Amoxicillin

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to OECD 473 guideline study

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2010

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

This study was conducted according to the IPCS guidelines [Albertini et al., 2000].

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Amoxicillin

EC Number:

248-003-8

EC Name:

Amoxicillin

Cas Number:

26787-78-0

Molecular formula:

C16H19N3O5S

IUPAC Name:

6-{[amino(4-hydroxyphenyl)acetyl]amino}-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): Amoxicillin (AMO)

Method

Species / strain

Species / strain / cell type:

primary culture, other: human peripheral blood lymphocytes (Whole blood from two healthy donors: one male and one female, nonsmokers, age: 24).

Details on mammalian cell type (if applicable):

- Type and identity of media: 2.5 mL chromosome medium B (Biochrom AG, F 5023) supplemented with 10 µg/mL of bromodeoxyuridine (Sigma, St. Louis, MO, B5002).

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

400, 600, 800, and 1000 µg/mL
The test concentrations were chosen based on the top concentration that resulted a 50% (LD50) reduction in mitotic index (MI) (1000 µg/mL).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Remarks:

(0.25 µg/mL)

Positive control substance:

mitomycin C

Remarks:

Without metabolic activation (-S9-mix)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

(DMSO)

Positive controls:

yes

Remarks:

(28 µg/mL)

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation (+S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
In Vitro Assay (Without S-9 Mix):
The cells were treated with 400, 600, 800, and 1000 µg/ml concentrations of AMO that dissolved in DMSO, for 24 hr (AMO was added 48 hr after initiating the culture) and 48 hr (AMO was added 24 hr after initiating the culture), respectively.
A negative control, a DMSO control (DMSO, 9 µl/ml), and a positive control (Mitomycin-C, 0.25 µg/ml) were also used.
The cells were exposed to colchicines (0.06 µg/ml) 2 hr before harvesting. The cells were treated with 0.4% KCl (37ºC) as the hypotonic solution and methanol: glacial acetic acid (3:1) as the fixative (at room temperature 22ºC ± 1ºC, fixative treatments were repeated three times). The staining of the air-dried slides was performed following the standard methods using 5% Giemsa stain for CA. The slides were irradiated with 30 W, 254 nm UV lamp at 15 cm distance in Sorensen buffer for 30 min, then incubated with 1 x SSC (standard saline citrate) at 60ºC for 50 min and stained with 5% Giemsa prepared with Sorensen buffer.

In Vitro Assay (With S9 Mix):
Cells were cotreated with 400, 600, 800, and 1000 µg/ml concentrations of AMO and 0.5 ml S9 mix for 3 hr (AMO and S9 mix was added 48 hr after initiating the culture).
A negative control, a DMSO control (DMSO, 9 µl/ml), and a positive control (cyclophosphamide monohydrate, 28 µg/ml) were also used.
The test chemical and S9 mix were removed from the culture by centrifugation 4 min at 2500 rpm. The pellet of the lymphocytes was washed twice with 2.5 ml RPMI 1640 medium and resuspended in complete medium (chromosome medium B), and the cultures were incubated for a total of 72 hr at 37ºC.

DURATION
- Preincubation period: 72 hours at 37ºC.
- Exposure duration: 24 and 48 hours (without S-9 mix) and 3 hours (with S-9 mix).

SPINDLE INHIBITOR (cytogenetic assays): The cells were exposed to colchicine (0.06 µg/mL) 2 hr before harvesting.
STAIN (for cytogenetic assays): 5% Giemsa stain

NUMBER OF CELLS EVALUATED: Chromosomal aberrations were evaluated in 100-well spread metaphases per donor (totally 200 metaphases per concentration).

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI) and proliferation index (PI).

OTHER EXAMINATIONS:
- Determination of polyploidy: yes.

Evaluation criteria:

The number of CAs was obtained by calculating the percentage of the metaphases from each concentration and treatment period that showed structural and/or numerical alterations. The CA was classified according to the ISCN (International System for Human Cytogenetic Nomenclature) [Paz-y-Miño et al., 2002]. Gaps were not evaluated as CA according to Mace et al. [1978]. CAs were classified as the structural and the numerical aberrations. Structural CAs were consisted of the chromatid type (breaks and exchanges) and the chromosome type (breaks, fragments, sister unions, dicentrics, translocations) abnormalities, whereas the numerical CAs were consisted of poliploid cells. Only the structural CAs were taken into consideration to determine the genotoxicity.

Statistics:

One-Way ANOVA (LSD test) was used for the statistical significance of all parameters. Concentration-response relationships were determined from the correlation and regression coefficients for the percentage of cells with Chromosomal aberration.

Results and discussion

Test results

Species / strain:

primary culture, other: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(AMO concentration-dependently decreased the proliferation index (PI) in the absence of the metabolic activation for 24-hr treatment period).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
AMO did not significantly decrease the PI when compared with both the negative and the solvent control for 24- and 48-hr treatment periods; however, AMO induced a concentration-dependent decrease in PI when it is present for the last 24 hr (24-hr treatment) of the culture period (r = 20,980, P < 0.05). The decrease occured in the PI in 24-hr treatment period was not significant as the decrease caused by the positive control, MMC.
On the other hand, 48-hr treatment with AMO did not lead to a marked decrease in the PI when compared with both the negative and the solvent controls; however, the positive control significantly reduced the PI. In 24-hr treated cultures, MI was generally found to be reduced when compared with the negative control. Forty-eight hour treatment with AMO induced significant decreases in the MI at certain concentrations when compared to the negative control. Similarly, in the presence of an S9 mix, the PI and MI did not significantly decrease in comparison with the negative and the solvent control.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1. Effect of Amoxicillin (AMO) on Chromosomal Aberration (CA) in Human Peripheral Lymphocytes for 24- and 48-hr Treatment Periods.

 

Test substance	Treatment		Structural CA		Polyploid cells	Percentage of cells with aberrations ± SE
	Time (hr)	Conc. (µg/mL)	Chromatid type	Chromosome type
N. control	--	--	6	--	1	3.00 ± 2.00 c3, c3^*
DMSO	24	9µL	3	2	1	2.50 ± 0.50 c3
MMC	24	0.25	44	47	1	36.50 ± 0.50
AMO	24	400	2	3		2.50 ± 0.50 c3
AMO	24	600	4	2	1	3.00 ± 0.00 c3
AMO	24	800	9	2		5.00 ± 2.00 c3
AMO	24	1000	9	1		4.50 ± 2.50 c3
DMSO	48	9 µL	8	--		4.00 ± 2.00 c3
MMC	48	0.25	173	86	1	64.50 ± 0.50
AMO	48	400	2	2		2.00 ± 1.00 c3
AMO	48	600	1	3		1.00 ± 0.00 c3
AMO	48	800	4	2		3.00 ± 2.00 c3
AMO	48	1000	8	2	1	5.00 ± 1.00 c3

 

Data are expressed as the mean values (±S.E) obtained from two donors; n = 2.

a: significant from negative control; b: significant from solvent control; c: significant from positive control.

c^*: Positive control significant from negative control for 48-hr.

a₁b₁c₁: P < 0.05; a₂b₂c₂: P < 0.01; a₃b₃c₃: P < 0.001.

 

 

 

Table 2. Effect of Amoxicillin (AMO) on Chromosomal Aberration (CA) in Human Peripheral Lymphocytes in the Presence of S9 Mix

 

Test substance	Treatment		Structural CA		Polyploid cells	Percentage of cells with aberrations ± SE
	Time (hr)	Conc. (µg/mL)	Chromatid type	Chromosome type
N. control	--	--	4	1		2.50 ± 1.50 c1
DMSO	3	9µL	4	--		2.00 ± 1.00 c1
CYP	3	28	15	5	1	10.00 ± 5.00
AMO	3	400	4	2		2.50 ± 0.50 c1
AMO	3	600	4	3		3.50 ± 0.50
AMO	3	800	8	3	1	5.50 ± 0.50
AMO	3	1000	6	3		4.50 ± 0.50

 

Data are expressed as the mean values (±S.E) obtained from two donors; n = 2.

a: significant from negative control; b: significant from solvent control; c: significant from positive control.

a₁b₁c₁: P < 0.05; a₂b₂c₂: P < 0.01; a₃b₃c₃: P < 0.001.

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative (with and without metabolic activation)

Amoxicillin did not induce chromosomal aberrations at 24- and 48- hr treatment periods in the absence and presence of the S9 mix when compared to the negative and the solvent control.

Executive summary:

Evaluation of the ability of amoxicillin (AMO) to induce chromosome aberrations in human peripheral blood lymphocytes was determinated according to equivalent or similar OECD 473 Guideline. The cells were treated with 400, 600, 800, and 1000 µg/mL AMO in the presence and absence of a metabolic activator (S9 mix). AMO concentration-dependently decreased the proliferation index (PI) in the absence of the metabolic activation for 24-hr treatment period. Mitotic index (MI) was generally found to have been reduced when compared with the negative control but not with the solvent control in cultures treated with AMO for 24 hr. AMO did not decrease the PI and MI in the presence of the metabolic activator. The test substance did not induce chromosomal aberrations in human peripheral blood lymphocytes both in the presence and absence of the metabolic activator.