Ampicillin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study does not completely meet requirements of the guideline 471 OECD (use of only four strains) but is considered as sufficiently reliable for risk assessment due to clear negative results.

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1986

Reference 2

Reference Type:

publication

Title:

Unnamed

Year:

1981

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(use of only four strains, results only presented as summarised tables)

Principles of method if other than guideline:

Method: other: pre-incubation modification of the Ames test.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2S,5R,6R)-6-{[(2R)-2-amino-2-phenylacetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate

Cas Number:

7177-48-2

Molecular formula:

C16H25N3O7S

IUPAC Name:

(2S,5R,6R)-6-{[(2R)-2-amino-2-phenylacetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid trihydrate

Details on test material:

- Name of test material (as cited in study report): Ampicillin trihydrate
- Analytical purity: 99.9%

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 was prepared from male Sprague-Dawley rats and Syrian hamsters that were induced with Aroclor 1254.

Test concentrations with justification for top dose:

0.050, 0.100, 0.300, 1, 2 and 3.300 µg/plate (TA 1535 and TA1537)
10, 53, 108, 353 and 1000 µg/plate (TA100 and TA98)

Vehicle / solvent:

DMSO was used as a solvent.

Controls

Untreated negative controls:

yes

Remarks:

Potassium chloride

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains,+S9), 4-nitro-o-phenylenediamine (TA98), sodium azide (TA100/TA1535), 9-aminoacridine (TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min at 37 °C
- Fixation time (start of exposure up to fixation or harvest of cells): 48 h

NUMBER OF REPLICATIONS: three plates

DETERMINATION OF CYTOTOXICITY
- Method: The test substance was initially tested with strain TA 100 in the presence and the absence of the metabolic activation systems, over a wide dose range with an upper limit of 10 mg/plate, or less when solubility problems were encountered . Toxicity was evidenced by one or more of the following phenomena : appearance of hispinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn . Nontoxic chemicals were tested in the initial experiment up to the 10 mg/plate dose level, or to a level determined by their solubility . Toxic chemicals were tested up to a high dose which exhibited some degree of toxicity . As a rule, at least one toxic dose was incorporated into the first mutagenicity test ; the repeat test(s) occasionally had the doses adjusted so that an apparent toxic dose was not reached .

Evaluation criteria:

The criteria used for data evaluation were:
1) mutagenic response : a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold.
2) nonmutagenic response : when no increase in the number of revertants was elicited by the chemical.
3) questionable response : when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible or when the response was of insufficient magnitude to support a determination of mutagenicity.

Results and discussion

Test results

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Concentrations were chosen so that the high dose exhibited some degree of toxicity.

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Results for the Salmonella study (Strain TA100)

Dose	No Activation		No Activation		No Activation		10% HLI		10% HLI		10% HLI		10% RLI		10% RLI		10% RLI
	(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg/Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	149	11.4	144	6.9	165	11.8	155	1.5	119	1.3	130	6.1	142	2	116	3.9	138	9.5
10			132	3.8	149	7.7			95	5.5	141	4.9			111	9.3	148	12.2
33			146	5.5	135	3.2			125	6.4	140	2			97	6.7	133	2.6
100	127	4.2	131	4.3	125	4.6	150	5.7	112	9	126	3.8	146	3	115	6.4	153	4.5
333	131	1.2	137	8.5	129	3.5	144	6.5	117	5.3	137	4.6	152	5.4	114	4.3	139	8.2
1000	124s	7.2	81s	1.5	97s	6.2	119s	8.5	74s	4.7	113	4.3	147s	7.8	81	4.7	123s	3.2
3333	56s	8.5					82s	10.1					64s	3.4
10000	23s	3.8					34s	2.6					32s	4.9
Positive Control	655	50.6	1260	21.2	1146	26.3	845	24.3	913	43.3	1090	17.2	885	51.9	499	35.2	865	40.6

 

Table 2. Results for the Salmonella study (Strain TA1535)

Dose	No Activation		No Activation		No Activation		10% HLI		10% HLI		10% HLI		10% RLI		10% RLI		10% RLI
	(Not Valid Test)		(Negative)		(Negative)		(Not Valid est)		(Negative)		(Negative)		(Not Valid Test)		(Negative)		(Negative)
Protocol	Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation		Preincubation
µg /Plate	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM	Mean	± SEM
0	32	2	42	3.3	24	3.3	24	1.5	10	1.3	15	0.9	29	0.7	13	1.5	19	0.9
0.03					24	0.9			11	2.4	11	1.2			15	1.8	12	2.4
0.1			30	2.2	27	2.9			11	2.3	12	0.9			17	2.1	16	1.8
0.3			38	3.3	26	5.2			12	3.6	12	0.6			13	2	12	2.1
1			37	1.7	25	2.1			12	0.9	10	3			13	4.6	14	3.3
2									4s	0.6	7s	1.5			9	1.8	6	2.4
3.3			17s	0.7	10s	3.9
6.6			t
100	t						t						t
333	t						t						t
1000	t						t						t
3333	t						t						t
10000	t						t						t
Positive Control	698	34.6	1002	53.7	898	15.9	47	0.7	86	9.7	76	5	71	8.9	59	3.3	56	0.7

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Executive summary:

Evaluation of the mutagenic activity of the test substance in the Salmonella typhimurium reverse mutation assay was determinated by pre-incubation method.

Four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) were used for the assay. The test was performed in the presence and absence of S9-mix. The dose range was determined in a prelimirary toxicity assay and was 0.05 to 1000 µg/plate. The test chemical was checked for toxicity to TA100 up to a concentration of 10 mg/plate or the limit of solubility, both in the presence and absence of S9-mix. The negative and strain-specific positive control values were valid indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.