Metformin hydrochloride

Endpoint summary

Description of key information

Metformin hydrochloride was tested in a chronic oral toxicity study in mice, which was performed according to OECD 452 and under GLP regulation. From this study, a NOAEL of 450 mg/kg bw/day was established based on effects on body weight and cystic tubular dilatation in the kidney.

In another non-GLP repeat study, rats were daily treated with metformine over a period of 26 weeks Based on the effects observed in this study (clinical signs, body weight reduction, kidneys), a NOAEL value of 300 mg/kg bw/day could be established. Despite not performed under GLP, this study is acceptable to support the assessment of the toxicity of Metformin hydrochloride.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1990-11-13 to 1992-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

adopted 12 May 1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.30 (Chronic Toxicity Studies)

Version / remarks:

01 July 1990

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Inc., Portage, Michigan
- Age at study initiation: 4 weeks
- Weight at study initiation: 23.9 - 32.7 g (M), 16.0 - 26.1 g (F)
- Fasting period before study: no
- Housing: two per cage of the same sex in stainless-steel, hanging, wire-mesh cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days prior to initiation of treatment

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Photoperiod (hrs dark / hrs light): 12/12 hours

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): Purina Certified Rodent Chow
- Storage temperature of food: room temperature in a sealed container

The test item was reported as 99.9% active but was adjusted to 100% purity for the purpose of dosage calculations. For each dietary level, the test material and basal feed were weighed on an appropriate balance (mg) in series with an Epson computer with printout tape. Each level was prepared by placing the weighed test material into approximately 200 g of basal feed and mixing in a Waring blender until an apparent homogeneous mixture was achieved.
The premixes were transferred to a Patterson-Kelly twin-shell mixer (fitted with an intensifier bar) containing the remaining required amount of basal feed. The diets were mixed for approximately 1 minute/kg with a minimum mixing time of at least 10 minutes.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability - Prior to initiation of compound administration, samples from the low- and high-dose formulations (590.4580 and 10362.2047 ppm) were analyzed to assess 0, 7, and 10-day stability at room temperature.

Homogeneity - Evaluation for homogeneity was performed on the low- and high-dose formulations prior to initiation of test material exposure. Duplicate samples from the top, middle, and bottom of the mixed batches were analyzed for concentration of the test material.

Concentration Analyses - Duplicate samples were taken of each dietary concentration from Weeks 1, 2, 3, 4, 8, 13, 26, 39, and 52. One sample of each level was analyzed for concentration of the test material. The other sample was retained frozen for possible analysis.

Analytical Method - Stability and routine concentration analyses were performed using methanol extraction and HPLC (Waters, Column: Waters Bondapak C18, 30 cm x 3.9 mm, UV detection: 230 nm, Injection volume: 5 µL, Flow: 1.5 mL/min, Mobile phase: 900 mL aqueous sodium pentanesulfonate solution (0.096%) + 100 mL methanol + 2 g potassium phosphate, pH adjustment to pH 3.5-4.0 with 50% o-phosphoric acid)

Duration of treatment / exposure:

52 weeks

Frequency of treatment:

daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1 (Control)

Dose / conc.:

150 mg/kg bw/day (nominal)

Remarks:

Group 2 (low dose)

Dose / conc.:

450 mg/kg bw/day (nominal)

Remarks:

Group 3 (mid dose)

Dose / conc.:

1 500 mg/kg bw/day (nominal)

Remarks:

Group 4 (high dose)

No. of animals per sex per dose:

Group 1: 0 mg/kg bw: 42 (m), 42 (f)
Group 2: 150 mg/kg bw: 42 (m), 42 (f)
Group 3: 450 mg/kg bw: 42 (m), 42 (f)
Group 4: 1500 mg/kg bw: 42 (m), 42 (f)

Control animals:

yes, concurrent no treatment

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly for Weeks 1 - 16, and once every fourth week thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Individual food consumption values were recorded at randomization, prior to treatment (Week -1), weekly for Weeks 1 - 16, and once every fourth week thereafter.


OPHTHALMOSCOPIC EXAMINATION: Yes
An indirect ophthalmoscopic examination was performed on all animals prior to treatment (week -1) and prior to their scheduled sacrifice (Weeks 13, 26 or 52), using 1% Mydriacyl® as the mydriatic agent.

- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Weeks 14, 27, and 53 on ten animals/sex/group
- Anaesthetic used for blood collection: No
- Parameters:
corrected leukocyte count (COR WBC)
hemoglobin (HGB)
leukocyte count (WBC)
erythrocyte count (RBC)
leukocyte differential
hematocrit (HCT)
platelet (PLATELET)
cell morphology

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Weeks 14, 27, and 53 on ten animals/sex/group
- Animals fasted: No
- How many animals: 10 / sex / group
- Parameters
alanine aminotransferase (ALT)
albumin (ALBUMIN)
albumin/globulin ratio (A/G)
alkaline phosphatase (ALK P)
aspartate aminotransferase (AST)
blood urea nitrogen (BUN)
gamma glutamyltransferase (GGT)
globulin (GLOBULIN)
glucose (GLUCOSE)
total bilirubin (T BILI)
total protein (T PROT)

URINALYSIS: Yes
- Time schedule for collection of urine: Weeks 14, 27, and 53 on ten animals/sex/group
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters
appearance (UTRANS, UCOLOR)
glucose (GLUCOSE)
ketones (KETONES)
microscopic examination of sediment
occult blood (OC BLD-U)
protein (PROTEIN)
specific gravity (SP GR)
urine volume (U VOL)

Sacrifice and pathology:

GROSS PATHOLOGY
All animals which were found dead or sacrificed in extremis were subjected to a gross postmortem examination. All surviving animals were weighed the day of scheduled necropsy, fasted overnight, weighed, anesthetized with sodium pentobarbital (injected intraperitoneally), exsanguinated, and grossly examined. Necropsies were performed on all animals by appropriately trained personnel using procedures approved by board-certified pathologists. A veterinary pathologist was available for confirmation of any unusual findings. Necropsies included examination of the following:
all orifices
carcass
cervical tissues and organs
cranial cavity
external surface of the body
external surface of the brain (at necropsy); the external surface of the spinal cord and cut surfaces of the brain and spinal cord were examined at the time of tissue trimming.
nasal cavity and paranasal sinuses
thoracic, abdominal and pelvic cavities and their viscera

ORGAN WEIGHTS
Organ Weights - At the Week 53 terminal sacrifice, the following organs (when present) were weighed after careful dissection and trimming of fat and other contiguous tissue:
adrenals (weighed postfixation)
brain (including brainstem)
heart
kidneys
liver (with gallbladder)
ovaries
pituitary (weighed postfixation)
prostate
spleen
testes
uterus
thyroids/parathyroids (weighed postfixation)

Paired organs were weighed separately. Organ-to-terminal-body-weight and organ-to-brain-weight ratios were calculated.

TISSUE PRESERVATION
The following tissues (when present) from each animal were preserved in 10% neutral-buffered formalin:
adrenals
aorta
brain with brainstem (medulla/ pons, cerebellar cortex, and cerebral cortex)
cervical spinal cord
colon, cecum, rectum
duodenum, jejunum, ileum esophagus
eyes
femur (including joint)
heart
kidneys
lesions
liver (with gallbladder)
lumbar spinal cord
lung
mammary gland (females only)
mandibular lymph node
masses and associated tissues
mesenteric lymph node
mid-thoracic spinal cord
ovaries
pancreas
pituitary
prostate
salivary glands (mandibular)
sciatic nerve
seminal vesicles
skeletal muscle
skin
spleen
stomach
sternum with bone marrow
testes with epididymides
thymus
thyroid/parathyroids
trachea
urinary bladder
uterus with vagina and cervix

HISTOPATHOLOGY
All preserved tissues were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically from the terminally-sacrificed animals in Groups 1 (control) and 4 (high-dose) and from all unscheduled deaths. In addition, all grossly visible tumors and lesions were microscopically examined from all animals. Histopathologic evaluation revealed abnormalities in the kidney at the high-dose level. As a result, the kidneys from all Group 2 and 3 animals were subjected to microscopic evaluation.

Statistics:

Mean absolute body weight data (Weeks 0 through 16, 20, 24, 28, 32, 36, 40, 44, 48, and 52); mean body weight change data (Weeks 0-28 and 0-52); mean absolute food consumption data (Weeks 1 through 16, 20, 24, 28, 32, 36, 40, 44, 48, and 52); total food consumption data (Weeks 1-28 and 1-52); clinical pathology data (except cell morphology gradings and urinalysis); and organ weight data of the control group were compared statistically to the data from the same sex of the treated groups. Statistical analyses were performed using Anova tests and related methods.

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs observed at weekly physical examinations were incidental in nature and without relation to administration of the test material. None of the deaths could be directly related to the administration of the test item. In some animals, systemic infectious processes were responsible for their death. The possible cause of death was not apparent morphologically in others.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

A total of six animals died during the study: one low dose male, one high dose male, two mid dose females and two high dose females. The mortality observed was evaluated as not treatment-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

A treatment-related depression of body weight was displayed for Group 4 males, as evidenced by significant decreases in mean absolute body weight values for Weeks 14, 15, 16, 20, 24, 28, 32, 36, 40, 48, and 52 (ranging from 5.3 to 7.6% of the concurrent control value). Significantly decreased mean body weight changes were also observed for the Group 4 males at Weeks 0-28 and 0-52 relative to the control data. In addition, the Group 4 females exhibited significantly decreased mean absolute body weight values for Weeks 14, 24, 40, and 52 (ranging from 5.9 to 10.9% of the concurrent control value).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Alterations of food consumption were not reflective of test item exposure since increases were spurious and not related to dose. Significantly increased mean food consumption values were exhibited for the Group 2 males at Weeks 4, 8, 10, 12, and 15; the Group 3 males at Week 5; the Group 4 males at Weeks 5 and 8; the Group 2 females at Weeks 6, 11, and 16; and the Group 4 females at Week 48 compared to respective controls. All other mean food consumption values were similar between treated and respective control groups.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no test material-related ophthalmoscopic abnormalities noted at the Week 13, 26, or 52 examinations.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The mean values for erythrocyte count and hemoglobin concentration were elevated in a dose-related manner for treated males, significantly for Group 4 males at Weeks 14, 27, and/or 53.
In addition, the mean hematocrit value was significantly increased for Group 4 (1,500 mg/kg bw/day) males at Week 27, but appeared to be dose-related only at Week 53 (without statistical significance). These changes were considered to be of negligible biologic importance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Some significant changes observed for a few of the serum chemistry parameters (Week 14: decreased blood urea nitrogen in Group 3 females,; decreased total protein and decreased total bilirubin in Group 4 males; Week 27: increased AST in Group 4 females) were not considered to be toxicologically relevant.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Urinalysis data were generally unremarkable between control and treated groups at all intervals.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

The mean brain weights were increased in males of Group 2 (low dose) and Group 3 (mid dose), however, not in females and not in the high dose males. This finding is considered to be incidental.
Increased organ / body weight ratios were observed in Group 4 males (kidneys, brain, Testes) and Group 4 females (kidneys, liver) which is secondary to reduced body weight and no direct effect on organs.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Dietary administration of the test item at dosage levels of 1500 mg/kg bw/day for 52 weeks to mice was associated with (1) an increased incidence and severity of cystic tubular dilatation of the kidney of male and female mice and (2) an increased incidence and severity of increased tubular vacuolization of the kidneys in male mice.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival was generally comparable between control and treated groups. A total of six animals died during the study: one Group 2 male, one Group 4 male, two Group 3 females and two Group 4 females. One Group 3 female that died during Week 30 was considered an accidental death. All other animals survived until their scheduled termination. The clinical signs noted during the study did not reveal any indication of a treatment effect.

BODY WEIGHT AND WEIGHT GAIN
Significantly decreased mean body weight values were exhibited for Group 4 males at Weeks 14, 15, 16, 20, 24, 28, 32, 36, 40, 48, and 52, ranging from 5.3 to 7.6% of the concurrent control values. Additional significant decreases in mean body weight change data for the Group 4 males during Weeks 0-28 and 0-52 were observed. In addition, the Group 4 females displayed lower, but usually nonsignificant, mean body weight values relative to respective controls beginning at approximately Week 14 and continuing for the remainder of the study. All statistically significant differences in mean food consumption data for the treated groups were considered to be unrelated to administration of the test material.


OPHTHALMOSCOPIC EXAMINATION
Ophthalmoscopic examinations failed to reveal any potential compound-related changes.

HAEMATOLOGY
Significant compound-related and dose-related increases were present in mean erythrocyte counts for the treated males of Group 4 at Weeks 14, 27, and 53; generally accompanied by increases in mean hemoglobin concentration and hematocrit also significant in Group 4. In addition, a slight, but significant decrease in the mean eosinophil count for the Group 4 males at Week 14 was present relative to controls.

CLINICAL CHEMISTRY & URINALYSIS
No indication of a compound effect was noted in the serum chemistry or urinalysis data.

ORGAN WEIGHTS & GROSS PATHOLOGY
No evidence of test-material-related alterations was present in the gross pathology data of the animals that died or were sacrificed during Weeks 14, 27, or 53. The significant increases observed in the mean organ-to-terminal-body-weight values for animals sacrificed at Week 53 were attributed to the corresponding decreased terminal body weight values. No microscopic correlation for the mean organ weight differences between control and treated groups could be determined.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histomorphologic evaluation revealed a compound-related increase in the severity and incidence of cystic tubular dilatation in the kidneys of the Group 4 males and females and an increased incidence and severity of tubular vacuolization in the kidneys of the Group 4 males. No other treatment-related changes were evident in any of the tissues examined.

Key result

Dose descriptor:

NOAEL

Effect level:

450 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 500 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Conclusions:

Based upon the results of this feeding study, daily administration of the test item to mice for at least 52 consecutive weeks was well tolerated at a dose of 450 mg/kg bw/day; while a histomorphologic alteration in the kidneys was observed in mice following at least 52 weeks of administration of 1500 mg/kg bw/day of the test item.

Executive summary:

Study Design

This study was designed to characterize the potential chronic toxicity of the test material when administered daily in the diet of Cr1:CD®-1 (ICR)BR mice for at least 52 consecutive weeks.

During Weeks 14 (first interim sacrifice) and 27 (second    interim    sacrifice),    ten    animals/sex/group/interval   were sacrificed. All surviving animals were sacrificed during Week 53. A total of 336 animals (168/sex) was utilized and assigned to the study as follows:

Group 1: 0 mg/kg bw: 42 (m), 42 (f)
Group 2: 150 mg/kg bw: 42 (m), 42 (f)
Group 3: 450 mg/kg bw: 42 (m), 42 (f)
Group 4: 1500 mg/kg bw: 42 (m), 42 (f)

During the course of the study, criteria evaluated for compound effect included survival; daily clinical signs; physical examinations (detailed clinical observations); ophthalmoscopic examinations (once pretreatment and during Weeks 13, 26, and 52); body weight and food consumption data; clinical pathology data; organ weight data; and gross and microscopic examinations of selected tissues.

Clinical pathology parameters evaluated included hematology, serum chemistry, and urinalysis during Weeks 14, 27, and 53 for ten animals/sex/group; following each bleed interval the animals were appropriately sacrificed.

Results

Survival was generally comparable between control and treated groups. A total of six animals died during the study: one Group 2 male, one Group 4 male, two Group 3 females and two Group 4 females. All other animals survived until their scheduled termination. The deaths observed were not considered to be treatment-related.

The clinical signs noted during the study did not reveal any indication of a treatment effect. Tissue masses discovered during the study were noted spuriously throughout the male groups with no relationship to dose level or test material. No tissue masses were evident among the control or treated females.

Significantly decreased mean body weight values were exhibited for Group 4 males at Weeks 14, 15, 16, 20, 24, 28, 32, 36, 40, 48, and 52, ranging from 5.3 to 7.6% of the concurrent control values. Additional significant decreases in mean body weight change data for the Group 4 males during Weeks 0-28 and 0-52 were observed. In addition, the Group 4 females displayed lower, but usually nonsignificant, mean body weight values relative to respective controls beginning at approximately Week 14 and continuing for the remainder of the study. All statistically significant differences in mean food consumption data for the treated groups were considered to be unrelated to administration of the test material.

Ophthalmoscopic examinations failed to reveal any potential compound-related changes.

Compound-related and dose-related increases were present in mean erythrocyte counts for the treated males at Weeks 14, 27, and 53; generally accompanied by increases in mean hemoglobin concentration and hematocrit. In addition, a slight, but significant decrease in the mean eosinophil count for the Group 4 males at Week 14 was present relative to controls. There were no other remarkable hematology findings, and no indication of a compound effect was noted in the serum chemistry or urinalysis data.

No evidence of test-material-related alterations was present in the gross pathology data of the animals that died or were sacrificed during Weeks 14, 27, or 53. The significant increases observed in the mean organ-to-terminal-body-weight values for animals sacrificed at Week 53 were attributed to the corresponding decreased terminal body weight values. No microscopic correlation for the mean organ weight differences between control and treated groups could be determined.

Histomorphologic evaluation revealed a compound-related increase in the severity and incidence of cystic tubular dilatation in the kidneys of the Group 4 males and females and an increased incidence and severity of tubular vacuolization in the kidneys of the Group 4 males. No other treatment-related changes were evident in any of the tissues examined.

Conclusion

Based upon the results of this study, daily administration of the test item to mice for at least 52 consecutive weeks was well tolerated at a dose level of 450 mg/kg bw/day; while a histomorphologic alteration in the kidneys was observed in mice following at least 52 weeks of administration of 1500 mg/kg bw/day of the test item.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1968 - 1969

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

yes

Remarks:

only 15 animals per sex and dose, exposure only 6 months, the identity of the test item is not completely clear (salt form not mentioned)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

other: Charles River Caesarean derived albino strain

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Weight at study initiation: 136 - 210 g (M), 133 - 160 g (F)
- Fasting period before study: no
- Housing: individually in elevated wire-mesh cages
- Diet: ad libitum (Purina laboratory chow)
- Water: ad libitum

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
The test item was incorporated into the basal diet on a weight-per-weight basis and thoroughly mixed in a twin-shell, Patterson-Kelley blender. Fresh diets were prepared weekly. Dosages were adjusted weekly on the basis of group mean body weight and food consumption determined for each sex at the end of the preceding week.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

6 month

Frequency of treatment:

7d/week

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Control

Dose / conc.:

120 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

900 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Group 1: 0 mg/kg bw: 15 (m), 15 (f)
Group 2: 120 mg/kg bw: 15 (m), 15 (f)
Group 3: 300 mg/kg bw: 15 (m), 15 (f)
Group 4: 600 mg/kg bw: 15 (m), 15 (f)
Group 5: 900 mg/kg bw: 15 (m), 15 (f)

Control animals:

yes, plain diet

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: weekly


OPHTHALMOSCOPIC EXAMINATION: Yes in Month 1 and 6
An indirect ophthalmoscopic examination was performed on all animals prior to treatment (week -1) and prior to their scheduled sacrifice, using 1% Mydriacyl® as the mydriatic agent.
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Month 1, 3 and 6
- Anaesthetic used for blood collection: No
- Parameters
corrected leukocyte count (COR WBC)
hemoglobin (HGB)
leukocyte count (WBC)
erythrocyte count (RBC)
leukocyte differential
hematocrit (HCT)
clotting time


CLINICAL CHEMISTRY: Yes
Blood urea nitrogen
fasting blood sugar
serum glutamic-pyruvic transaminase (GPT)
serum alkaline phosphatase
total serum bilirubin
total serum protein
serum elektrophoresis

URINALYSIS: Yes
- Time schedule for collection of urine: Month 6
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters
appearance (UTRANS, UCOLOR)
occult blood (OC BLD-U)
glucose (GLUCOSE)
protein (PROTEIN)
ketones (KETONES)
specific gravity (SP GR)
microscopic examination of urine volume (U VOL)
sediment

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
brain, pituitary glands, eye, thoratic spinal cord, thyroid, lung, heart, liver, spleen, kidney, adrenal, stomach, pancreas, small intestine, large intestine, mesenteric lymph node, urinary bladder, testis, prostate, seminal vesicle, ovary, uterus, skin, bone (rib junction), bone marrow (sternum), nerve with muscle, mammary tissue, and any unusual lesions

ORGAN WEIGHTS
- prior fixation: heart, liver, spleen, pancreas, kidneys, testes, prostate, seminal vesicles, uterus
- after fixation: thyroid, adrenals, ovaries
Organ-to-terminal-body-weight ratios were calculated.

HISTOPATHOLOGY: Yes
from 5 males and 5 females from Group 1 (Control) and Group 5 (900 mg/kg bw/day):
Brain (three sections), pituitary, eye, thyroid, lung, heart, liver, spleen, kidney, adrenal, stomach, pancreas, small intestine, large intestine, mesenteric lymph node, urinary bladder, testis, prostate, seminal vesicle, ovary, uterus, bone marrow, mammary tissue, and unusual lesions

from 5 males and 5 females from Group 2 (120 mg/kg bw/day), Group 3 (300 mg/kg bw/day) and Group 4 (600 mg/kg bw/day):
thyroid, liver, kidney, adrenal pancreas, testis, and ovary

Statistics:

Standard statistical methods applied for the analysis of growth rate, total food consumption, terminal body weights, organ weights, and organ/bw ratios.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The rats in the two lower test groups (120 and 300 mg/kg bw/day) showed no compound effect wich regard to physical appearance and behavior.
Signs of espiratory involvement (wheezing, a discharge from the nose or eyes, or reddened eyelids) or alopecia on ehe legs, paws, or body were noted in a few instance among the rats in the control group and all four test groups. During the first three months of the study, the majority of the rats in the 600 and 900 mg/kg bw/day test groups maintained a generally normal appearance and behavior similar to those of the controls. During the latter part of the study, several males and females in the 600 mg/kg bw/day group and about one-third of the males and the majority of the females in the 900 mg/kg bw/day group exhibited a hunched posture and showed very poor weight gains. In a few instances, staining of the abdominal fur by urine or sores on the tail were noted among these animals.

Mortality:

no mortality observed

Description (incidence):

Survival was 100% in all groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Growth for the males in the 120 mg/kg bw/day test group was comparable with that for the male control. Growth suppression to a dose-related degree was evident in the three higher male test groups. In the 300 mg/kg bw/day male group, growth through week 7 was comparable with the control. Statistical evaluation revealed the growth rate from week 7 until termination for the 300 and 600 mg/kg bw/day males and that for the high level (900 mg/kg bw/day) males throughout the study to be significantly lower than the male controls.
The reduction of body weight at the end of the study for the 300 mg/kg bw/day group was 2.4% for the males and 10.7% for the females. This is not considered to be adverse.
A dose-related decrease in growth rate was evident in all four female test groups. Growth suppression was marked in the 900 mg/kg bw/day females but, by statistical analysis, not significantly different from that for the female controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for the 600 mg/kg bw/day male and 900 mg/kg male and female groups was somewhat lower than that for the respective controls but not to a significant degree, which is not considered adverse.
Food consumption was comparable among the remaining test groups and the controls.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No treatment-related effects observed.

Haematological findings:

no effects observed

Description (incidence and severity):

The hematological values for the test groups showed no significant differences from concrol values and no dose-related trends. Coagulation time determinations at one month for the 900 mg/kg female group were somewhat higher than the concrols but were comparable at the three- and six-month interval. Except for occasional high or low individual values obtained for both control and test rats, the hematological values were generally within normal range for laboratory rats and comparable among the various test groups and the controls.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Alkaline phosphatase (at each interval) and serum glutamic-pyruvic transaminase determinations (at six months) for the high dose males (900 mg/kg bw/day) were somewhat lower compared with the male control but still within normal limits. An overall increase in blood sugar was evident at six months in all groups including the controls, but the values were highest for the 900 mg/kg bw/day male group. The mean sugar value for the 600 mg/kg bw/day male group was also increased due to one unusually high individual value. However, there is no statistical significance between these means and those of the controls, except for a specific comparison between the females at the 600 mg/kg level and the female controls, which comparison produces a significant t-test. The remaining biochemical values for the test groups were within normal limits and comparable with the respective controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The results of urine analyses for the test groups were within normal limits and comparable with the respective controls.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Observations at necropsy revealed no consistent gross alterations in the organs of the test rats. Significantly increased organ/body weight ratios determined for the rats at the two higher test levels were considered to
reflect the suppressed growth in these groups. There were no other meaningful differences between the organ weight data for the test groups and those for the controls.
The significantly decreased thyroid and spleen weights for the high level male group were not considered to be meaningful since to compound-induced, histologic alterations were found in these organs.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopically, the kidneys of the high level females (900 mg/kg bw/day) revealed a slightly increased degree of cytoplasmic vacuolation in the epithelial lining of the deeper cortical tubules compared with that noted in controls and lower level test rats. As degenerative or necrotic changes associated with vacuolated cells were not observed in the affected female rats, this observation is considered not relevant/adverse.
The remaining tissues examined from the test rats were comparable with those from the concrols. Lesions of incidental or spontaneous diseases were observed; however, the severity and degree of these changes were not meaningfully influenced by the experimental regimen.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Survival was 100% in all groups.
The rats in the 120 and 300 mg/kg test groups showed no compound effect with regard to physical appearance and behavior. About one-third of the :hales and the majority of the females in the high level group (900 mg/kg) and several rats in the 600 mg/kg group- developed a hunched position during the latter part of the study and showed very poor weight gains. In several instances, staining of the abdominal fur by urine was noted among these animals.

BODY WEIGHT AND WEIGHT GAIN
Growth for the males in the 120 mg/kg test group was comparable with that for the male control. Growth suppression to a dose-related degree was evident in the three higher male test groups. In the 300 mg/kg female group, growth through :week 7 was comparable with the control. Statistical evaluation revealed the growth race from week 7 until termination for the 300 and 600 mg/kg males and that for the high level (900 mg/kg) males throughout the study to be significantly lower than the male controls. A dose-related decrease in growth rate was evident in all four female test groups. Growth suppression was marked in the 900 mg/kg females but, by statistical analysis, not significantly different from that for the female controls.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food consumption for the 600 mg/kg male and 900 mg/kg male and female groups was somewhat lower than that for the respective controls but not to a significant degree. Food consumption was comparable among the remaining test groups and the controls.


OPHTHALMOSCOPIC EXAMINATION
Gross ophthalmoscopic examination at initiation of the study revealed minor eye abnormalities in seven male rats on the study. At the terminal examination, moderate opacity of the lens was noted in the left eye of one of these rats (male Rat No. 73-916 - Group No. 4, 600 mg/kg); the eyes of the other six rats had a normal gross appearance. The following gross eye abnormalities were observed in one female and three male rats at the terminal examination only; at initiation, no gross eye changes had been noted in these animals.


HAEMATOLOGY
The hematological values for the test groups showed no significant differences from those for the control groups. At termination (six-month sacrifice), elevated blood sugar values were obtained for all groups including the control; however, the highest group mean values were determined for the 600 and 900 mg/kg males. The results of urine analyses were comparable among the test and control groups.

CLINICAL CHEMISTRY
Alkaline phosphatase (at each interval) and SG-PT determinations (at six months) for the high level males (900 mg/kg) were somewhat lower ccmpared with the male control but still within normal limits. An overall increase in blood sugar was evident at six months in all groups including the controls, but the values were highest for the 900 mg/kg male group. The mean sugar value for the 600 mg/kg male group was also increased due to one unusually high individual value. However, there is no statistical significance between these means and those of the controls, except for a specific comparison between the females at the 600 mg/kg level and the female controls, which comparison produces a significant t-test. The remaining biochemical values for the test groups were within normal limits and comparable with the respective controls.

URINALYSIS
The results of urine analyses for the test groups were within normal limits.

ORGAN WEIGHTS
The significantly increased organ/body weight ratios determined for the males and females at the 600 and 900 mg/kg levels and, in some instances, also for the 300 mg/kg female group are, most likely, a reflection of the significantly suppressed body weights in these groups. The weights of the involved organs were comparable with the controls or only slightly lower than the control values. The significantly decreased thyroid and spleen weights for the high level male group were not considered to be meaningful since to compound-induced, histologic alterations were found in these organs.

Key result

Dose descriptor:

NOEL

Effect level:

120 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Conclusions:

Based on the results of this 6-months oral toxicity study with Dimethyldiguanide (Metformin) in rats, it is concluded that a dose of 120 mg/kg bw/day is a NOEL for Dimethyldiguanide and that a dose of 300 mg/kg bw/day is the NOAEL due to minimal effects on the body weight of females.

Executive summary:

Study Design

This study was designed to characterize the potential chronic toxicity of a test material, Dimethyldiguanide (Metformin), when administered daily in the diet of albino rats for 6 months.
A total of 60 animals (15/sex) was utilized and assigned to the study as follows:

Group 1: 0 mg/kg bw/day: 15 (m), 15 (f)
Group 2: 120 mg/kg bw/day: 15 (m), 15 (f)
Group 3: 300 mg/kg bw/day: 15 (m), 15 (f)
Group 4: 600 mg/kg bw/day: 15 (m), 15 (f)

Group 9: 900 mg/kg bw/day: 15 (m), 15 (f)

Criteria for evaluation of a compound effect were physical appearance and behavior, body weight and body weight development, food consumption, survival, clinical laboratory values, organ weights, and gross and microscopic pathology.

Results

The rats in the 120 and 300 mg/kg bw/day test groups showed no compound effect with regard to physical appearance and behavior. About one-third of the males and the majority of the females in the high level group (900 mg/kg bw/day) and several rats in the 600 mg/kg bw/day group developed a hunched position during the latter part of the study and showed very poor weight gains. In several instances, staining of the abdominal fur by urine was noted among these animals.

Growth from Week 7 until termination for the 300 mg/kg bw/day was slightly reduced to max. 10%. Body weight development for 600 mg/kg bw/day male groups and that for the 900 mg/kg bw/day males throughout the study was significantly suppressed. Growth in the 900 mg/kg females was markedly suppressed but not to a statistically significant degree. A dose-related decrease in growth rate was also evident in the three remaining female test groups. Food consumption for both sexes at the 600 and 900 mg/kg test levels was lower than, but not significantly different from, that of the controls.

Survival was 100% in all groups.

The hematological values for the test groups showed no significant differences from those for the control groups. At termination (six-month sacrifice), elevated blood sugar values were obtained for all groups including the control; however, the highest group mean values were determined for the 600 and 900 mg/kg males. The results of urine analyses were comparable among the test and control groups.

Observations at necropsy revealed no consistent gross alterations in the organs of the test rats. Significantly increased organ/body weight ratios determined for the rats at the two higher test levels were considered to reflect the suppressed growth in these groups. There were no other meaningful differences between the organ weight data for the test groups and those for the controls.

Microscopically, the kidneys of the high level females (900 mg/kg) revealed a slightly increased degree of cytoplasmic vacuolation in the epithelial lining of the deeper cortical tubules compared with that noted in controls and lower level test rats. Degenerative or necrotic changes associated with vacuolated cells were not observed in the affected female rats and the significance of the slightly increased vacuolation is not known. The remaining tissues examined from the test rats were comparable with those from the controls. Lesions of incidental or spontaneous diseases were observed; however, the severity and degree of these changes were not meaningfully influenced by the experimental regimen.

Conclusion

It is concluded that the 120 mg/kg bw/day dose level in rats is a "no-effect" level (NOEL) for Dimethyldiguanide and that a minimal effect occurred at the 300 mg/kg/day level, mainly body weight suppression. Consequently, a dose of 300 mg/kg bw/day is regarded as the NOAEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

good quality

System:

urinary

Organ:

kidney

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, metformin hydrochlorid must not be classified with regard to specific target organ toxicity after repeated administration of the test item.