Registration Dossier
Registration Dossier
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EC number: 214-230-6 | CAS number: 1115-70-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The genotoxic potential of merformin hydrochloride was investigated in three in vitro tests including a bacterial reverse gene mutation test (Ames test), a mammalian gene mutation test (mouse lymphoma assay) and an in vitro chromosomal aberration test in human lymphocytes. All tests were performed according to international accepted guidelines and under GLP regulation. Metformin hydrochlorid did not show any mutagenic or clastogenic potential in any test with or without metabolic activation.
Link to relevant study records
- 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
- 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
- Target gene:
- HIS Operon
- Species / strain / cell type:
- S. typhimurium TA 1535
- Details on mammalian cell type (if applicable):
- his G46 rfa uvrB
- Species / strain / cell type:
- S. typhimurium TA 1537
- Details on mammalian cell type (if applicable):
- his C3076 rfa uvrB
- Species / strain / cell type:
- S. typhimurium TA 1538
- Details on mammalian cell type (if applicable):
- his D3052 rfa uvrB
- Species / strain / cell type:
- S. typhimurium TA 98
- Details on mammalian cell type (if applicable):
- his D3052 rfa uvrB pKM 101
- 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
- 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
- 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
- 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
- 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
- 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
- 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
- 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.
- 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
- Study period:
- 1991-10-08 to 1992-01-23
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted 26 May 1983
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- lymphocytes:
- Details on mammalian cell type (if applicable):
- Human lymphocytes from male donors
- Metabolic activation:
- with and without
- Metabolic activation system:
- Aroclor induced rat S9
- Test concentrations with justification for top dose:
- 9.8 - 5000 µg/mL
- Vehicle / solvent:
- Sterile distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- treatment / recovery series 1: 24 / 0 hours
- treatment / recovery series 2: 3 / 21 hours
SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 metaphases
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index - Evaluation criteria:
- The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test (Fisher 1950).
- Statistics:
- Fischer's Test
- Key result
- Species / strain:
- lymphocytes: primary culture from humans
- Remarks:
- male donors
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Conclusions:
- Metformin hydrochloride has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
- Executive summary:
A study was performed to assess the ability of Metformin hydrochloride to induce chromosomal aberrations in human lymphocytes cultured in vitro.
Cultured human lymphocytes, stimulated to divide by addition of phytohaemagglutinin, were exposed to the test substance both in the presence and absence of S-9 mix derived from rat livers. Solvent and positive control cultures were also prepared. After the appropriate treatment time cell division was arrested using colchicine, the cells harvested and slides prepared so that metaphase figures could be examined.
In order to assess the toxicity of Metformin hydrochloride to cultured human lymphocytes the mitotic index of all cultures treated with the test substance and the solvent control was calculated. Based on these data the dose levels selected for the metaphase analysis were 19.5, 156 and 313 µg/mL in the absence of S-9 mix and 625, 2500 and 5000 µg/mL in its presence.
In the absence of S-9 mix, statistically significant increases in the proportion of aberrant cells were observed following treatment with 156 and 313 µg/mL of test substance. However, these increases lie within the historical control range, and were therefore not considered to be treatment-related.
In the presence of S-9 mix Metformin hydrochloride caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations at any dose level when compared with the solvent control.
Both positive control compounds caused large statistically significant increases in the proportion of aberrant cells.
It is concluded that Metformin hydrochloride has shown no evidence of clastogenic activity in this in vitro cytogenetic test system.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1991-10-08 to 1992-03-03
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted 04 April 1984
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- RPMI 1640. obtained from a suitable supplier (Imperial Laboratories). This medium. supplemented with 0.1% Synperonic F68 (Serva). 0.011% sodium pyruvate (Sigma). 200 mM glutamine (Sigma). 50 µg/ml gentamicin (Biological Industries) and buffered with 2 mg/ml sodium bicarbonate is referred to as R0p.
RPMI 1640 (Dutch modification).obtained from Imperial Laboratories This medium is HEPES and bicarbonate buffered and supplemented with gentamicin as above. The resulting medium is referred to as R0 (HEPES).
R0p supplemented with 10% HiDHS (supplied by Imperial Laboratories), referred to as R10p. was used for general cell culture. e.g. when growing cells up from frozen stocks.
R10p from which growing L5178Y cells had been removed was used as conditioned medium.
R0 (HEPES) containing S% HiDHS, designated R5 (HEPES). was used as the treatment medium.
R0p in which the amount of Synperonic F68 bad been reduced to 0.02% and supplemented with
30% HiDHS. referred to as R30p, formed the basis of the cloning medium. This was semisolidified by the addition of 4% Noble agar (Difco- in 0.9$ saline).
Selective medium consisted of cloning medium containing 4 µg/lml TFT (Sigma). - Metabolic activation:
- with and without
- Metabolic activation system:
- S-9 rat (Aroclor induced)
- Test concentrations with justification for top dose:
- 1st series: 500 - 5000 µg/mL
2nd series: 500 - 5000 µg/mL - Vehicle / solvent:
- sterile distilled water 20 µL/mL
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- other: 20-methylcholanthrene
- Details on test system and experimental conditions:
- L5178Y mouse lymphoma cells were obtained from Sussex University. These cells are heterozygous at the thymidine kinase locus. TK +/-. Spontaneous thymidine kinase deficient mutants. TK:+/- were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate, thymidine, hypoxanthine and glycine two days prior to storage at -196°C. The cells were stored in polypropylene ampoules in heat-inactivated donor horse serum (HiDHS) containing 10% dimethytsulphoxide (DMSO} and used within seven days of recovery from frozen stock. R10p medium was used for routine expansion of cultures.
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate (main), singel (pretest)
- Number of independent experiments: 2
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 2x10^5 cells per mL
- Test substance added in medium; in agar (plate incorporation); in suspension
TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: no
- Exposure duration/duration of treatment: 3 hours
- Harvest time after the end of treatment (sampling/recovery times): 2 days
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 48 h
- Analysis after 11-12 days incubation
- Method used: microwell plates for the mouse lymphoma assay.
- If a selective agent is used: trifluorothymidine: 4 µg/mL
- Criteria for small (slow growing) and large (fast growing) colonies: none
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: relative survival (RS)
METHODS FOR MEASUREMENTS OF GENOTOXICIY
- OTHER:
After the plates bad been incubated for approximately 11 - 12 days. colonies of L5178Y cells growing on the plates were counted using an Optomax V image analyser (Analytical Measuring Systems.
Cambridge). The results reported were baseci on colonies with a diameter of > 100 µm. - Evaluation criteria:
- The criteria for a positive response were:
At least a two-fold increase in mutant frequency in treated cultures relative to the concurrent control.
The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.
Evidence of a dose relationship over at least two dose levels, in any increase in mutant frequency.
Demonstration of reproducibility in any increase in mutant frequency.
The observed increases in mutant frequency must lie outside the historical control range. - Statistics:
- Standard statistical methods have been applied for data processing.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- True negative controls validity:
- not examined
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- It is concluded that Metformin hydrochloride did not demonstrate mutagenic potential in this in vitro gene mutation assay.
- Executive summary:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 476.
Metformin hydrochloride was tested for mutagenic potential in the mouse lymphoma thymidine kinase assay. Four independent tests were carried out, two in the absence of exogenous metabolic activation (S9 mix) and two in the presence of S-9 mix. In the preliminary toxicity test on Metformin hydrochloride, treatment with 50 - 5000 µg/mLin the presence and absence of S-9 mix resulted in relative growth in suspension of 110 - 77% and 106-76 % respectively compared to the solvent controls. Concentrations used in the main test were based upon this data. In the absence of S-9 mix treatment of cells with 500 - 5000 µg/ml in Test 1 and Test 2 resulted in mean cell growths in suspension of 108 - 85% and 96 - 73% respectively. Cultures treated with 2000, 3000, 4000 and 5000 µg/ml in Test l and Test 2 were cloned in soft agar to permit measurement of the levels of viability and induced mutation. The resulting cell survival levels were 77 – 69 % in Test 1 and 100 – 65 % in Test 2 relative to the controls. Increases in mutant frequency indicative of a positive response were not observed in either test after treatment with Metformin hydrochloride. EMS, the positive control, induced highly significant increases in mutant frequency in both tests. In the presence of S-9 mix treatment of cells with 500- 5000 µg/mL in Test 1 and Test 2 resulted in mean cell growths in suspension of 109 - 86% and 101 - 80% respectively. Cultures treated with 2000, 3000. 4000 and 5000 µg/mL in Test l and Test 2 were cloned in soft agar to permit measurement of the levels of viability and induced mutation. The resulting cell survival levels were 93 – 77 % in Test 1 and 106 - 87% in Test 2 relative to the controls. The criteria used to determine a positive response as described in Evaluation of Results section, were not fulfilled in either test on Metformin hydrochloride in the presence of S-9 mix. 20- Methylcholanthrene, the positive control, induced highly significant increases in mutant frequency in both tests.
Referenceopen allclose all
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 |
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 |
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 |
Metformin hydrochloride was tested for mutagenic potential in the mouse lymphoma thymidine kinase assay. Four independent tests were carried out, two in the absence of exogenous metabolic activation (S9 mix) and two in the presence of S-9 mix. In the preliminary toxicity test on Metformin hydrochloride, treatment with 50 - 5000 µg/mLin the presence and absence of S-9 mix resulted in relative growth in suspension of 110 - 77% and 106-76 % respectively compared to the solvent controls. Concentrations used in the main test were based upon this data. In the absence of S-9 mix treatment of cells with 500 - 5000 µg/ml in Test 1 and Test 2 resulted in mean cell growths in suspension of 108 - 85% and 96 - 73% respectively. Cultures treated with 2000, 3000, 4000 and 5000 µg/ml in Test l and Test 2 were cloned in soft agar to permit measurement of the levels of viability and induced mutation. The resulting cell survival levels were 77 – 69 % in Test 1 and 100 – 65 % in Test 2 relative to the controls. Increases in mutant frequency indicative of a positive response were not observed in either test after treatment with Metformin hydrochloride. EMS, the positive control, induced highly significant increases in mutant frequency in both tests. In the presence of S-9 mix treatment of cells with 500- 5000 µg/mL in Test 1 and Test 2 resulted in mean cell growths in suspension of 109 - 86% and 101 - 80% respectively. Cultures treated with 2000, 3000. 4000 and 5000 µg/mL in Test l and Test 2 were cloned in soft agar to permit measurement of the levels of viability and induced mutation. The resulting cell survival levels were 93 – 77 % in Test 1 and 106 - 87% in Test 2 relative to the controls. The criteria used to determine a positive response as described in Evaluation of Results section, were not fulfilled in either test on Metformin hydrochloride in the presence of S-9 mix. 20- Methylcholanthrene, the positive control, induced highly significant increases in mutant frequency in both tests.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
Metformin hydrochloride was not genotoxic in an in vivo micronucleus test in mice treated with 2000 mg/kg bw performed under GLP regulation and according to OECD guideline.
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1991
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- other: Mouse MN test OECD 474
- Species:
- mouse
- Strain:
- CD-1
- Details on species / strain selection:
- Strain: SPF outbred CD1
Source: Charles River UK
Weight: 22-24 g
Age: 35 d - Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River UK
- Age at study initiation: 35 d
- Weight at study initiation: 22-24 g
- Assigned to test groups randomly: yes, under following basis: separated by sex
- Fasting period before study: overnight prior to and for 2 hours after dosing
- Housing: grouped separated by sex plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum) ad libitum:
- Acclimation period: 4 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): n.d.
- Air changes (per hr): 30
- Photoperiod (hrs dark / hrs light): 12/12 h
- Route of administration:
- oral: gavage
- Vehicle:
- Water
- Duration of treatment / exposure:
- single dose
- Frequency of treatment:
- 1
- Post exposure period:
- 24, 48, 72 h after dosing
- Dose / conc.:
- 2 000 mg/kg bw/day
- Dose / conc.:
- 0 mg/kg bw/day
- No. of animals per sex per dose:
- Neg. Control: 15 f, 15 m
2000 mg/kg Metformin HCl: 20 f, 20 m
Pos. Control: 5 f, 5 m - Control animals:
- yes
- Positive control(s):
- 12 mg/kg bw Mitomycin C
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- - direct bone marrow smears prepared on slides
- Methanol fixation (10 min)
- Giemsa staining - Evaluation criteria:
- MN incidence per 1000 PCEs per animal
- Statistics:
- Standard statistical methods have been applied for data processing.
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- It is concluded that Metformin hydrochloride has not shown any evidence of causing chromosome damage in this in vivo test.
- Executive summary:
This study was performed according to GLP and the methods applied are fully compliant with OECD TG 474. It is concluded that Metformin hydrochloride has not shown any evidence of causing chromosome damage in this in vivo test.
Reference
Summary of results - group totals/means for the entire experiment and results of the statistical analysis
| Samplng Time |
Treatment |
Dose / (mg/kg) |
Ratio p/n (mean) § |
Incidence mnp (mean) § |
Incidence mnn (total) |
| 24 hour |
Vehicle control |
- |
0.847 |
0.2 |
0.2 |
| Metformin HCl |
2000 |
1.059ns |
0.9ns | 0.2 |
|
| Mitomycin C |
12 |
0.759ns | 40.3 *** | 0.4 |
|
| 48 hour |
Vehicle control | - |
1.146 |
1.0 |
0.2 |
| Metformin HCl | 2000 |
0.936ns | 0.6ns | 0.4 |
|
| 72 hour |
Vehicle control | - |
1.228 |
0.8 |
0.6 |
| Metformin HCl | 2000 |
1.165ns | 0.2ns | 0.4 |
p/n Ratio of polychromatic to normocbromatic erythrocytes
mnp Number of micronucleaied cells observed per 1000 polycbromatic
erythrocytes
mnn Number of micronucleated cells observed per 1000 normochromatic
erythrocytes
§ Results of statistical analysis using Wilcoxon's sum of ranks test
(one-sided):
ns P > 0.05
*** P < 0.001
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Additional information from genetic toxicity in vitro:
In this dossier, the endpoint genetic toxicity is addressed by
substance-specific information from in vitro and in vivo experimental
results.
a) in vitro studies
The endpoint genetic toxicity in vitro is addressed with the
experimental studies as shown below:
1. Bacterial Reverse Mutation Assay (in vitro, Ames, GLP) LPA
161/89819: not mutagenic Further in vitro tests have been performed as
follows:
2. Mammalian Cell Mutation Assay (in vitro, MLA, GLP) LPA 178/911534:
not mutagenic
3. Human Lymphocyte Metaphase Analysis (in vitro, CA, GLP) LPA
179/911645: not clastogenic
Overall, there was no positive response in any assay performed thus
showing that test material is not mutagenic in vitro.
b) in vivo studies
In addition, further in vivo studies have been performed as
shown below:
1. Mouse Micronucleus Test (in vivo MNT, GLP) LPA180/911635: not
mutagenic
Overall, there was no positive response. Thus test material is not
mutagenic in vivo.
Conclusion
The test material does not need to be classified according to
CLP-Regulation (EC) No 1272/2008.
Justification for classification or non-classification
The test material does not need to be classified according to CLP-Regulation (EC) No 1272/2008.
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