[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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

not specified

Type of assay:

mammalian erythrocyte micronucleus test

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 88001
- Source: Toyama Chemical Industry Co., Ltd.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
Stability of PIPC in physiogical saline up to the time of its use was confirmed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid: The powder was dissolved in physiological saline.

FORM AS APPLIED IN THE TEST (if different from that of starting material): solution

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan.
- Age at study initiation: 9 weeks.
- Weight at study initiation: 24.5 - 29.0 g in the preliminary study and 24.0 - 29.0 g in the main study.
- Assigned to test groups randomly: yes
- Housing: polycarbonate cages.
- Diet (e.g. ad libitum): standard feed (CE-2, CLEA Japan) ad libitum.
- Water: tap water ad libitum.
- Acclimation period: 1 week.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 30 - 70
- Air changes (per hr): 10 changes/hour or more
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

intravenous

Vehicle:

- Vehicle(s)/solvent(s) used: distilled water.
- Justification for choice of solvent/vehicle:
- Concentration of test material in vehicle:
- Lot/batch no. (if required):
- Purity:

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

625 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

2 500 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 males per dose per group.

Control animals:

yes, concurrent no treatment

Positive control(s):

mitomycin C (MMC, Kyowa Hakko Kogyo)
- Route of administration: intraperitoneal
- Doses / concentrations: 2 mg/kg bw; prepared in distilled water and diluted with physiological saline.

Examinations

Tissues and cell types examined:

polychromatic erythrocytes, femoral bone marrow cells.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: in order to determine the maximum dose and sampling period, a preliminary test of single-dose toxicity via intravenous route was conducted based on the method of Hayashi et al. (1984) (see 'attached background material'). The LD50 of the test item was found to be > 5000 mg/kg bw, and this value was taken as the top dose for the main study.

TREATMENT AND SAMPLING TIMES:
- Treatment: a single-dose of 625, 1250, 2500 or 5000 mg/kg test item in physiological saline was administered to the animals by injection in the tail vein. The dose volume was 12.5ml/kg bw for the top dose and 10ml/kg for the other doses. The animals were further divided in three groups of 6 mice each, according to sampling time.
- Sampling: The femoral marrow cells were sampled 24, 48 or 72h post-administration. Animals were sacrificed by cervical dislocation, one femur was excised and bone marrow cells were washed out using a smal amount (0.5-1.0 ml) of fetal bovine serum. After centrifugation at 1000 rpm for 5 minutes, the supernatant was discarded by decantation.

DETAILS OF SLIDE PREPARATION: Using a Pasteur pipette, a uniform cell suspension was prepared with a small amount of serum remaining in the centrifuge tube. One drop of this suspension was dropped onto the slide glass and smeared. After drying at room temperature, it was fixed with methanol for 5 minutes. Then, it was dyed with 3% Giemsa solution in Sörensen buffer (pH 6.8), washed with 0.004% citric acid solution in distilled water, rinsed with distilled water and air-dried.

METHOD OF ANALYSIS: The preparations were coded and examined blindly, a 100x oil immersion lens. Red blood cells (RBC) were distinguished into polychromatic erythrocytes (PCE) and normochromatic erythrocytes (NCE), and 1000 PCE per mouse were scored and the frequency of micronucleated polychromatic erythrocytes (MNPCE) recorded.

CITOTOXICITY: As a measure of cytotoxicity, the proportion of PCE and of NCE in 1000 cells per mouse and per day was determined.

Evaluation criteria:

The results were judged according to Hayashi (1984) (see 'attached background material'). A result was deemed negative when MNPCE appearance was 10 or less, and positive if it was 18 or more. Evaluation of the mutagenic potential of the test item was made by further considering the dose dependence.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): The frequency of polychromatic erythrocyte with micronuclei was 0.02 - 0.10%. No dose-dependent increase was observed.
- Ratio of PCE/NCE (for Micronucleus assay): the PCE/NCE ratio was in the range 39.9 - 47.7%, and tended to decrease with increasing doses.
- Appropriateness of dose levels and route: In the PIPC 5000 mg/kg administration group of the preliminary test, 1 animal died 2 days after administration, but no effects were observed in the other animals of the group, and no significant increase in the MNPCE rate was observed. In the main study, 4 out of 6 animals in the high dose group died after administration, although no significant increase in the MNPCE was observed either. Due to these deaths, the data in the 5000 mg/kg bw dose group was removed from the evaluation.

Any other information on results incl. tables

Table 1. The frequency of micronucleated PCE/NCE and gained body weight after a single administration (i.v.) of test item (preliminary test).

Piperacillin Dose (mg/kg/d)	Sampling time [frequency (%) of MNPCE, (ratio (%) PCE/NCE; gained body weight (g) (+ increase, - decrease))]			Row Average
	24 h	48 h	72 h
625	0.1 (41.3, + 0.09) 0.0 (45.7, + 0.76)	0.4 (37.7, + 1.44) 0.0 (45.7, + 0.76)	0.1 (47.0, + 0.80) 0.0 (55.9, + 1.14)	0.17 (46.6, + 0.41)
1250	0.0 (34.8, + 0.46) 0.0 (50.5, + 1.06)	0.0 (36.0, - 1.05) 0.1 (49.9, + 1.29)	0.0 (61.8, + 0.28) 0.1 (50.1, + 0.60)	0.22 (46.4, + 0.15)
2500	0.2 (53.6, - 0.08) 0.0 (40.8, + 0.31)	0.2 (41.8, + 0.59) 0.1 (44.5, + 0.08)	0.2 (41.1, + 0.12) 0.1 (48.1, + 0.29)	0.15 (40.3, - 0.18)
5000	0.4 (32.9, - 1.38) 0.2 (43.4, - 2.37)	0.0 (20.5, - 2.04) 0.4 (33.0, - 5.12)	0.0 (43.2, - 0.10) Death	0.13 (42.2, + 0.78)
Col. Average	0.11 (43.2, -0.28)	0.15 (38.6, -0.51)	0.03 (49.6, +0.45)

Table 2. Result of micronucleus test with CD-1 male mice 48h after a single administration (i.v.) of test item (main test).

Chemical	Dose (mg/kg/d)	Frequency MNPCE (%) (total number; Min/Max)	PCE/RBC(%) (Min/Max)	Judgement
Saline (NC)	-	0.03 ± 0.05^a (2; 0.0/0.1)	52.9 ± 8.43 (43.5/63.3)	-
Test item	625	0.07 ± 0.08 (4; 0.0/0.2)	47.7 ± 5.31 (40.7/56.4)	-
	1250	0.10 ± 0.13 (6; 0.0/0.3)	42.8 ± 11.2 (21.9/55.8)	-
	2500	0.02 ± 0.04^b (1; 0.0/0.1)	39.9 ± 12.2 (19.5/53.8)	-
MMC (PC)^c	2	3.80 ± 1.20 (228; 2.3/5.1)	52.0 ± 13.0 (35.4/71.8)	+

a: Mean ± SD of 6 animals; b: The presented data were obtained from 5 animals, because low ratio (<10%) of PCE; c: positive control, 24h after i.p. administration.

Applicant's summary and conclusion

Conclusions:

Under test conditions, no consistent increases in the micronucleus frequency were observed; the frequency of polychromatic erythrocyte with micronuclei was 0.02 - 0.10%, and the PCE/NCE ratio was in the range 39.9 - 47.7%. Based on the test results, the test substance is considered non-mutagenic.

Executive summary:

An in vivo micronucleus assay was conducted to determine the mutagenic potential of the sodium salt of piperacillin in male CD-1 mice, by a method similar to OECD 474. 6 male NIH mice per group were treated with the test item at concentrations of 625, 1250 and 2500 mg/kg, based on the results of a preliminary toxicity study. Negative and positive controls (mytomicin C) were run in parallel. The animals were sacrificed at 24, 48 or 72 h post administration, one femur was excised and bone marrow cells were washed out, from which two smears were prepared, fixed with ethanol and stained with Giemsa solution. 1000 polychromatic erythrocytes (PCE) per mouse were scored to determine the frequency of micronucleated polychromatic erythrocytes (MNPE). As a measure of cytotoxicity, the ratio of PCE/NCE was also determined. Under test conditions, no consistent increases in the micronucleus frequency were observed; the frequency of polychromatic erythrocyte with micronuclei was 0.02 - 0.10%, and the PCE/NCE ratio was in the range 39.9 - 47.7%. Based on the test results, the test substance is considered non-mutagenic.