		F	27.7 ± 2.2	15.1 ± 3.8*	9.1 ± 1.9**	12.9 ± 2.9**	16.9 ± 3.1*
	Eosino.	M	1.9 ± 0.51	1.4±0.37	1.9 ± 0.46	1.7 ± 0.42	2.0 ± 0.31
		F	2.8 ± 0.48	1.3±0.47	2.1 ± 0.51	2.0±0.49	1.1±0.40
Differential	Baso.	M	0	0	0	0	0
count (%)		F	0	0	0	0	0
	Lymph.	M	63.9 ± 4.7	76.9 ± 5.6	79.4±3.6*	78.1 ± 3.1*	80.4 ± 1.0**
		F	66.3 ± 2.0	81.7 ± 3.4**	84.9 ± 1.8**	82.0 ± 2.9**	79.1 ± 2.9
	Mono.	M	4.3±0.87	2.6 ± 0.75	2.3 ± 0.64	2.9±0.96	2.6 ± 0.43
		F	3.2 ± 0.98	1.9 ± 0.51	3.9±0.34	3.1±0.46	2.9 ± 0.70

Table 6. Biochemical examinations in rats treated intravenously with T-1220 for 1 month.

Dose/day	Sex	Control	T-1220			ABPC
Item		(saline)	500 mg/kg	1000 mg/kg	2000 mg/kg	2000 mg/kg
GOT (Karmen unit)	M	111 ± 8.2	122 ± 6.2	113 ± 6.4	88 ± 3.1*	97 ± 6.2
	F	107 ± 4.4	112 ± 9.0	98 ± 7.7	90 ± 4.0*	100 ± 6.5
GPT (Karmen unit)	M F	37 ± 1.5 37 ± 1.3	41 ± 2.4 37 ± 1.3	44 ± 3.0 40 ± 1.1	37 ± 1.6 35 ± 0.7	38 ± 2.2 32 ± 1.4*
ALP (K-A unit)	M	38 ± 3.4	36 ± 2.7	33 ± 4.0	32 ± 3.6	30 ± 1.0*
	F	26 ± 1.7	28 ± 3.7	23 ± 2.3	24 ± 3.2	23 ± 1.4
LDH (Wroblewski unit)	M F	920 ± 7.4 940 ± 6.4	940 ± 7.7 960 ± 7.1	990 ± 4.3** 950 ± 25.4	930 ± 11.7 990 ± 7.9*	910 ± 12.7 990 ± 4.0*
Ch-E (ipH)	M	0.31 ± 0.01	0.31 ± 0.01	0.32 ± 0.02	0.32 ± 0.01	0.35 ± 0.02
	F	0.94 ± 0.06	0.94 ± 0.05	0.92 ± 0.03	0.85 ± 0.12	0.78 ± 0.10
BUN (mg/dl)	M	20.6 ± 0.64	22.8 ± 0.87	22.8 ± 1.40	20.4 ± 0.68	24.3 ± 1.47*
	F	21.4 ± 0.64	25.3 ± 1.47	21.8 ± 0.94	20 0 ± 0.91	29.1 ± 2.72*
Bilirubin (mg/dl)	M	0.3 ± 0.03	0.3 ± 0.03	0.3 ± 0.03	0.3 ± 0.03	0.3 ± 0.02
	F	0.3 ± 0.02	0.3 ± 0.02	0.2 ± 0.02	0.2 ± 0.03	0.2 ± 0.02
Choi, (mg/dl)	M	78 ± 2.9	69 ± 2.9*	78 ± 3.1	71 ± 1.9	70 ± 1.6*
	F	68 ± 3.0	65 ± 2.6	70 ± 2.7	67 ±1.9	73 ± 2.4
Total protein (g/dl)	M	5.7 ± 0.12	5.6 ± 0.08	5.8 ± 0.20	5.8 ± 0.07	5.4 ± 0.12
	F	5.8 ± 0.06	5.7 ± 0.12	6.0 ± 0.12	5.9 ± 0.08	5.6 ± 0.10
A/G	M	1.07 ± 0.03	1.09 ± 0.01	1.03 ± 0.02	1.04 ± 0.01	1.05 ± 0.01
	F	1.03 + 0.01	1.01 ± 0.01	1.05 ± 0.02	1.03 ± 0.02	1.04 ± 0.01
Glucose (mg/dl)	M	77 ± 7.0	93 ± 2.3*	91 ± 3.9	92 ± 2.6	89 ± 2.4
	F	81 ± 1.1	90 ± 4.3	85 ± 2.0	76 ± 4.0	87 ± 1.4
Na (mEq/1)	M	140 ± 1.5	144 ± 1.3	141 ± 1.2	138 ± 0.9	137 ± 2.1
	F	132 ± 3.3	133 ± 1.7	135 ± 2.0	139 ± 2.4	131 ± 1.0
K (mEq/1)	M	5.8 ± 0.20	5.6 ± 0.06	5.5 ± 0.16	5.7 ± 0.12	5.6 ± 0.35
	F	5.4 ± 0.08	5.5 ± 0.17	5.2 ± 0.13	5.1 ± 0.14	5.4 ± 0.19

Table 7 Organ weight in rats treated intravenously with T-1220 for 1 month.

	Dose/day	Sex	Control Control	T—1220			ABPC
Item (g)			(saline)	500 mg/kg	1000 mg/kg j	2000 mg/kg j	2000 mg/kg
Heart		M	1.47 ± 0.079	1.10±0.040**	1.16±0.087*	1.07 ± 0.026**	1.08 ± 0.068**
		F	0 85 ± 0.028 I	0.83±0.021	0.89±0.035	0.84 ± 0.042	0.85 ± 0.029
Lung		M	1.33±0.056	1.28±0.060	1.36±0.111	1.22±0.053	1.23±0.073
		F	1.14±0 073	1.04±0.021	1.03±0.034	1.07±0.039	1.04 ± 0.032
Liver		M	11.68±0.335	11.06±0.396	11.72±0.473	10 00±0.552	9.58 ± 0.353**
		F	7.09 ± 0.265	6.96±0.201	7.47±0.496	6.46 ± 0.245	6.72 ± 0.221
	left	M	1.17 ± 0.023	1.10±0.038	1.13 ± 0.045	1.08±0.056	1.16±0.048
		F	0.73±0.022	0.72±0.017	0.79±0.031	0.78±0.037	0.84 ± 0.022**
Kidney	right	M	1.16 ± 0.040	1.10 ± 0.033	1.14±0.051	1.07±0.060	1.22±0.046
		F	0.75 ± 0.021	0.76 ± 0.029	0.81±0.034	0.81±0.042	0.87±0.034*
Spleen		M	0.68±0.018	0.63 ± 0.031	0.63±0.014	0.59 ± 0.033	0.57±0.023
		F	0.54±0.021	0.53±0.021	0.55±0.050	0.47 ± 0.028	0.49±0.017
Thymus		M	0.49±0.019	0.46±0.025	0.48±0.025	0.42±0.021	0.40±0.024
		F	0.47 ± 0.039	0.44 ± 0.028	0.46±0.040	0.47 ± 0.047	0.50 ± 0.049
Thyroids		M	0.02±0.003	0.01±0.002	0.02 ± 0.003	0.02±0.002	0.02±0.003
		F	0.02 ± 0.003	0.02 ± 0.002	0.01±0.002	0.02 ± 0.003	0.01 ± 0.002
Adrenals		M	0.06 ± 0.006	0.06±0.005	0.06 ± 0.006	0.06 ± 0.008	0.05±0.004
		F	0.07±0.006	0.07±0.005	0.07±0.006	0.06±0.005	0.07 ± 0.008
Submaxillary glands		M	0.60 ± 0.037	0.52±0.041	0.55 ± 0.023	0.49 ± 0.022	0.47±0.012**
		F	0.40 ± 0.019	0.37 ± 0.019	0.43 ± 0.023	0.35 ± 0.010	0.38±0.018
Testis	left	M	1.65±0.052	1.56±0.054	1.66±0.023	1.60 ± 0.043	1.57±0.029
	right	M	1.64 ± 0.055	1.52 ± 0.043	1.66±0.022	1.58 ± 0.031	1.58 ± 0.038
Ovaries		F	0.14±0.011	0.13±0.012	0.15 ± 0.009	0.13 ± 0.014	0.12±0.012

Significantly different from control *p<0. 05 **p<0. 01 Mean±S.E. ( n = 7)

Table 8. Relative organ weight per 100g bw in rats treated intravenously with T-1220 for 1 month.

	Dose/day	Sex	Control	T-1220			ABPC
Item (g%)			(saline)	500 mg/kg	1000 mg/kg	2000 mg/kg	2000 mg/kg
Heart		M	0.468±0.0255	0.368±0.0176**	0.370±0.0243*	0.377±0.0198*	0.376±0.0201*
		F	0.413 ± 0.0140	0.397 ± 0.0143	0.412±0.0178	0.413 ± 0.0176	0.424±0.0188
Lung		M	0.425±0.0173	0.425±0.0160	0.434 ± 0.0397	0.424±0.0115	0.427 ± 0.0185
		F	0.551 ± 0.0311	0.500 ± 0.0167	0.476±0.0213	0.526 ± 0.0143	0.519±0.0108
Liver		M	3.717 ± 0.1037	3.670±0.0713	3.735 ± 0.0973	3.469 ± 0.1018	3.322±0.0585**
		F	3.431 ± 0.1132	3.320±0.0305	3.425±0.1066	3.166±0.0612	3.342±0.0687
	left	M	0.372±0.0077	0.366 ± 0.0060	0.361 ± 0.0131	0.375 ± 0.0109	0.402±0.0097*
		F	0.354±0.0070	0.342 ± 0.0054	0.365 ± 0.0101	0.382±0.0108	0.420±0.0157**
Kidney	right	M	0.371 ± 0.0127	0.365±0.0056	0.364±0.0130	0.371 ± 0.0103	0.424 ± 0.0088**
		F	0.363 ± 0.0068	0.361±0.0101	0.372 ± 0.0112	0.398 ± 0.0131*	0.434±0.0205**
Spleen		M	0.216±0.0055	0.208 ± 0.0093	0.201 ± 0.0020	0.203±0 0045	0.199 ± 0.0057
		F	0.263±0.0106	0.254 ± 0.0046	0.249±0.0128	0.228 ± 0.0092*	0.246±0.0032
Thymus		M	0.155±0.0063	0.152±0.0064	0.152 ± 0.0071	0.146±0.0088	0.140±0.0073
		F	0.230 ± 0.0182	0.209±0.0091	0.213±0.0120	0.230±0.0193	0.247±0.0220
Thyroids		M	0.007±0.0009	0.005 ± 0.0006	0.005±0.0009	0.006±0.0008	0.006±0.0011
		F	0.010±0.0016	0.008±0.0010	0.007 ± 0.0011	0.009 ± 0.0013	0.007±0.0010
Adrenals		M	0.018±0.0020	0.018±0.0014	0.018±0.0018	0.020±0.0024	0.019±0.0019
		F	0.035 ± 0.0038	0.034 ± 0.0018	0.034±0.0026	0.032±0.0027	0.037±0.0033
Submaxillary glands		M	0.191±0.0107	0.174±0.0131	0.175±0.0044	0.172±0.0091	0.163±0.0027
		F	0.195±0.0094	0.178 ± 0.0093	0.200±0.0106	0.173±0.0038	0.187±0.0073
Testis	left	M	0.527±0.0187	0.519±0.0175	0.531±0.0132	0.559 ± 0.0167	0.545 ± 0.0110
	right	M	0.523±0.0195	0.505±0.0150	0.529 ± 0.0119	0.554±0.0166	0.548±0.0093
Ovaries		F	0.067±0.0054	0.062±0.0044	0.069±0.0048	0.064±0.0064	0.062±0.0050

Applicant's summary and conclusion

Conclusions:

The test item was found to have an intravenous NOAEL of 1000 mg/kg bw in rats.

Executive summary:

A 30-day repeated dose toxicity study via intravenous route was performed on Wistar SPF rats (no guideline available, no GLP), to determine the toxicity of the analogue substance sodium salt of piperacillin. The test item was orally administered to 15 male and 15 female rats per dose, at doses of 0 (control), 500, 1000 and 2000 mg/kg test item. Aminobenzylpenicillin (ABPC) was used as a positive control. All animals were thoroughly observed immediately after administration for the onset of any toxic signs and once daily thereafter. Survival, feed intake, and body weight were monitored. At the end of the study, the animals were sacrificed and haematology, clinical chemistry, urianalysis, gross necropsy, and histopathology was performed on 7 males and 7 females per group. During the study, mortality was observed at the highest dose tested, plus slight but significant changes in clinical signs, clinical biochemistry and organ weights. Based on the study results, the test item was found to have an intravenous NOAEL of 1000 mg/kg bw in rats.