Metformin hydrochloride

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: FDA TAD 4.02

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

A stock solution of Metformin HCl was prepared by adding 1000 mg of test chemical to 500 mL ABC reagent water (= 2000 mg/L) in a Class A volumetric flask (pH ca. 6.6). A dilution of this stock solution was analyzed by high performance liquid chromatography (HPLC) method described below, to confirm the nominal concentration.

A dilution series of the test chemical was prepared by adding 40.0 mL of the stock solution to a 360.0 mL sterile ABC reagent water blank, yielding a 1:10 dilution. The resulting solution of the test chemical was then further diluted by dispensing 40.0 mL into 360.0 mL of ABC reagent water. This process was repeated for each subsequent dilution, resulting in a series of 1:10 dilution steps, yielding a dilution series with the following nominal concentrations of the test chemical: 2000, 200, 20, 2, and 0 mg/L.

Double strength agar media (double the normal concentration recommended for plate media) was dispensed in 15-mL aliquots into screw-cap tubes and autoclaved for 25 minutes to ensure sterility. The double strength agar media used to prepare the pour plates were stored at room temperature. On the day test media were prepared, the agar tubes were autoclaved for 5 minutes to melt the agar and then were placed in a warm water bath set to keep the agar molten.

Triplicate test plates containing the target concentration of the Metformin HC1 were made by adding 15.0 mL of the appropriate stock dilution to the melted agar tubes containing 15 mL of the double strength agar media. Each tube was inverted three times and the contents were poured into a sterile labeled Petri plate. This resulted in a 1:2 dilution of each Metformin HCl solution and yielded plated media with the following nominal concentrations of Metformin HCI: 1000, 100, 10, and 1 mg/L. Triplicate series of control plates, containing no test material, were also made by adding 15 mL of sterile ABC reagent water to agar tubes and dispensing to sterile Petri plates.

Three plates were used for each concentration for each species.

Test organisms (species):

other: Pseudomonas fluorescens, Bacillus megaterium, Aspergillus clavatus, Penicillium canescens, Chaetomium globoswn,Anbaena flos-aquae, Azotobacter chroococcum

Details on inoculum:

All microbes were obtained from American Type Culture Collection (ATCC, Rockville, MD). The genus and species of each organism used in the test and its source are listed in Table I. These test organisms are known to be present in soil (3 and 4). The organisms were shipped by ATCC as lyophilized pellets. The test organisms were all successfully cultured on agar based medium (see Table II for organism specific medium). The bacterial cultures were incubated at 26 ± 2 °C in the dark. The fungi cultures except Chaetomium globosum were incubated in the dark. Chaetomium globosum was incubated under continuous fluorescent light at 26 ± 2 °C. After eight days other fungi were also incubated under continuous fluorescent light at 26 ± 2 °C. The blue-green alga was also incubated under continuous fluorescent light at 26 ± 2 °C.

Test type:

static

Water media type:

not specified

Limit test:

no

Total exposure duration:

6 d

Test temperature:

26 ± 3 °C

pH:

6.5 - 7

Nominal and measured concentrations:

Nominal concentrations of 1000, 100, 10, 1, and 0 mg/L metformin HCl in agar media

Details on test conditions:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ABC reagent water is the product of a multiple-step deionization and purification process that begins by passing feed water over cation, anion, and unibed ion-exchange resin beds (Culligan Corporation; Northbrook, IL). Feed water going into the Culligan system for delivery to the LABCONCO purifier is ABC well water purified by reverse osmosis, while feed water going into the Culligan system for delivery to the Millipore purifier is potable water supplied by Boone County Rural Water District #9. The deionized water is plumbed directly to either a Millipore (Bedford, MA) Milli-Q, or a LABCONCO (Kansas City, MO) Waterpro PS system for further purification. The two systems are equivalent four-stage purifiers, comprising carbon, ion-exchange, and organic adsorption cartridges. Both the Millipore and the LABCONCO systems deliver water that is 16-18 megohm • cm in resistivity. As water leaves the purifiers, it passes through a final 0.2 µm hollow fiber filter. The filtrate is referred to as ABC reagent water.


OTHER TEST CONDITIONS
The test organisms were each inoculated onto plates containing the various concentrations of the test chemical and control plates containing no test chemical. The bacterial test organism plates were incubated in a 26 ± 3 °C incubator shielded from light. The fungi test organism plates were incubated in a 26 ± 3 °C incubator under continuous fluorescent light. The blue-green alga was incubated at a temperature of 26 ± 3 °C under continuous fluorescent light. Visual observations of growth were made after an incubation period sufficient to yield adequate growth (see Figures 1-7) on the control plates.

The test microorganisms were inoculated onto the test plates as stated below. After the agar media solidified, triplicate series of plates containing the five test chemical concentrations were inoculated with the test organisms. Broth cultures of each test organism served as the inoculum source. The inoculum was spotted onto eight sections of the plate, making eight spots per plate of which at least six spots were counted. The plates were labeled with sticker labels, and using a permanent pen the study number, date, initials, concentration of test compound, replicate A, B or C, and the name of the inoculated organism were included on the label. The plates were then incubated until growth was observed on the control plates.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

TEST CONCENTRATIONS
The test chemical was used at nominal concentrations of 1000, 100, 10, 1 and 0 (control or untreated) mg/L.

RANGE FINDING STUDY
An additional stock solution of Metformin HCl was prepared by adding 400 mg of test chemical to 250 mL ABC reagent water (= 1600 mg/L) in a Class A volumetric flask (pH ca. 6.7). Dilutions of this stock solution was analyzed by high performance liquid chromatography (HPLC) method described below, to confirm the nominal concentration. For the Azotobacter testing concentrations the following procedure was used to make the dilutions. A dilution series of the test chemical was prepared by adding 100.0 mL of the stock solution to a 100.0 mL sterile ABC reagent water blank, yielding a 1:2 dilution. The resulting solution of the test chemical was then further diluted by dispensing 100.0 mL into 100.0 mL of ABC reagent water. This yielded a dilution series with the following nominal concentrations of the test chemical: 1600, 800, and 400 mg/L. For the Anabaena testing concentrations the following procedure was used to make the dilutions. A dilution series of the test chemical was prepared by adding 20.0 mL of the stock solution to a 180.0 mL sterile ABC reagent water blank, yielding a 1:10 dilution. The resulting solution of the test chemical was then further diluted by dispensing 100.0 mL into 100.0 mL of ABC reagent water. This process was repeated for the subsequent dilution, resulting in 1:2 dilution steps. This yielded a dilution series with the following nominal concentrations of the test chemical: 160, 80, and 40 mg/L. The test plates (in triplicate) containing the target concentration of the Metformin HCl were prepared as described on page 14 (para 5). This yielded plated media with the following nominal concentrations of Metformin HC1: 800, 400, 200, 80, 40, and 20 mg/L. Triplicate series of control plates, containing no test material, were also made by adding 15 mL of sterile ABC reagent water to agar tubes and dispensing to sterile Petri plates. Three plates were used for each concentration for each species.

Duration:

6 d

Dose descriptor:

NOEC

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Details on results:

Analysis Stock Solutions:

The HPLC method was checked for linearity and reproducibility. Concentrations tested were: 50, 125, 250, and 500 µg/mL. The standard curve was linear with a correlation coefficient of 0.99977 (Figure 8). Retention times for these four concentrations ranged from 4.62 minutes to 4.72 minutes (Figures 9-12). The concentration of stock solutions were determined to be 2011.8 and 1658.1 mg/L, against a nominal concentrations of 2000 and 1600 mg/L respectively, confirming the homogeneity and concentration of the stock solutions. Retention times of Metformin HCl stock solutions of 2000 and 1600 mg/L were 4.68 and 4.71, respectively.

Observations of Growth Inhibition:

Nominal concentrations of 1000, 100, 10, 1 and 0 mg/L of the test chemical were prepared from the stock solution and tested for microbial inhibition. The stock solution was incorporated in to the test media to arrive at these concentrations. No inhibition was observed for the test organisms Pseudomonas fluorescens, Bacillus megaterium, Aspergillus clavatus, Penicillium canescens, and Chaetomium globosum at any concentration of Metformin HCl tested up to and including 1000 mg/L Metformin HCl. Azotobacter chroococcum was inhibited at 1000 mg/L. Anabaena flos-aquae was inhibited at 100 mg/L. A further test series was conducted at the nominal concentrations of 800, 400, 200, and 0 mg/L for Azotobacter, and 80, 40, 20, and 0 mg/L for Anabaena. Inhibition was observed for the test organisms Azotobacter chroococcum and Anabaena flos-aquae with MICs of 800 and 100 mg/L, respectively.

Validity criteria fulfilled:

yes

Conclusions:

The test chemical Metformin HCl showed no inhibitory effect on Pseudomonas, Bacillus, Aspergillus, Penicillium and Chaetomium at the maximum concentration of 1000 mg/L. Hence no MIC could be determined for these species. However, the bacteria Azotobacter showed inhibition at 800 mg/L metformin HCl, and the blue-green alga Anabaena showed inhibition at 100 mg/L metformin HCl.

Executive summary:

Study Design

In this study the test item, metformin HCl, was evaluated for potential inhibitory effects on the growth of representative microbial species usbing a method described in the FDA TAD 4.08 (Microbial Growth Inhibition).

Metformin HCl was incorporated into an agar medium in a series of five concentrations. Triplicate series of the test plates were inoculated with pure cultures of microorganisms. After an appropriate incubation period, the presence or absence of microbial growth on the agar surface was noted. Absence of visual growth was used as an indication of possible inhibitory effects, and for each organism a minimum inhibitory concentration (MIC) value was attempted. The MIC is defined as the lowest concentration of the test chemical that inhibits the growth of the test microorganism.

Results

Nominal concentrations of 1000, 100, 10, 1, and 0 mg/L Metformin HCl in agar media were prepared and the growth of microorganisms on the appropriate media tested. No inhibition was observed for Pseudomonas fluorescens, Bacillus megaterium, Aspergillus clavatus, Penicillium canescens, and Chaetomium globosum at any concentration of metformin HCl tested up to and including 1000 mg/L. Inhibition was observed for the test organism, Azotobacter chroococcum, at a concentration of 1000 mg/L. Inhibition was observed for the test organism Anabaena, flos-aquae, at a concentration of 100 mg/L. The MICs for Azotobacter and Anabaena were narrowed down to 800 and 100 mg/L, respectively in an experiment conducted over a narrower range of concentrations.

Conclusion

The test item showed no inhibitory effect on Pseudomonas, Bacillus, Aspergillus, Penicillium and Chaetomium at the maximum concentration of 1000 mg/L. Hence no MIC could be determined for these species. However, the bacteria Azotobacter showed inhibition at 800 mg/L metformin HCl, and the blue-green alga Anabaena showed inhibition at 100 mg/L metformin HCl.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: only abstract available

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

GLP compliance:

not specified

Test organisms (species):

activated sludge

Test type:

not specified

Water media type:

freshwater

Total exposure duration:

3 h

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

233 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

Activated Sludge, Respiration Inhibition (3h): EC50 = 233 mg/L
Activated Sludge, COD removal (24h): EC50 > 250 mg/L
Activated Sludge, Nitrification Inhibition: EC50 = 48 to 65 mg/L

Conclusions:

In this activated sludge respiration inhibition assay, the test material shows an EC50 of 233 mg/L

Description of key information

The test chemical Metformin HCl showed no inhibitory effect on Pseudomonas, Bacillus, Aspergillus, Penicillium and Chaetomium at the maximum concentration of 1000 mg/L. Hence no MIC could be determined for these species. However, the bacteria Azotobacter showed inhibition at 800 mg/L metformin HCl, and the blue-green alga Anabaena showed inhibition at 100 mg/L metformin HCl. In an activated sludge respiration inhibition assay, the test material shows an EC50 of 233 mg/L