Metformin hydrochloride

Biodegradation in water: screening tests

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biodegradation in water: ready biodegradability

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:

equivalent or similar to guideline

Guideline:

EPA OTS 796.3260 (Ready Biodegradability: Modified Sturm Test)

Qualifier:

according to guideline

Guideline:

other: FDA TAD 3.11

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

sewage, domestic, non-adapted

Details on inoculum:

The inoculum used in this study consisted of activated sludge and secondary effluent collected from the Columbia Wastewater Treatment Plant on June 23, 1994. These inocula were aerated, blended, settled, and then filtered through glass wool. The filtrate was retained. The inoculum was added to the test solutions at a concentration of 1% blended and filtered activated sludge and 1 % blended and filtered secondary effluent.

Duration of test (contact time):

28 d

Initial conc.:

10 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

CO2 evolution

Remarks:

and other 14C-organic volatiles

Details on study design:

Three separate media, designated control, reference, and test were prepared and added to each corresponding reaction vessel. A total of 9 systems (3 replicates x 3 treatments) were prepared. Each of the reaction flasks was connected to a series of traps. The organic 14C-volatiles produced in the test systems were trapped in the ethylene glycol traps. The CO2 produced in the test systems and reference systems was trapped in the 1 N KOH traps. The test chemical medium was prepared by adding 8.55 mL of a 2.00 mg/mL nonradiolabeled metformin HCl solution (equivalent to 4.964 mg C), 0.250 mL of 0.502 mg/mL of 14C-metformin HCl solution (equivalent to 0.036 mg C), 5 mL of activated sludge inoculum, and 5 mL of secondary effluent inoculum into 500 mL of mineral salt solution. The reference chemical medium was prepared by adding 6.125 mL of a 2.00 mg/mL nonradiolabeled dextrose solution (equivalent to 4.899 mg C), 0.080 mL of 14C-glucose (equivalent to 0.100 mg C), 5 mL of activated sludge inoculum, and 5 mL of secondary effluent inoculum into a 500 mL volumetric flask, and bringing the contents to volume with mineral salt solution. The blank control medium was prepared by adding 5 mL of activated sludge inoculum and 5 mL of secondary effluent inoculum into a 500 mL volumetric flask and bringing the contents to volume with mineral salt solution. Three 110 mL volumes of each stock solution prepared as above were added to their corresponding study flasks. Then 10.0-mL aliquots were taken from each study flask for future analyses (pH measurement, LSC, microbial). A portion of the 3 test 10.0-mL aliquots was filtered and analyzed by HPLC. The applied 14C-activity of test and reference chemicals were verified by analyzing triplicate 0.5-mL aliquots of test and reference medium. The pH of all reaction media were measured using pH paper (Baxter S/Pm 0-14.0 pH Indicator strips).

Microbial Evaluation:

Microbial activity was determined by conducting aerobic bacterial count analysis at the study's initiation and termination. Plate count agar (Difco Laboratories, Detroit, MI) was used for the microbial evaluation. A standard microbial plate count was performed on a sample of each of the test solutions (test, reference, and control) as described in the previous section "Initiation". At termination, standard microbial plate count was performed for each of the three test systems. Also, standard microbial plate count was performed for a composite of the reference systems and a composite of the control systems. A dilution scheme that consisted of 10'2, 10', and 10's dilutions of the solutions were used. The plates were prepared and incubated in an environmental chamber at approximately 21 °C.


Sampling Procedure

Samples were taken for 14C-volatile and'4CO2 analysis on days 1, 3, 7, 14, 21, and 28. Both ethylene glycol and KOH trapping vials were sampled and refilled with 20 mL of the appropriate trapping solutions. Triplicate aliquots (0.5-mL for KOH and 1-mL for ethylene glycol) of the trapping solutions were taken for LSC analysis.

Study Termination

On day 28, the pH of test media were measured with an Orion pH meter (Boston, MA). Triplicate 0.5-mL aliquots of the media were taken for LSC analysis. A portion of each test media was filtered and analyzed by HPLC.

Reference substance:

other: 14C-glucose-UL

Key result

Parameter:

% degradation (CO2 evolution)

Value:

0.6

Sampling time:

28 d

Details on results:

After the 28 days of incubation, 80.5% of the applied reference compound (14C-glucose-UL) was mineralized to CO2, verifying that the microbial inoculum was viable and active. For the test chemical 14C-metformin HCl, approximately 0.6% of the applied 14C-activity was mineralized to 14CO2 during the same incubation period.

At the end of study, the degradation of parent test compound was monitored by high-performance liquid chromatography (HPLC). Results showed that the test compound 14C-metformin HCl was not biotransformed (degraded) during the 28-day incubation period.

The overall 14C-mass balance was taken as a summation of the 14C-activity in CO2 produced, 14C-activity in organic volatiles produced, and 14C-activity remaining in the water medium. The 14C-mass balance of 14C-metformin HCl test chemical and 14C-glucose reference chemical averaged 99.8 ± 2.8% and 85.1 ± 8.3%, respectively.

The test results indicated that 14C-metformin was not biodegradable under the test conditions.

Validity criteria fulfilled:

yes

Interpretation of results:

under test conditions no biodegradation observed

Conclusions:

The test results indicated that 14C-metformin was not readily biodegradable under the test conditions.

Executive summary:

Study Design

The test chemical, ¹⁴C-metformin HCl, was tested for biodegradability in water at a dosing concentration of 10 mg C/L. Production of ¹⁴O₂and other ¹⁴C-organic volatiles was measured during the 28-day test period. A reference chemical (¹⁴C-glucose) at a dosing concentration of 10 mg C/L was tested concurrently to verify the viability of the microbial inoculum.

The study was conducted in the dark at a temperature range of 21 ± 1°C. The percent biodegradability was calculated as a function of the ¹⁴CO₂ production in the test systems as compared to the amount of applied ¹⁴C-activity.

Results

After the 28 days of incubation, 80.5% of the applied reference compound (¹⁴C-glucose-UL) was mineralized to ¹⁴CO₂, verifying that the microbial inoculum was viable and active. For the test chemical ¹⁴C-metformin HCl, approximately 0.6% of the applied ¹⁴C-activity was mineralized to ¹⁴CO₂during the same incubation period.

At the end of study, the degradation of parent test compound was monitored by high-performance liquid chromatography (HPLC). Results showed that the test compound ¹⁴C-metformin HCl was not biotransformed (degraded) during the 28-day incubation period.

The overall ¹⁴C-mass balance was taken as a summation of the ¹⁴C-activity in CO₂ produced, ¹⁴C-activity in organic volatiles produced, and ¹⁴C-activity remaining in the water medium. The ¹⁴C-mass balance of ¹⁴C-metformin HCl test chemical and ¹⁴C-glucose reference chemical averaged 99.8 ± 2.8% and 85.1 ± 8.3%, respectively.

Conclusion

The test results indicated that ¹⁴C-metformin was not biodegradable under the test conditions.

Reference 2

Endpoint:

biodegradation in water: inherent biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 302 C (Inherent Biodegradability: Modified MITI Test (II))

Deviations:

yes

Remarks:

The test was run for 95 days

GLP compliance:

yes

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge: Werdhölzli WWTP (Zurich, Switzerland)

Duration of test (contact time):

95 d

Initial conc.:

38.5 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

Metformin hydrochloride was added at 38.5 mg MET-HCl/L (corresponding to 30 mg MET/L) to OECD 302 C medium containing 100 mg (dry matter) rinsed activated sludge/L.
Note only one activated sludge was used as the inoculum, from Werdhölzli WWTP (Zurich, Switzerland) treating predominantly domestic wastewaters.
Biochemical oxygen demand (BOD) was measured with OxiTop®-C measuring heads (WTW Wissenschaftlich-Technische Werkstätten, Weilheim, Germany).
Ten such vessels with 200 mL working volume (520 mL total volume) were set up: four test vessels (three as required by the guideline plus one as a standby for redundancy/safety reasons); two blank replicates containing inoculum and medium; two procedure control replicates containing inoculum, medium and 100 mg sodium benzoate/L; one abiotic sterile control replicate containing MET, deionized water and 0.2 mM HgCl2 as a sterilizing agent to prevent microbial decomposition; and one toxicity control replicate (which is not normally performed for the OECD 302 C test) containing inoculum, test medium, MET and sodium benzoate as biodegradable reference item.
In addition, four further test vessels with 1800 mL working volume (2000 mL total volume) without BOD measurement were set up with the same sludge, for weekly substance analytics: two replicates containing inoculum, test medium and MET as test systems, plus two replicates containing inoculum and test medium only as blank. All 14 test vessels were stirred using magnet bars and kept in a climate-controlled room at 22±1 °C in the dark for 95 days.
Substance analytics were set up for both MET and GUU, to elucidate the fate of both compounds, following the method recently described by Trautwein et al. [2014]. HPLC analysis of MET and GUU was performed weekly using the same method described further below for the OECD 301F test. The four additional vessels were also used for determination of DOC using a Shimadzu TOC-Analyzer TOC-L CSH (Shimadzu, Reinach, Switzerland), nitrite (photometry according to guidelines of EDI, the Swiss Federal Department of Home Affairs, Berne, Switzerland) and nitrate (Metrohm 930 Compact Ion Chromatography Flex system, Metrohm, Zofingen, Switzerland).

Reference substance:

benzoic acid, sodium salt

Key result

Parameter:

% degradation (DOC removal)

Value:

> 95

Sampling time:

95 d

Details on results:

Biodegradation of MET as measured by average blank-corrected BOD/ThOD in the three test vessels proceeded in five phases (see Figure):
First, a lag phase with an insignificant 0e5% BOD/ThOD (NO3) until day 8; second, an initial degradation phase from day 8 to 18, when degradation increased to 25e30%; third, a plateau at that level without further significant oxygen uptake from day 18 to 42; fourth, a second degradation phase from day 42 to 64, when 70% BOD/ThOD (NO3) was reached; and last, a second plateau phase from day 64 to 95, with BOD/ThOD (NO3) undulating between 62 and 71%.
The toxicity control showed the same pattern as the averaged three test vessels, with 45% combined degradation reached during day 4 and 65% on day 14; from day 49 to 95, the total degradation showed a plateau around 85-93% (not shown).
In the parallel set of test vessels without BOD measuring units (see Figure), the blank-corrected DOC was stable for the first seven days, then showed a decrease to approximately 60% of the starting level in parallel with the initial BOD degradation phase, followed by a plateau from day 14 to 35 with slightly increasing DOC levels, then a second decrease from day 35 to 42 (one week earlier than the second degradation phase in the BOD vessels) to <10% of initial
DOC, finally a slower decrease from day 42 to 56 to DOC values < LOQ, corresponding to >95% removal by DOC loss. Blankcorrected metformin concentrations showed an initial decline by nearly 10% of initial from day 0 to 7, then a strong decrease to < LOQ from day 7 to 14, with a minor peak of <5% of initial from days 35 to 56, then consistently < LOQ until day 95. Blank-corrected GUU concentrations started with 0 (Nitrite remained below LOQ on most sampling days (except day 14, not shown). Nitrate formation showed an opposite pattern to MET and GUU degradation; from day 7 onwards, nitrate increased to reach a plateau around day 21, this was followed by a second increase from day 35 to reach a second plateau approximately on day 56 (Fig. 2b)
Based on the measurements, full nitrification proceeded slower than the degradation of either MET or GUU.

Results with reference substance:

The positive control, sodium benzoate, reached 71% degradation by BOD/ThOD in 7 days, evidencing awell degrading AS, and reached a plateau between 85 and 90% on day 20.

Validity criteria fulfilled:

yes

Interpretation of results:

inherently biodegradable

Conclusions:

This prolonged inherent biodegradation test strongly suggests not only primary transformation of metformin to guanyl urea, but also subsequent full mineralization of GUU, with both degradation phases starting after a clear lag phase.

Executive summary:

This prolonged inherent biodegradation test strongly suggests not only primary transformation of metformin to guanyl urea, but also subsequent full mineralization of guanyl urea, with both degradation phases starting after a clear lag phase.

Description of key information

Based on a GLP-conform ready biodegradability test, metformin HCL is considered to be not readily biodegradable under the test conditions. In a prolonged inherent biodegradation test according to OECD 302C, complete mineralization of metformin HCL and its primary degradation product guanyl urea was observed with both degradation phases starting after a clear lag phase.

Key value for chemical safety assessment

Biodegradation in water:

inherently biodegradable

Type of water:

freshwater