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Diss Factsheets

Toxicological information

Repeated dose toxicity: oral

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Metformin hydrochloride
EC Number:
214-230-6
EC Name:
Metformin hydrochloride
Cas Number:
1115-70-4
Molecular formula:
C4H11N5.ClH
IUPAC Name:
N,N-dimethylimidodicarbonimidic diamide hydrochloride
Test material form:
solid: bulk

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

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

Applicant's summary and conclusion

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.