Metformin hydrochloride

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

Study period:

1989-01-19 to 1990-01-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Metformin hydrochloride

EC Number:

214-230-6

EC Name:

Metformin hydrochloride

Cas Number:

1115-70-4

Molecular formula:

C4H11N5.ClH

IUPAC Name:

N,N-dimethylimidodicarbonimidic diamide hydrochloride

Test material form:

solid: bulk

Method

Target gene:

HIS Operon

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G46 rfa uvrB

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C3076 rfa uvrB

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

his D3052 rfa uvrB

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D3052 rfa uvrB pKM 101

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G46 rfa uvrB pKM 101

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9 : isolated from livers of Aroclor 1254 pretreated rats
- method of preparation of S9 mix:
All steps were at 0 - 4°C usinq sterile solutions and glassware. The livers were placed in beakers containinq 0.15 M potassium chloride. After weighinq, the livers were transferred to a beaker containing 0.15 M KCl (3 ml KCl per 1g liver), minced with a sterile scalpel and homogenised in an Ultra Turrax homoqeniser. This homogenate was centrifuqed for 10 minutes at 9000g and the supernatant divided into aliquots were stored at -80°C.
- quality controls of S9: tested with the carcinoqen 7,12-dimetbylbenzanthracene before use

Test concentrations with justification for top dose:

Dose Range Finder: 5, 50, 500, 5000 µg/plate
Main Test: 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration: 3 days

SELECTION AGENT (mutation assays): HIS

NUMBER OF REPLICATIONS: 3
NUMBER OF INDEPENDENT EXPERIMENTS: 2

Evaluation criteria:

Number of revertant colonies was compared to those of the solvent controls.
The substance is considered mutagenic if:
a) an increase in revertant colony number of at least twice the concurrent solvent control is obtained and
b) there exists evicendence of a positive dose response relationsship and
c) reproducibility of the results is obtained

Statistics:

no

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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 nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Any other information on results incl. tables

Summary 1st Series

 

Metabolic Activation	Test compound	Concentration / [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	TA 1538
Without activation	Water		20 ± 2.6	123 ± 3.5	7 ± 1.0	9 ± 5.6	5 ± 1.4
	Test material	50.0	20 ± 6.0	115 ± 2.6	10 ± 0.0	9 ± 2.9	6 ± 1.5
		150	16 ± 1.0	113 ± 8.5	7 ± 1.5	8 ± 1.0	5 ± 1.0
		500	20 ± 4.0	113 ± 23.4	8 ± 3.1	9 ± 0.6	9 ± 1.5
		1500	19 ± 3.5	126 ± 8.4	7 ± 3.2	5 ± 3.1	2 ± 0.0
		5000	19 ± 6.1	121 ± 8.5	6 ± 1.7	5 ± 2.6	7 ± 4.7
	ENNG	5.00		340 ± 34.7	341 ± 129.0
	ENNG	3.00
	9 AC	2.00				633 ± 111.9
	NF	20.0	365 ± 1.0				31 ± 6.1
			TA 98	TA 100	TA 1535	TA 1537	TA 1538
With activation	Water		19 ± 6.4	113 ± 18.6	9 ± 2.3	10 ± 3.6	10 ± 3.6
	Test material	50.0	17 ± 4.4	120 ± 9.3	6 ± 4.0	8 ± 3.6	7 ± 4.6
		150	16 ± 5.5	117 ± 14.2	8 ± 1.7	8 ± 5.0	13 ± 3.8
		500	20 ± 6.0	134 ± 10.0	10 ± 4.0	6 ± 2.1	10 ± 4.0
		1500	18 ± 2.6	134 ± 8.9	7 ± 3.1	5 ± 0.0	9 ± 5.5
		5000	13 ± 3.1	117 ± 27.5	8 ± 1.2	6 ± 1.5	12 ± 4.0
	2-AA	0.50	266 ± 10.7				302 ± 32.7
	2-AA	1.00		909 ± 52.6
	2-AA	2.00			174 ± 30.6	170 ± 9.2

 

 

Summary 2nd Series

Metabolic Activation	Test compound	Concentration / [µg/plate]	Revertants per plate (Mean ± SD)
			TA 98	TA 100	TA 1535	TA 1537	TA 1538
Without activation	Water		18 ± 4.0	90 ± 14.8	10 ± 2.0	11 ± 2.5	5 ± 1.4
	Test material	50.0	20 ± 0.0	104 ± 10.0	10 ± 6.7	10 ± 2.6	6 ± 1.5
		150	18 ± 4.7	108 ± 16.1	8 ± 2.1	11 ± 1.5	5 ± 1.0
		500	19 ± 6.7	107 ± 9.9	9 ± 4.7	12 ± 4.2	9 ± 1.5
		1500	19 ± 1.0	103 ± 2.1	10 ± 3.1	10 ± 2.6	2 ± 0.0
		5000	19 ± 3.1	99 ± 19.1	8 ± 3.2	10 ± 2.5	7 ± 4.7
	ENNG	5.00		340 ± 34.7	341 ± 129.0
	ENNG	3.00
	9 AC	2.00				633 ± 111.9
	NF	20.0	365 ± 1.0				31 ± 6.1
			TA 98	TA 100	TA 1535	TA 1537	TA 1538
With activation	Water		19 ± 6.4	113 ± 18.6	9 ± 0.6	12 ± 1.5	15 ± 4.0
	Test material	50.0	17 ± 4.4	120 ± 9.3	9 ± 1.5	12 ± 3.0	9 ± 3.5
		150	16 ± 5.5	117 ± 14.2	12 ± 2.0	9 ± 1.5	14 ± 2.3
		500	20 ± 6.0	134 ± 10.0	9 ± 1.5	11 ± 1.0	11 ± 2.9
		1500	18 ± 2.6	134 ± 8.9	8 ± 2.6	11 ± 6.4	10 ± 4.5
		5000	13 ± 3.1	117 ± 27.5	10 ± 1.2	8 ± 1.7	9 ± 3.6
	2-AA	0.50	217 ± 16.2				159 ± 17.4
	2-AA	1.00		602 ± 31.1
	2-AA	2.00			144 ± 7.2	141 ± 7.1

 

Key to Positive controls

ENNG             N-ethyl-N'-nitro-N-nitrosoguanidine 2-AA               2-aminoanthracene 9 AC               9-aminoacridine NF                  2-nitrofluorene

Applicant's summary and conclusion

Conclusions:

It is concluded that, when tested at dose levels up to 5000 µg/plate in water, Metformin Hydrochloride was not mutagenic in this bacterial test system.

Executive summary:

Study Design

In this in vitro assessment of the mutagenic potential of Metformin Hydrochloride, histidine dependent auxotrophic mutants of Salmonella typhimurium (strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100) were exposed to the test material, diluted in water which was also used as a negative control.
Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254-induced rats.

Results

In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Other dose levels used in the mutation assays were: 1500, 500, 150, 50 µg/plate.
The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.
No evidence of mutagenic activity was seen at any dose level of Metformin Hydrochloride in either mutation test.

Conclusion

It is concluded that, when tested at dose levels up to 5000 µg/plate in water, Metformin Hydrochloride was not mutagenic in this bacterial test system.