[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 vitro

Administrative data

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

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 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

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: 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

Method

Target gene:

Histidine-requiring gene in four strains of S. typhimurium, and tryptophan requiring gene in E. coli WP2 uvrA.

Species / strain

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.

Controls

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).

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.