Toxicity and Toxicokinetics of

Metformin in Rats
- Journal club
Submitted by :
Miss.M.Pooja
M.Pharm (Pharmacology Dept)
Under the guidance of :
Dr.S.Mohana lakshmi
M.Pharm, Ph.D,
Professor, Dept of Pharmacognosy

Introduction
Type 2 diabetes :

Pathophysiology

Drug introduction
Metformin:

Materials and methods

Metformin HCl
Concentrations of 0,20,60,90 or 120 mg/mL in
0.01%
methylcellulose/0.1%
polyoxyethylene
sorbitan monooleate (Tween 80).

Experimental animals:
Male and female Crl:CD(SD) rats (charles river lab).
Temperature of 1826 C; relative humidity of 30
70%; 12-h light/dark cycle.
Males - 245348 g & females -180240 g.

Experimental design:
12/group/sex & additional 3 animals/sex/group
dose volume of 10 mL/kg.

In-life observations- clinical observations :

Body weight
Food consumption

Bioanalytical methods:
High performance liquid chromatography (HPLC)
with Tandem mass spectrometry (MS/MS)
Precipitation with acetonitrile that contained the
internal standard [ H ].
Chromatography was performed using a Varian
Pursuit C18 502.0 mm i.d., 3 m analytical
column.
2

The mobile phases (A) 100% acetonitrile (B)

0.26% heptaflurobutyric acid buffer (with 0.77 g
ammonium acetate added/liter).
The method used an isocratic gradiant (83A:17B
v/v) and flow rate of 0.6 mL/min with a run time
of 1.5 min.
Detection by tandem mass spectrometry was
based on precursor ion transitions of m/z 130 to
60. The calibration range was 19.5 to19,525
ng/mL.
Quality control standards were prepared at 55,
1796and 15, 620 ng/mL and were stored and
analyzed with the study samples.
8

Toxicokinetics :

Blood samples were collected from a jugular vein of

toxicokinetic animals into tubes containing sodium
fluoride/potassium oxalate at 0.5, 1, 2, 4, 8, 12 and 24 h
after administration of metformin on days 1, 26 and 89.
Plasma sample + equivalent volume of 0.1 M ammonium
acetate (pH 4.0, adjusted with acetic acid), analyzed for
metformin.
Plasma concentration values of the 24-h samples were used
as plasma concentration values for time zero on day 26 and
day 89.
Systemic exposure determined by calculating the area
under the plasma concentration time curve (AUC).
Maximum observed peak plasma concentration (Cmax) &
9
time at which it was observed (Tmax) determined.

 Ophthalmic

examination:

Ophthalmoscopy examinations.
Clinical

pathology :

Samples for hematologic analysis.

Samples for coagulation
Samples for serum clinical chemistry
Urine chemistry analysis.
Terminal

procedures :

Complete necropsies
Microscopic examinations.
10

Samples for hematologic analysis.

Hematocrit, Hemoglobin
Red blood cell count, Platelet count,
Absolute reticulocyte count, Absolute
differential leukocyte count,
Total leukocyte count, Red blood cell
distribution width
Mean corpuscular volume, Mean corpuscular
hemoglobin and
Mean corpuscular hemoglobin concentration.

11

Samples for serum clinical chemistry

Alanine aminotransferase, Alkaline
phosphatase
Aspartate aminotransferase, Albumin,
Calcium, Chloride,Cholesterol, Creatinine,
Glucose, Inorganic phosphorus, Lactate,
potassium,
Sodium, Triglycerides, Total protein, Total
bilirubin, Urea
Nitrogen, TCO2 (HCO3) and Beta
hydroxybutyric acid (-HBA).

12

Urine chemistry analysis

creatinine
excretion(total
amount
per
collection period), sodium excretion, chloride
excretion, potassium excretion, protein
excretion, glucose excretion,
calcium excretion, phosphorus excretion,
sodium/creatinine ratio,
chloride/creatinine ratio, potassium/creatinine
ratio, protein/creatinineratio,
glucose/creatinine ratio, calcium/creatinine
ratio and phosphorus/creatinine ratio.

13

Results

Mortality and in-life observations

14

15

Toxicokinetics

16

17

Clinical pathology

18

Urinalysis

19

Pathology

20

21

Discussion

600 mg/kg/day exhibited clinical signs of toxicity that

included a hunched posture and/or thin appearance,
few/nonformed feces, rough/red/yellow haircoat and
partially closed eyes.
In females, mortality occurred at doses 900 mg/kg/day. So
doses 600 mg/kg/day were considered adverse.
Doses 600 mg/kg/day was associated with effects on
glucose and lactate metabolism. Specifically metformininduced decrease in gluconeogenesis, glucose absorption
and lactate utilization coupled with an increase in glucose
utilization.
Minimal metabolic acidosis characterized by increased
serum lactate and beta-hydroxybutyric acid and decreased
serum bicarbonate and urine pH.
22

Minimally decreased urine glucose excretion

and minimal to mild increase in serum
triglycerides, betahydroxybutyric acid and
serum transaminases.
Epithelial inflammation and/or necrosis and
duct epithelial hypertrophy with or without
decreased cytoplasmic eosinophilic granules.
increased minimal necrosis with inflammation
of the parotid salivary gland in males given
metformin at 1200 mg/kg/day was considered
adverse

23

Oral administration of metformin at doses of 200,

600,900 and 1200 mg/kg/day in male and female
Crl:CD(SD) rats resulted in the mortality of 4 females
given 900 mg/kg/day.
Increased minimal necrosis with inflammation of the
parotid salivary gland for males given 1200
mg/kg/day.
clinical signs (hunched/thin appearance and frequent
nonformed feces) associated with moribund condition
at higher doses in both male and female rats given
600 mg/kg/day
No observable adverse effect level (NOAEL) was
200 mg/kg/day
mean day 89 AUC024 and Cmax (gender averaged)
24
at this dose was 41.1 g h/mL and 10.3 g/mL.

Conclusion :
Administration of 900 mg/kg/day resulted in
moribundity/mortality and clinical signs of
toxicity.
Males given 1200 mg/kg/day- minimal
necrosis with minimal to slight inflammation
of the parotid salivary gland.
600 mg/kg/day - body weight loss and
clinical signs.
No significant sex differences in mean
AUC024 or Cmax.
No observable adverse effect level (NOAEL)
was 200 mg/kg/day.

25

Thank you

26