Metformin hydrochloride

Long-term toxicity to fish

Link to relevant study record(s)

Reference

Endpoint:

fish early-life stage toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Nov 2007 - Jan 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0.15, 0.44, 1.3, 4.0, and 12 mg/L
- Sampling method: For the analysis of the test item concentrations in the test media, duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test (Day 0) and thereafter at least once the week during the test. To determine the maintenance of the test item concentrations during the test medium renewal periods of 48 and 72 hours, duplicate samples were taken from the aged test media of all test concentrations three times during the test. Two of these stability controls corresponded to the test medium renewal period of 48 hours and one of it to the renewal period of 72 hours. The stability samples were taken from the actual test itself by pouring together the volumes of the four replicate test vessels per treatment. The highest test concentration of nominal 12 mg/I, was determined as the overall NOEC in this test. From this test concentration, the duplicate samples from the freshly prepared test medium were measured from all sampling dates during the test. From the stability controls, the duplicate samples from all three sampling dates were analyzed. From the control samples, one of the duplicate samples was analyzed from four sampling dates during the test period.
- Sample storage conditions before analysis: Immediately after sampling, all samples were deep frozen at about -20°C.

Vehicle:

no

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: West Aquarium GmbH, 37431 Bad Lauterberg / Germany

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of eggs: 60/concentration divided in 4 replicates
- Method of collection of fertilised eggs: mesh
- Subsequent handling of eggs: --


TEST ORGANISM
- Common name: Zebra fish
- Strain: Brachydanio rerio
- Source: West Aquarium GmbH, 37431 Bad Lauterberg / Germany

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): In the laboratories a brood batch of at least 200 individuals was held.
- Method of collection of fertilised eggs: Glass dishes covered with a mesh were placed on the bottom of the aquarium one day prior to test start for the collection of the newly fertilized eggs used in the test. The mesh separated the fertilized eggs (sinking to the bottom into the glass dishes) from the parent fish. Spawning started in the morning after the light had been turned on. The eggs were exposed in the test media within about 2 hours after fertilization.
- Subsequent handling of eggs: NA

POST-HATCH FEEDING
Day 5 -- Day 13: Live ciliate (raised at RCC) and dry-food powder
Day 13 - test end: Freshly hatched artemia larvae (raised at RCC) and dry-food

Food was given at laest twice each working day (ad libitum while minizing the surplus). On weekends, food was given at least once per day. Fish were not fed 24 hours prior to the end of the test to allow clearance of the digestive tract before weighing the fish.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

34 d

Hardness:

125 mg CaCO3

Test temperature:

26.6-26.9°C

pH:

between 7.3 and 7.5

Dissolved oxygen:

During the test period, the dissolved oxygen concentration (Table 8) in the control and the test concentrations was at least 7.4 mg/L, corresponding to an oxygen saturation of 91%.

Nominal and measured concentrations:

Nominal: The highest test concentration of nominal 12 mg/I, was determined as the overall NOEC in this test. From this test concentration, the duplicate samples from the freshly prepared test medium were measured from all sampling dates during the test. From the stability controls, the duplicate samples from all three sampling dates were analyzed. From the control samples, one of the duplicate samples was analyzed from four sampling dates during the test period.

Measured: The EMD 89502 (Metformin hydrochloride) concentration in the freshly prepared test medium of nominal 12 mg/L ranged from 78 to 110% of the nominal value. In the aged test medium at the end of the test medium renewal periods the test concentration was between 103 to 112% of the initially measured concentration.

Details on test conditions:

A semi-static test design was chosen to keep the test item concentrations in the test media as constant as possible during the test period. The test media were renewed three times per week (every two and three days). At these dates the eggs, larvae and surviving fish were transferred into clean test vessels with freshly prepared test medium.

Four replicates were used for each test concentration and the control. From the start until Day 7 of the test, glass vessels (10 cm in diameter) with about 200 mL test medium were used. Thereafter, when the larvae had developed to juvenile fish and had started feeding on nauplia larvae, glass beakers with about 1 liter test medium volume were used (10 cm in diameter, 14 cm height). After the fish became bigger in size, they were transferred into glass beakers with about 2 liter (at Day 17 of the test) and 5 liter test medium volume (at Day 28 of the test), respectively.

The corresponding four replicate test vessels of each treatment were positioned in a temperature-regulated water bath. All test vessels were labeled with the study number and all necessary additional information to ensure unmistakable identification. The test media were slightly aerated during the test.

At the start of the test (Day 0), 60 fertilized eggs were exposed to each test concentration and the control. The eggs were randomly distributed to the replicate test vessels of the different treatments (15 eggs per replicate). The loading rate of the eggs (biomass per volume of test medium) in each test vessel was much lower than 0.5 g/liter,

The instantaneous loading rate of the test fish at the end of the test period was calculated to be in maximum 0.06 g fish wet weight/liter (calculation based on the volume of the 5 liter test beakers). This maximum value (calculated in the test concentration of 12 mg/L, replicate C with highest loading rate) is far below the loading rate of in maximum 1.0 g fish wet weight/liter as recommended by the test guideline.

Test Duration
34 days: 4 days post fertilization (Day 0 to Day 4) plus 30 days post-hatch (Day 0 post hatch equals Day 4 post fertilization)

Light conditions:
A 16-hour light to 8-hour darkness photoperiod (with a 30-minute transition period), light intensity at light period approximately 420 to 500 Lux.

Feeding:
Day 0 - Day 5 (embryo stage and newly hatched larvae): none
Day 5 -- Day 13: Live ciliate and dry-food powder'
Day 13 - test end: Freshly hatched artemia larvae and dry-foods
Food was given at laest twice each working day (ad libitum while minizing the surplus). On weekends, food was given at least once per day. Fish were not fed 24 hours prior to the end of the test to allow clearance of the digestive tract before weighing the fish.

Dead eggs, dead larvae or fish were removed at least on each working day. Faeces and surplus food were removed regularly from the test vessels.

TEST SYSTEM
- Emybro cups (if used, type/material, size, fill volume):
- Test vessel: From the start until Day 7 of the test, glass vessels (10 cm in diameter) with about 200 mL test medium were used. Thereafter, when the larvae had developed to juvenile fish and had started feeding on nauplia larvae, glass beakers with about 1 liter test medium volume were used (10 cm in diameter, 14 cm height). After the fish became bigger in size, they were transferred into glass beakers with about 2 liter (at Day 17 of the test) and 5 liter test medium volume (at Day 28 of the test), respectively.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume:
- Aeration: slightly
- Type of flow-through (e.g. peristaltic or proportional diluter): NA
- Renewal rate of test solution (frequency/flow rate): three times per week (every two and three days)
- No. of fertilized eggs/embryos per vessel: 15
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): NA
- Biomass loading rate: The instantaneous loading rate of the test fish at the end of the test period was calculated to be in maximum 0.06 g fish wet weight/liter (calculation based on the volume of the 5 liter test beakers). This maximum value (calculated in the test concentration of 12 mg/L, replicate C with highest loading rate) is far below the loading rate of in maximum 1.0 g fish wet weight/liter as recommended by the test guideline.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water was used in the study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations:

CaCl x 2H2O: 147 mg/L
MgSO4 x 7H2O: 61.5 mg/L
NaHCO3: 32.5 mg/L
KCl: 2.9 mg/L

- Total organic carbon: NA
- Particulate matter: NA
- Metals: NA
- Pesticides: NA
- Chlorine: NA
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: 4:1
- Salinity: NA
- Culture medium different from test medium: NA
- Intervals of water quality measurement: Water temperature, oxygen concentration and pH were measured in all test concentrations and the control at the start of the test and one times the week thereafter in the freshly prepared and/or aged test media. These parameters were measured alternately in one of the replicate test beakers per treatment. Additionally, the water temperature was monitored continuously by a data logger in the control. The appearance of the test media was checked each working day. Observations were recorded once a week.

OTHER TEST CONDITIONS
- Adjustment of pH: NA
- Photoperiod: A 16-hour light to 8-hour dark photoperiod with a 30-minute transition period was applied
- Light intensity: 420 to 500 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : development and hatching rate, time to hatch / development rate, development and survival of larvae and juvenile fish, fish length and fish weight

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY : NA

POST-HATCH DETAILS
- Begin of post-hatch period: Day 0 post hatch equals Day 4 post fertilization
- No. of hatched eggs (alevins)/treatment released to the test chamber: NA
- Release of alevins from incubation cups to test chamber on day no.: NA

FERTILIZATION SUCCESS STUDY
- Number of eggs used: NA
- Removal of eggs to check the embryonic development on day no.: NA

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Egg development nad hatching rate

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: time to hatch / development rate

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: survival of larvae and juvenile fish

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

length

Key result

Duration:

34 d

Dose descriptor:

NOEC

Effect conc.:

>= 12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

weight

Remarks on result:

other: wet and dry weight

Details on results:

Egg development and hatching rate:

In the control and at all test concentrations hatching was completed at Day 4 post fertilization. The mean hatching rate in the control was 90.0 ± 3.3% and consequently the hatching rate in the control was sufficiently high under the test conditions. At all test concentrations up to and including the highest test item concentration of nominal 12 mg/L, the mean hatching rates were in the range of 91.7 ± 5.5% to 95.0 ± 5.5% and thus even slightly higher compared to the control. Therefore, the NOEC for egg development and hatching rate was determined to be >= 12 mg/L nominal. The LOEC was > 12 mg/L.

Time to hatch 1 development rate of eggs:

According to a Williams tests (one sided smaller, a = 0.05), the development rate in all test concentrations up to and including 12 mg/L was not statistically different to the control. Therefore, the NOEC for hatching time and development rate of the eggs was determined to be >= 12 mg/L nominal. The LOEC was > 12 mg/L.

Survival of larvae and juvenile fish:

The survival of the fish larvae and juvenile fish is shown in Table 3. The mean survival rates of the fish in the control until the end of the test was 96 ± 3.7%. This demonstrates the suitability of the test conditions (the validity criteria for post hatch success is >= 70%). During the test, sporadic mortality of larvae and fish was observed at all test concentrations up to and including 12 mg/L. Since this low mortality rate was also observed to the same extent in the control, it was not considered as toxic effect of the test item but as natural mortality rate. Furthermore, the mean survival rate in these test concentrations was clearly above the validity criteria for post hatch success. All fish which survived until the end of the test were healthy and showed normal behavior. However, a high mortality was observed in one replicate (C) of the test concentration of nominal 0.44 mg/L. This mortality was not attributed to a toxic effect of the test item, but as accidental for unknown reasons. The mean survival rates in all test concentrations were in the range of 72.9 ± 31.5% to 94.9 1 5.6%. No concentration-effect relationship was observed. According to a Williams tests (one sided smaller, a = 0.05), the mean survival rates in all test concentrations up to and including the highest test concentration of 12 mg/L was not statistically different to the control. Therefore, the NOEC for survival of the test fish was determined to be >= 12 mg/L. The LOEC was > 12 mg/L.

Fish length and body weight:

The fish body length, fish wet weight and fish dry weight were not statistically significantly reduced compared to the control up to and including the highest test concentration of 12 mg/L (Williams test, one sided smaller, alpha = 0.05). Thus, the NOEC for fish body length, and the fish dry weight were determined to be be >= 12 mg/L. The LOEC was > 12 mg/L.

Reported statistics and error estimates:

Determination of the NOEC: The mean survival rates, the mean development rates, the mean fish length, and the mean fish wet weight in the different test concentrations were evaluated for significant differences to the control by the multivariate Williams-test after one-way analysis of variance (ANOVA). For the mean hatching rates and the mean dry weight a statistical evaluation was not necessary due to the clear results.

Validity criteria fulfilled:

yes

Conclusions:

Summarizing the NOECs for each of the test parameters assessed, the overall NOEC of EMD 89502 (Metformin hydrochloride) for early life stages of zebra fish was determined to be >= 12 mg/L since up to and including this highest test concentration no toxic effect was observed on the eggs, larvae or fish. The overall LOEC was determined to be > 12 mg/L.

Executive summary:

Study Design

The toxicity of the test item to zebra fish (Brachydanio rerio) was investigated in a semi-static early-life stage toxicity test according to the OECD Guideline for Testing of Chemicals, No. 210, "Fish, Early-life Stage Toxicity Test", 1992.

Freshly fertilized eggs of zebra fish were exposed in this semi-static test to test media containing the test item at nominal concentrations of 0.15, 0.44, 1.3, 4.0, and 12 mg/L under defined conditions. Additionally a control was tested in parallel. The test media were renewed every two and three days during the test period of 34 days.

At the start of the test, 60 eggs each (divided into 4 replicates) were distributed to the test concentrations and the control. The eggs, larvae and juvenile fish were observed for toxic effects on their development, growth and survival.

Results

The test item concentration in the freshly prepared test medium of nominal 12 mg/L ranged from 78 to 110% of the nominal value. In the aged test medium at the end of the test medium renewal periods the test concentration was between 103 to 112% of the initially measured concentration. Thus, metformin HCl was stable during the test medium renewal periods of 48 and 72 hours.

The mean measured test concentration (calculated as the arithmetic mean over all measurements) was 12.7 mg/L, corresponding to 106% of nominal. The biological results are related to the nominal test item concentration of 12 mg/L.


The biological results at test end are given for each test parameter assessed:

Parameter	NOEC / [mg/L]	LOEC / [mg/L]
Egg development and hatching rate	≥12	>12
Time to hatch / development rate:	≥12	>12
Survival of larvae and juvenile fish	≥12	>12
Fish length	≥12	>12
Fish wet weight	≥12	>12
Fish dry weight	≥12	>12

Summarizing the NOECs for each of the test parameters assessed, the overall NOEC of metformin HCl for early life stages of zebra fish was determined to be >= 12 mg/L since up to and including this highest test concentration no toxic effect was observed on the eggs, larvae or fish.

Conclusion

Summarizing the NOECs for each of the test parameters assessed, the overall NOEC of EMD 89502 (Metformin hydrochloride) for early life stages of zebra fish was determined to be >= 12 mg/L since up to and including this highest test concentration no toxic effect was observed on the eggs, larvae or fish. The overall LOEC was determined to be > 12 mg/L.

Description of key information

Summarizing the NOECs for each of the test parameters assessed, the overall NOEC of EMD 89502 (Metformin hydrochloride) for early life stages of zebra fish was determined to be >= 12 mg/L since up to and including this highest test concentration no toxic effect was observed on the eggs, larvae or fish. The overall LOEC was determined to be > 12 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

12 mg/L

Additional information

Summarizing the NOECs for each of the test parameters assessed, the overall NOEC of EMD 89502 (Metformin hydrochloride) for early life stages of zebra fish was determined to be >= 12 mg/L since up to and including this highest test concentration no toxic effect was observed on the eggs, larvae or fish. The overall LOEC was determined to be > 12 mg/L.