Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 262-811-8 | CAS number: 61477-96-1
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In-vitro gene mutation in bacteria: Weight of evidence. Two studies available, both performed according to Ames (1975), similar to OECD 471 (non-GLP). The analogue substance, piperacillin sodium salt, was not mutagenic with or without metabolic activation. Based on the available information for the read-across approach, the target substance, piperacillin acid, is deemed non-mutagenic.
In-vitro cytogenicity study in mammalian cells: Weight of evidence. Chromosome aberration study in Chinese Hamster Lung (CHL) cells, method similar to OECD 473 (non-GLP). No significant increases were observed in the incidence of chromosomal aberrations as compared with solvent controls. The analogue substance, piperacillin sodium salt, was not mutagenic with or without metabolic activation. Based on the available information for the read-across approach, the target substance, piperacillin acid, is deemed non-mutagenic.
In-vitro gene mutation study in mammalian cells: Data waiving (study scientifically not necessary / other information available). In accordance with Column 2 of REACH Annex VIII, the study does not need to be conducted since there is adequate data from reliable in vivo studies.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 177-700
- 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 - Target gene:
- Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2 uvrA.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Remarks:
- S. typhimurium TA 100, TA 98, TA 1535, TA 1537 strains dispensed from Dr. Ames, BN (University of California), and 5 strains of E. coli WP 2 uvrA strains dispensed from Dr. Ishikan (National Sanitary Experiment Station) were used as assay strains.
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Preliminary studies were conducted using 5 strains at doses of 0.16, 0.8, 4.0, 20, 100 µg/plate. Since TA 100 and TA 98 strains did not show antimicrobial activity even at 100 µg/plate, additional tests were conducted at doses of 5000, 1000, 200 and 40 µg/plate. Based on these results, the maximum dose was 500 µg/plate for TA100 strain, 1000 µg/plate for TA 98 strain, 10 µg/plate for TA1535 strain, 5.0 µg/plate for TA1537 strain and WP2 strain.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water.
- Justification for choice of solvent/vehicle: the test item is a white powder easily soluble in water. - Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- 2-nitrofluorene
- N-ethyl-N-nitro-N-nitrosoguanidine
- other: 2-aminoanthracene
- Remarks:
- all in DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation.
- The test was carried out according to the preincubation method of Yahagi (1975), which improved the medhod of Ames et al. (1975): 0.1 ml of the test substance solution and 0.1 ml of the bacterial suspension in 0.5 ml of S9 Mix or 100 mM sodium phosphate buffer (pH 7.4) was added to a small test tube and cultured at 37ºC for 20 minutes with shaking. This test tube was mixed with 2 ml of a top agar kept at about 45ºC, then layered on a minimum glucose agar plate medium and cultured at 37ºC for 2 days.
DURATION
- Preincubation period: 20min
- Exposure duration: 48h
NUMBER OF REPLICATIONS: 3
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth. The presence or absence of growth inhibition of the test bacteria was examined with a stereoscopic microscope. - Rationale for test conditions:
- In the preliminary tests, no observations of insolubility of the test item or clear inhibitory or toxic effect of the test item were detected at the doses tested.
- Evaluation criteria:
- The number of revertant colonies was measured using a colony analyzer (CA-7, Toyo) and the presence or absence of growth inhibition was examined with a stereoscopic microscope. The average number of colonies per plate was calculated and evaluated according to the criteria in the guideline of toxicity test method (Ministry of Health and Welfare Ministry of Pharmaceutical Affairs Review, 1984).
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: no.
RANGE-FINDING/SCREENING STUDIES: Preliminary studies were conducted using 5 strains at doses of 0.16, 0.8, 4.0, 20, 100 µg/plate. Since TA 100 and TA 98 strains did not show antimicrobial activity even at 100 µg/plate, additional tests were conducted at doses of 5000, 1000, 200 and 40 µg/plate. Based on these results, the maximum dose was 500 µg/plate for TA100 strain, 1000 µg/plate for TA 98 strain, 10 µg/plate for TA1535 strain, 5.0 µg/plate for TA1537 strain and WP2 strain. In TA 100 and TA 98 strains, no antibacterial action was observed even at the highest dose of PIPC, so a confirmatory test at a high dose was conducted.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: relative total growth. Reduction of the number of revertant colonies was observed in TA 1535, TA 1537 and WP2 strains at the highest dose of PIPC, but at 1000 μg/plate of TA 100, no antibacterial action was observed, or at 500 μg/plate of TA 98 strain. Therefore, TA 100 and TA 98 strains were further tested at higher doses. - Conclusions:
- No biologically significant increase in the number of revertants was noted in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.
- Executive summary:
The test item was studied for potential mutagenic activity using the Bacterial Reverse Mutation Assay, by a method similar to OECD 471 (non-GLP). Four histidine requiring strains of S. typhimurium (TA1535, TA 1537, TA 98, TA 100) and tryptophan requiring E.coli WP2uvrA were tested in triplicate, with and without metabolic activation (post mitochondrial supernatant S9 mix), at 5 concentrations per strain 1000 µg/plate based on the results of a range-finding test. Vehicle and positive controls were run in parallel. Under the experimental conditions applied, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.
- Endpoint:
- in vitro gene mutation study in bacteria
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Principles of method if other than guideline:
- The proceudres used were according to the method of Ames: Ames, B.N., McCann, J., Yamasaki, E., Mutat. Res. 31, 347 (1975); Green, M.H.L., Muriel, W.J., Mutat. Res. 38, 3 (1976); Brusick, D., Andrews, H., Mutat. Res. 26, 491 (1974).
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Toyama Chemical Co. - Target gene:
- Histidine dependent gene for S. typhimurium, tryptophan dependent gene for E. coli and S. cerevisiae.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- S. typhimurium TA 1538
- Species / strain / cell type:
- E. coli WP2 uvr A
- Species / strain / cell type:
- Saccharomyces cerevisiae
- Remarks:
- D4
- Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 homogenate plus cofactors, the S9 was prepared from livers of Sprague Dawley adult male rats injected peritoneally with Aroclor 1254 five days prior to sacrifice.
- Test concentrations with justification for top dose:
- 0.01, 0.1, 1, 10, 50, 100, 500, 1000 µg/plate.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 1-methyl3-nitro-1-nitrosoguanidine (MNNG)
- Remarks:
- TA1535, TA100, WP2, D4 without metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene (AA)
- Remarks:
- TA1537 without metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: Nitrofluorene (NF)
- Remarks:
- TA1538 and TA98 without metabolic activation
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- All strains but D4 with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: Not specified.
DURATION
- Exposure duration: S. typhimurium and E. coli 48h while the D4 yeast strain 3-5 days.
NUMBER OF REPLICATIONS: Triplicate plates
DETERMINATION OF CYTOTOXICITY
- Method: Reduction or complete absence of revertant colonies.
OTHER
Following exposure to the test article, the S. typhimurium and E. coli culture plates were incubated at 37 °C for 48 h while the D4 yeast strain was incubated at 30ºC (nonactivation) and 37ºC (activation) for 2 days for the bacterial colonies or 3-5 days for S. cerevisiae before the colonies were scored. Appropriate posivie controls were run concurrently. - Evaluation criteria:
- The results were judged significant when the mean number of revertant colonies in the treated cultures was at least 2-fold greater than the negative control.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 50.0 μg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 50.0 μg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 50.0 μg/plate
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- 1.0 μg/plate without S-9; 50 μg/plate with S-9.
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Saccharomyces cerevisiae
- Remarks:
- D4
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Definition of acceptable cells for analysis:
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES:
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture:
- Indication whether binucleate or mononucleate where appropriate:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Cytotoxicity was evidenced by the reduction or complete absence of revertant colonies. - Conclusions:
- No biologically significant increase in the number of revertants was noted in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.
- Executive summary:
The ability of the sodium salt of piperacillin to induce mutation was assessed by the bacterial reverse mutation test (Ames test), performed according to the method described by Ames, similar to OECD 417 (no GLP). Five histidine dependent strains of S. typhimurium (TA 1535, TA 1537, TA 1538, TA 98, TA 100), E.coli WP2 uvrA and Saccharomyces cerevisiae D4 were exposed to five or more concentrations of the test item up to 1000 μg/plate, depending on their toxicity, in the presence and absence of S9 rat liver metabolic activation. Under the experimental conditions used, the test item did not induce an increase in the number of revertants in any strain at any dose level, either with or without metabolic activation. Therefore, the test item was not mutagenic.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- 100 metaphases observed.
- Principles of method if other than guideline:
- According to the method of Ishikan (1983).
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 177-700
- 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 - Species / strain / cell type:
- other: Chinese hamster lung cells (CHL)
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: CHL cells from Dainippon Pharmaceutical Co.
MEDIA USED
- Type and identity of media including CO2 concentration: Eagle's MEM culture solution with 0.12% NaHC03 and 10% bovine fetal calf serum (CS, Osaka University) maintained at 5% CO2, 37ºC, high humidity. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Three doses of 2.5, 5.0 and 10 mM with and without S9 Mix, plus an additional dose of 1.25 mM in the test with S9 Mix. The doses for the test were based on the results of a preliminary study, where no significant toxicity (growth inhibition) was observed up to 20 mM test item, and the top dose was set at 10 mM.
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: physiol. saline
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- physiological saline
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-dimethylnitrosamine
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
- In the direct method, 5 ml of cell suspension (4 E3 cells/ml) was seeded in a dish (6 cm diameter) and cultured for 3 days. Then, 100μL of test item solution in physiological saline was added and incubated for 24 or 48 hours. Two hours before collecting the cells, 100μL of 10μg/mL colcemid was added. After detaching the cells with 2 ml of 0.25% trypsin solution, they were incubated in 75 mM KCl hypotonic solution at 37ºC for 15 minutes. The cells were fixed on slides and stained.
- In the metabolic activation method, 5 ml of cell suspension (8 E3 cells/ml) was seeded and cultured for 3 days. Then, 1 ml of S9 Mix and 120 μL of test item solution in physiological saline were added. After treatment for 6 hours by the monolayer culture method, the cells were washed with Dulbecco's phosphate buffer solution and further cultured for 18 hours. Chromosome specimens were prepared by the same method as the direct method.
- Cell density at seeding: 4E3 cells/ml without metabolic activation and 8E3 cells/ml with metabolic activation.
DURATION
- Exposure duration: 24-48h
- Expression time (cells in growth medium): 0 in the direct method, 18h in the metabolic activation method.
SPINDLE INHIBITOR (cytogenetic assays): colcemid (100μL of 10μg/mL solution)
STAIN (for cytogenetic assays): not specified.
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 200
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): A hundred well-spread metaphases were observed under the microscope. Structural abnormalities and numerical aberrations of chromosomes were evaluated separately. The abnormalities were classified into gaps, breaks, exchanges, ring formations, fragmentations, etc., and cells having even one of these abnormalities were recorded as abnormal cells. Regarding the numerical evaluation, the incidence of polyploidy in the cells as well as the number of cells with structural chromosomal aberrations was recorded on each culture plate.
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth; cell-growth inhibition was estimated using a cell densitometer (Monocellater, Olympus Co., Ltd)
OTHER EXAMINATIONS:
- Determination of polyploidy: yes. The polyploid cells were removed from the determination of structural abnormality. - Evaluation criteria:
- For structural abnormalities, the results were considered to be negative if the incidence was 4% or less (-), equivocal if it was between 4% and 8% (±), and positive if it was 8% or higher (+). For numerical abnormalities, an incidence of poliploidy below 5% was considered negative (-), equivocal (±) at 5-10%, and positive above 10%. Evaluation of the mutagenic potential of the test item was made by further considering the dose dependence.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- The incidences of structural- and numeral-aberration were 0-3% in the absence or presence of mammalian metabolic activation system, and no significant increases were observed in the incidence of chromosomal aberrations as compared with solvent controls.
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
- Effects of osmolality:
- Evaporation from medium:
- Water solubility:
- Precipitation:
- Definition of acceptable cells for analysis:
- Other confounding effects:
RANGE-FINDING/SCREENING STUDIES:
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells:
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: [complete, e.g. CBPI or RI in the case of the cytokinesis-block method; RICC, RPD or PI when cytokinesis block is not used]
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells] - Conclusions:
- The incidences of structural and numeral aberrations were 0-3% in the absence or presence of mammalian metabolic activation system, and no significant increases were observed in the incidence of chromosomal aberrations as compared with solvent controls. Therefore, the test item is not mutagenic.
- Executive summary:
A chromosome aberration study in cultured Chinese hamster lung cells (CHL) was performed to determine the mutagenic potential of the sodium salt of piperacillin, by a method similar to OECD 473 (non-GLP). A preliminary toxicity test was carried out to determine the maximum dose. Since no significant inhibition was observed up to 20 mM test item the maximum dose was set at 10 mM. Based on this, the cells were exposed to three doses of 2.5, 5.0 and 10 mM test item for 24 and 48h, without metabolic activation. Using a second method, cells were exposed to doses of 1.25, 2.5, 5.0 and 10 mM for 6h and incubated further 18h, with and without metabolic activation. Concurrent solvent(negative) and positive controls were run. 100 metaphases were evaluated, and the incidence of polyploid cells as well as of cells with structural chromosomal aberrations such as chromatid or chromosome gaps, breaks, exchanges, ring formations, fragmentations and others, was recorded on each culture plate. The incidences of structural and numeral aberrations were 0-3% in the absence or presence of mammalian metabolic activation system, and no significant increases were observed in the incidence of chromosomal aberrations as compared with solvent controls.Therefore, the test item was not mutagenic under test conditions.
Referenceopen allclose all
Table 1. Reverse mutation test on PIPC in S. typhimurium and E. coli strains with or without S9 mix.
Chemical |
Dose (μg/plate) |
S9 mix |
Revertants/plate |
Dose (μg/plate) |
|
|||||||||||||
TA100 |
TA98 |
TA1535 |
TA1537 |
WP2uvrA |
||||||||||||||
DW |
… |
- |
122 |
101 |
92 (105a) |
27 |
33 |
31 (30) |
… |
5 |
11 |
3 (6a) |
4 |
2 |
5 (4) |
13 |
10 |
16 (13) |
PIPC |
5 |
- |
79 |
104 |
93 (92) |
…b |
0.05 |
… |
2 |
2 |
4 (3) |
19 |
24 |
13 (19) |
||||
|
10 |
- |
99 |
83 |
77 (86) |
38 |
38 |
35 (37) |
0.10 |
2 |
5 |
10 (6) |
1 |
5 |
3 (3) |
17 |
12 |
17 (15) |
|
25 |
- |
84 |
89 |
80 (84) |
35 |
38 |
22 (32) |
0.25 |
9 |
7 |
6 (7) |
4 |
2 |
6 (4) |
15 |
12 |
9 (12) |
|
50 |
- |
76 |
74 |
90 (80) |
27 |
32 |
33 (31) |
0.50 |
6 |
5 |
5 (5) |
4 |
3 |
6 (4) |
14 |
18 |
16 (16) |
|
100 |
- |
71 |
84 |
98 (84) |
31 |
21 |
43 (32) |
1.0 |
6 |
7 |
10 (8) |
0 |
5 |
2 (2) |
15 |
13 |
15 (14) |
|
250 |
- |
90 |
93 |
77 (87) |
38 |
23 |
38 (33) |
2.5 |
6 |
6 |
1 (4) |
2 |
4 |
3 (3) |
21 |
13 |
16 (17) |
|
500 |
- |
53 |
70 |
58 (60) |
24 |
43 |
22 (30) |
5.0 |
1 |
2 |
1 (1) |
3 |
0 |
2 (2) |
9 |
9 |
3 (7) |
|
1000 |
- |
… |
15 |
25 |
23 (21) |
10.0 |
0 |
0 |
1 (0) |
… |
… |
||||||
ENNG |
3.0 |
- |
1215 |
1157 |
1088(1153) |
… |
2.0 |
… |
… |
464 |
398 |
373 (412) |
||||||
2NF |
1.0 |
|
… |
153 |
165 |
178 (165) |
5.0 |
2263 |
3471 |
2396 (2710) |
… |
… |
||||||
9AA |
… |
|
|
|
80 |
… |
286 |
229 |
244 (253) |
… |
||||||||
DW |
… |
+ |
84 |
97 |
70 (84) |
29 |
30 |
33 (31) |
… |
4 |
2 |
2 (3) |
3 |
6 |
7 (5) |
9 |
12 |
14 (12) |
PIPC |
5 |
+ |
82 |
82 |
108 (91) |
… |
0.05 |
… |
7 |
2 |
2 (4) |
14 |
11 |
13 (13) |
||||
|
10 |
+ |
94 |
85 |
90 (90) |
38 |
38 |
38 (38) |
0.10 |
2 |
4 |
2 (4) |
4 |
8 |
3 (5) |
17 |
18 |
19 (18) |
|
25 |
+ |
103 |
95 |
94 (97) |
40 |
29 |
23 (31) |
0.25 |
8 |
7 |
8 (5) |
3 |
10 |
6 (6) |
14 |
22 |
18 (18) |
|
50 |
+ |
83 |
101 |
97 (94) |
33 |
29 |
36 (33) |
0.50 |
3 |
6 |
5 (6) |
5 |
8 |
2 (5) |
16 |
14 |
… (15)c |
|
100 |
+ |
78 |
74 |
93 (82) |
38 |
34 |
29 (34) |
1.0 |
4 |
7 |
6 (5) |
3 |
2 |
3 (3) |
15 |
10 |
15 (13) |
|
250 |
+ |
69 |
67 |
64 (67) |
32 |
27 |
30 (30) |
2.5 |
5 |
10 |
8 (3) |
1 |
2 |
1 (1) |
7 |
8 |
13 (9) |
|
500 |
+ |
74 |
74 |
90 (79) |
19 |
23 |
35 (26) |
5.0 |
7 |
3 |
2 (1) |
0 |
3 |
0 (1) |
11 |
7 |
6 (8) |
|
1000 |
+ |
… |
29 |
23 |
36 (29) |
10.0 |
0 |
0 |
0 (0) |
… |
… |
||||||
2AA |
0.5 |
+ |
… |
199 |
260 |
253 (237) |
2.0 |
121 |
101 |
115 (112) |
104 |
122 |
94 (107) |
… |
||||
|
1.0 |
+ |
828 |
900 |
933 (907) |
… |
20 |
… |
… |
331 |
330 |
289 (317) |
||||||
*a: mean of 3 plates; b: not tested. Abbreviations: DW = distilled water, PIPC = piperacillin, ENNG = N-ethyl-N’-nitro-N-nitrosoguanidine, 2NF = 2-nitrofluorene, 2AA = 2-aminoanthracene.
Except for the metabolic activation method in strain TA 1535, no increase in the number of revertant mutant colonies was observed more than 2-fold as compared with the solvent control group with or without metabolic activation in the strains tested. In the metabolic activation method with TA1535 strain, the number of revertant colonies increased from 2.0 to 2.7 times in the solvent control group at 0.25, 1.0 and 2.5 ug / plate, but there was no dose correlation.
Table 1. Effect of piperacillin in the microbial assay. Each datum represents the mean revertant colonies from triplicate plates.
Concentration (μg/plate) |
No. of colonies/plates |
||||||
TA 1535 |
TA 1537 |
TA 1538 |
TA 98 |
TA 100 |
WP 2 |
D4 |
|
Without S-9 mix Solvent control 0.01 0.1 1.0 10.0 50.0 100.0 500.0 1000.0 Positive control |
27 25 24 28 10 T - - - 2167 |
20 22 20 15 3 T - - - 476 |
24 23 19 16 6 T - - - 2669 |
45 - - 50 47 - 24 42 27 1490 |
464 - - 444 465 - 492 432 406 3142 |
24 22 16 T T T - - - 297 |
61 - - 42 52 - 57 55 47 870 |
With S-9 activation Solvent control 0.01 0.1 1.0 10.0 50.0 100.0 500.0 1000.0 Positive control |
37 32 33 29 8 T - - - 329 |
16 14 15 16 5 T - - - 302 |
19 20 21 20 12 T - - - 302 |
38 - - 42 38 - 27 25 24 1585 |
384 - - 434 422 - 426 474 406 2591 |
16 28 19 19 11 3 - - - 32 |
34 - - 31 42 - 37 35 40 53* |
T = toxic; Positive controls (non-activation): TA 1535, TA 100, WP2, D4 – MNNG 10μL/plate, TA 1537 – AA 10μL/plate, TA 1538, TA 98 – NF 10μL/plate; Positive controls (activation): AA 10 μL/plate for all strains, except *D4 – DMN (no known positive control that works with this strain in the activation plate assay.
Table 1. Chromosomal aberration test on PIPC in CHL cells.
Chemical |
S9 Mix |
Time** (h) |
Dose (mM) |
Scored cell No. |
Chromosomal aberrant (%) |
Judgement |
Polyploid (%) |
Judgement |
|||||||
|
|
|
|
|
Ctg |
Ctb |
Cte |
Csb |
Cse |
other |
TAG |
TA |
|
|
|
Saline PIPC
MMC |
- - - - - |
24-0 24-0 24-0 24-0 24-0 |
- 2.5 5.0 10 0.1* |
200 200 200 200 200 |
0 0 0 0 1.5 |
0 0.5 0 0 16 |
0 0 0 0 20 |
0 0 0 0 0 |
0 0 0 0 1 |
0 0 0 0 0 |
0 0.5 0 0 33.5 |
0 0.5 0 0 32.5 |
- - - - + |
2 0 0 0 0.5 |
- - - - - |
Saline PIPC
MMC |
- - - - - |
48-0 48-0 48-0 48-0 48-0 |
- 2.5 5.0 10 0.1* |
200 200 200 200 200 |
0 1 0 0.5 1 |
0 0 0 0 19 |
0 0 0 0 30.5 |
0 0 0 0 0 |
0 0 0 0 1.5 |
0 0 0 0 0.5 |
0 1 0 0 43.5 |
0 0 0 0 43 |
- - - - + |
0.5 0 0.5 0 0.5 |
- - - - - |
Saline PIPC
MMC |
- - - - - - |
6-18 6-18 6-18 6-18 6-18 6-18 |
- 1.25 2.5 5.0 10 0.1* |
200 200 200 200 200 200 |
0 0 0.5 0 0 0 |
0 0 0 0 0 0 |
0.5 0 0 0 0 0 |
0 0 0 0 0 0 |
0 0 0 0 0 0 |
0 0 0 0 0 0 |
0.5 0 0.5 0 0 0 |
0.5 0 0 0 0 0 |
- - - - - - |
0 0.5 0.5 0 0 1 |
- - - - - - |
Saline PIPC
MMC |
+ + + + + + |
6-18 6-18 6-18 6-18 6-18 6-18 |
- 1.25 2.5 5.0 10 0.1* |
200 200 200 200 200 200 |
0 0 0 0 0 1 |
0 0 0 0 0 24 |
0 0 0 0.5 0 41.5 |
0 0 0 0 0 0 |
0 0 0 0 0 8 |
0 0 0 0 0 0.5 |
0 0 0 0 0 55 |
0 0 0 0 0 54.5 |
- - - - - + |
0.5 0.5 0 0 1.6 0.5 |
- - - - - - |
*μg/ml; **Treatment time – Expression time; Abbreviations: ctg: chromatid and chromosomal gap; ctb: chromatid break; cte: chromatid exchange; csb: chromosomal break; TAG: total cells which have chromosomal aberrants including ctg; TA: total cells which have chromosomal aberrants excluding ctg; PIPC: piperacillin, MMC: mitomycin C; DMN: N-nitrosodimethylamine.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus. Weight of evidence. In vivo micronucleus assay in male CD-1 mice. Method similar to OECD 474 (non-GLP). No consistent increases in the micronucleus frequency were observed. The test item, piperacillin sodium salt, has no mutagenic potential. Based on the available information for the read-across approach, the target substance, piperacillin acid, has no mutagenic potential.
In vivo mammalian somatic cell study: cytogenicity / chromosome aberration. Weight of evidence. In vivo mammalian bone marrow chromosome aberration test in male Sprague-Dawley rats. Method similar to OECD 475 (non-GLP). The test item did not induce any significant increase in the number of chromosomal aberrations in the treated animals relative to controls. The test item, piperacillin sodium salt, has no mutagenic potential. Based on the available information for the read-across approach, the target substance, piperacillin acid, has no mutagenic potential.
In vivo mammalian germ cell study: cytogenicity /
chromosome aberration. Weight of evidence. Rodent dominant lethal
assay in male Sprague-Dawley rats. Method similar to OECD 478
(non-GLP). Therefore, the test item does not show evidence of
genotoxicity in the germ cells of the treated male rats. The test
item, piperacillin sodium salt, has no mutagenic potential. Based on the
available information for the read-across approach, the target
substance, piperacillin acid, has no mutagenic potential.
Link to relevant study records
- 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
- 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
- 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 - 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 - 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: - Dose / conc.:
- 625 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 250 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 500 mg/kg bw/day (nominal)
- 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. - 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.
- 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. - 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.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
- Deviations:
- yes
- Remarks:
- samples only taken at one time, mitotic index not determined, only 100 metaphases per animal examined.
- GLP compliance:
- no
- Type of assay:
- mammalian bone marrow chromosome aberration test
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 275 - 315 g - Route of administration:
- intravenous
- Vehicle:
- - Vehicle(s)/solvent(s) used: distilled water.
- Amount of vehicle (if gavage or dermal): 1ml/100g bw, infusion rate 1 ml/min. - Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- one dose per day
- Post exposure period:
- 1 day
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 500 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 10 males per dose group.
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- triethylenemelamine (TEM)
- Route of administration: intraperitoneal
- Doses / concentrations: 0.3 mg/kg/d. - Tissues and cell types examined:
- Femoral bone marrow cells.
- Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES: 24 h after the last dose, colchicine was administered intraperitoneally (0.4 mg/kg) and and animals were sacrificed 2.5 h later. Femoral bone marrow cells were harvested.
DETAILS OF SLIDE PREPARATION: After hypotonic treatment (1% sodium citrate), the cells were fixed in Carnoy's fixative and dropped onto microscopic slides.
METHOD OF ANALYSIS: 100 metaphases per animal were analysed for chromosomal aberrations. - Statistics:
- The data were analysed using the Student t-test.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels: the test item did not induce any significant increase in the number of chromosomal aberrations in the treated animals relative to controls. The majority of the aberrations were mainly chromatid breaks with one incidence of chromosomal break. One animal in the high dose group had multiple fragments, gaps and polyploids. Because these observations were not seen in any other animals in the group, they were considered anomalous and therefore were not included in the calculation of the total number of aberrations.
- Appropriateness of dose levels and route: When given to rats intravenously at doses up to 1500 mg/kg/d for 5 days, the test item did not produce adverse effects to the animals. - Conclusions:
- The test item did not induce any significant increase in the number of chromosomal aberrations in the treated animals relative to controls. Therefore, it is not mutagenic under test conditions.
- Executive summary:
An in vivo Mammalian Bone Marrow Chromosomal Aberration Test was conducted to determine the mutagenic potential of the sodium salt of piperacillin, by a method similar to OECD 475. 10 male Sprague-Dawley rats per group were treated intravenously with the test item at concentrations of 125, 500, 1500 mg/kg bw for 5 days. Concurrent negative and positive controls were run in parallel. 24h after the last dose, colchicine (0.4 mg/kg) was administered intreperitoneally and the animals were sacrificed 2.5 later. Femoral bone marrow cells were harvested, and prepared in slides. 100 metaphases per animal were analysed for chromosomal aberrations. Under test conditions, the test item did not produce any adverse effects at the highest dose tested, nor induce any significant increase in the number of chromosomal aberrations in the treated animals relative to controls. Therefore, the test item is not mutagenic.
- Endpoint:
- in vivo mammalian germ cell study: cytogenicity / chromosome aberration
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 478 (Genetic Toxicology: Rodent Dominant Lethal Test)
- Deviations:
- yes
- GLP compliance:
- no
- Type of assay:
- rodent dominant lethal assay
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Weight at study initiation: 320 - 355 g - Route of administration:
- intravenous
- Vehicle:
- - Vehicle(s)/solvent(s) used: distilled water.
- Amount of vehicle (if gavage or dermal): 1ml/100g bw, infusion rate 1-3 ml/min. - Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- one dose per day
- Post exposure period:
- 8 weeks
- Dose / conc.:
- 250 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:
- 10 males per dose group.
- Control animals:
- yes, concurrent no treatment
- Positive control(s):
- triethylenemelamine (TEM)
- Route of administration: intraperitoneal
- Doses / concentrations: 0.05 mg/kg/d. - Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES: Two days after administration of the final dose, each male was housed for 5 days with a virgin Sprague Dawley female rat (average weight 210-260 g). They were mated in this manner with new groups of virgin females for a total of 8 weeks. All female rats were sacrificed on day 13 from mid week of the mating period, corresponding to 11-15 days of gestation. At autopsy, the number of pregnant animals, the numver of live implantations, early and late post-implantation deaths and the total number of implantations were recorded.
METHOD OF ANALYSIS: Reproductive data were transformed using the square root transformation and were analysed using the Sutdent t-test to evaluate each treatment group against its control mean.
- The Dominant Lethal factor is estimated as: (post-implantation deaths/total implantations per female) x 100. - Statistics:
- The data were analysed using the Student t-test.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF DEFINITIVE STUDY
- The pregnancy rate varied from 70-100% for all female rats in the tests. Sporadic decreases in total implantations and live implantations and increases in early postimplantation deaths were observed in weeks 2 and 5. However, these could be attributed to one or two females which had either low total implantation in that week or had a high number of early postimplantation deaths. These incidences were not dose related and were considered unrelated to the treatment.
- Appropriateness of dose levels and route: piloerection was observed in the group receiving the 1000 mg/kg bw dose; piloerection, sedation, increased respiration, staggered gait and loss of righting reflex where observed in the group receiving the 2000 mg/kg bw dose. No effects were observed in the lower doses. - Conclusions:
- No significant reduction in the average number of implants per female was observed in comparison with control matings. Therefore, the test item does not show evidence of genotoxicity in the germ cells of the treated male rats.
- Executive summary:
An in vivo Rodent Dominant Lethal Test was conducted to determine the mutagenic potential of the sodium salt of piperacillin in male germ cells, by a method similar to OECD 478. 10 male Sprague-Dawley rats per group were treated intravenously with the test item at concentrations of 250, 500, 1000 or 2000 mg/kg bw for 5 days. Concurrent negative and positive controls were run in parallel. Two days after administration of the final dose, each male was housed for 5 days with a virgin Sprague-Dawley female. They were mated in this manner with new groups of females for a total of 8 weeks. All females were sacrificed on day 13 from mid week of the mating perdiod and the uteri contents were examined. Under test conditions, piloerection was observed in the group receiving the 1000 mg/kg bw dose; piloerection, sedation, increased respiration, staggered gait and loss of righting reflex where observed in the group receiving the 2000 mg/kg bw dose. No effects were observed in the lower doses. The pregnancy rate varied from 70-100% for all female rats in the tests. Sporadic decreases in total implantations and live implantations and increases in early postimplantation deaths were observed in weeks 2 and 5. However, these could be attributed to one or two females which had either low total implantation in that week or had a high number of early postimplantation deaths. These incidences were not dose related and were considered unrelated to the treatment. No significant reduction in the average number of implants per female was observed in comparison with control matings. Therefore, the test item does not show evidence of genotoxicity in the germ cells of the treated male rats.
Referenceopen allclose all
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 ± 0.05a (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 ± 0.04b (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.
Table 1. Evaluation of piperacillin in the chromosomal aberration analysis using rat bone marrow preparations.
Treatment |
Dose (mg/kg/d) |
No. of metaphases |
Total no. of aberrations |
Chromatid break |
Chromosome break |
Ring |
Di/poly-centric |
Trans |
Tri/quadri-radial |
Stk. Chr. |
Cplx. Aberr. |
Mltp. Aberr. |
Chrtd. Gap. |
Poly-pld. |
Control |
0 |
961 |
3 |
3 |
0 |
0 |
0 |
0 |
0 |
11 |
0 |
0 |
5 |
1 |
Piperacillin |
125 500 1500 |
854 862 763 |
2 4 2 |
1 2 1 |
0 1 0 |
0 0 0 |
0 0 0 |
0 0 0 |
0 0 0 |
8 13 15 |
0 0 0 |
0 0 1 |
2 0 2 |
2 4 2 |
TEM |
0.3 |
644 |
61 |
12 |
6 |
1 |
3 |
6 |
2 |
18 |
12 |
19 |
5 |
9 |
*Piperacillin was administered i.v. once a day and TEM i.p. for 5 consecuteive days. Abbreviations:Ring: ring chromosome; Di/policentric: dicentric or polycentric chromosomes; Trans: tranclocation; Tri/quadrial: triradial o quadriradials; Stk. Chr.: sticky chromosome; Cplx./aberr.: complex aberrations; Mltp. Aberr.: multiple aberration; Chrtd Gap: chfomatid gap; Polypld: polyploid.
Table 1. Dominant lethal assay in rats treated with the test item.
Treatment |
Dose (mg/kg bw/d) |
Week No. |
|||||||
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
||
Pregnancy Rate |
|||||||||
Control Piperacillin
TEM |
0 250 500 1000 2000 0.05 |
80 100 100 100 70 100 |
100 100 100 100 100 100 |
100 100 100 100 100 100 |
100 100 100 100 90 70 |
90 90 90 90 80 100 |
90 100 100 100 100 100 |
100 90 90 100 100 100 |
100 100 90 100 100 100 |
Total implantation/pregnancy |
|||||||||
Control Piperacillin
TEM |
0 250 500 1000 2000 0.05 |
13.6 14.0 13.7 14.0 13.7 10.9* |
13.3 14.5 12.9 14.6 13.0 9.8* |
13.8 13.7 13.4 13.1 12.5 5.3* |
13.4 14.5 12.2 14.5 14.3 7.9* |
14.7 12.4 14.0 11.7* 13.9 14.4 |
14.6 14.0 13.1 13.1 13.5 12.1* |
13.3 13.1 12.3 13.7 14.3 13.2 |
13.7 13.6 14.3 13.2 13.7 14.2 |
Early deaths/pregnancy (a) |
|||||||||
Control Piperacillin
TEM |
0 250 500 1000 2000 0.05 |
0.9 1.4 1.0 0.4 0.4 9.5* |
0.5 0.9 0.7 1.9* 1.0 8.4* |
0.8 0.4 0.9 1.0 0.8 5.2* |
0.6 0.7 0.7 0.9 1.0 6.4* |
0.3 0.6 1.8* 0.8 0.6 2.4* |
0.3 0.7 0.9 1.4 1.5 1.1 |
1.4 0.6 1.3 0.3* 1.0 0.9 |
1.1 0.2 0.3 0.7 1.9 1.2 |
(a) Late deaths were infrequent and judged to be insufficient for statistical analysis and are not shown on table.
* Statistically different from control at p ≤ 0.05.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro data: Weight of evidence.
Two Ames tests were performed to determine the mutagenic potential of
the analogue substance piperacillin sodium salt, by a method similar to
OECD 471 (non-GLP) on various strains of bacteria, i.e. S.
typhimurium (TA98, TA100, TA1535, TA1537, TA1538), E.coli WP2
uvrA and yeast S. cerevisiae D-1, exposed to five or more
concentrations of the test item up to 1000 μg/plate,
gave negative results with and without S9 metabolic activation (positive
and negative controls were run in parallel and the proposed validity
criteria were met). Furthermore, an in vitro mammalian chromosome
aberration test on CHL cells, by a method similar to OECD 473
(non-GLP) was also negative for piperacillin sodium salt with or without
metabolic activation. CHL cells were exposed to 3-4 doses up to 10 mM
test item, along with positive and solvent controls, by using two
exposure methods, with and without metabolic activation. 100
metaphases were evaluated, and the incidence of polyploid cells as well
as of cells with structural chromosomal aberrations was recorded on each
culture plate. The incidences of structural and numeral aberrations were
0-3% by both methods, and no significant increases were observed in the
incidence of chromosomal aberrations as compared with controls. Based
on the available information, the test item, piperacillin sodium salt,
is considered to be non-mutagenic. Based on the available information
for the read-across approach, the target substance, piperacillin acid,
is deemed non-mutagenic.
In vivo data: Weight of evidence:
An in vivo mammalian erythrocyte micronucleus test, a mammalian bone marrow chromosome aberration test and a rodent dominant lethal assay were performed on the analogue substance piperacillin sodium salt, and all report negative results.
- The micronucleus assay was conducted in male CD-1 mice, by a method similar to OECD 474 (non-GLP). 6 male 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 (mitomicyn 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). 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.
- The Mammalian Bone Marrow Chromosomal Aberration Test was conducted by a method similar to OECD 475 (non-GLP). 10 male Sprague-Dawley rats per group were treated intravenously with the test item at 3 concentrations up to 1500 mg/kg bw for 5 days. Concurrent negative and positive controls were run in parallel. 24h after the last dose, colchicine was administered intraperitoneally and the animals were sacrificed 2.5 later. Femoral bone marrow cells were harvested, and prepared in slides. 100 metaphases per animal were analysed for chromosomal aberrations. Under test conditions, the test item did not produce any adverse effects at the highest dose tested, nor induce any significant increase in the number of chromosomal aberrations in the treated animals relative to controls. Therefore, the test item is not mutagenic.
- An in vivo Rodent Dominant Lethal Test was conducted to determine the mutagenic potential of the sodium salt of piperacillin in male Sprague-Dawley rat germ cells, by a method similar to OECD 478. 10 males per group were treated intravenously with the test item at 5 concentrations up to 2000 mg/kg bw for 5 days. Concurrent negative and positive controls were run in parallel. Two days after the final dose, each male was mated with virgin Sprague-Dawley females for a total of 8 weeks. All females were sacrificed on day 13 from mid-week of the mating period and the uteri contents were examined. The pregnancy rate varied from 70-100% for all female rats in the tests. No significant reduction in the average number of implants per female was observed in comparison with control mating. Therefore, the test item does not show evidence of mutagenicity.
Based on the available information, the test item, piperacillin sodium salt, is considered to be non-mutagenic. Based on the available information for the read-across approach, the target substance, piperacillin acid, is deemed non-mutagenic.
Justification for classification or non-classification
Based on the available data (negative results in vitro and in
vivo), the test item is not classified for mutagenicity in accordance
with CLP Regulation (EC) No. 1272/2008.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
