Registration Dossier
Registration Dossier
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EC number: 214-230-6 | CAS number: 1115-70-4
Endpoint summary
Administrative data
Key value for chemical safety assessment
- Toxic effect type:
- dose-dependent
Effects on fertility
Description of key information
Possible effects of Metformin hydrochloride on fertility and reproductive toxicity was investigated in a well described 1-generation study in rats, performed under GLP regulation. In this study, no effects on male and female fertility or reproductive toxicity was observed up to 600 mg/kg bw/day, i.e. the highest dose tested. Moreover, no adverse effects on reproductive toxicity were observed in a perinatal study in rats. In repeat dose studies, no adverse effects were observed on reproductive organs from rodents treated orally up to 52 weeks with metformin hydrochloride.
Link to relevant study records
- Endpoint:
- one-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1984-04-16 to 1984-11-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]
- Version / remarks:
- 26 May 1983
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: FDA Segment I study
- Qualifier:
- according to guideline
- Guideline:
- other: ICH S5 Detection of Toxicity to Reproduction for Medicinal Products
- Version / remarks:
- March 1994
- Deviations:
- no
- Principles of method if other than guideline:
- The design of the the study follows the Guidelines for Reproductive Toxicity Testing of Medicinal Products. The parameters investigated reflects the requirements of OECD 421 and OECD 415.
- Principle of test: Fertility Study in Rats
- Short description of test conditions: The potential effect of Metformin on fertility and reproductive performance was evaluated in Sprague-Dawley rats. Twelve males and 24 females were given daily oral doses of 120, 300 or 600 mg/kg body weight of the drug, beginning nine and two weeks, respectively before mating. Daily dose administration continued to study termination at weaning. A control group, also consisting of 12 males and 24 females, was given distilled-deionized water under the requisite experimental conditions.
- Parameters analysed / observed:The animals were observed daily, and body weight recorded weekly prior to and during cohabitation.
The dams were observed daily for physical and behavioral abnormalities during gestation, parturition and lactation. Individual body weights were recorded on gestation days 0, 6, 14 and 19. Approximately one-half of the pregnant females were euthanized on gestation day 14 and subjected to gross necropsy. The number of implantations, viable fetuses, resorptions and corpora lutea were recorded. Remaining females were allowed to deliver. Dam body weights were recorded on lactation days 0, 7, 14 and 21. All litters were observed daily to determine the viability, general appearance and physical development of the offspring.
Offspring body weights were determined on lactation days 0, 7, 14 and 21. The sex ratio of each litter was determined on days 0, 4, 14, and 21 of lactation. The dams and their offspring were subjected to
gross necropsy on day 21 postpartum or, for dams not delivering litters, at least 23 days after the final day of cohabitation.
All lesions or physical anomalies were recorded. The genitourinary tract of the dams was carefully examined for identification and quantification of implantation scars. The sex of each pup was confirmed by gonadal inspection. Males were euthanized at conclusion of the cohabitation period and examined grossly. The testes of all males were weighed and examined microscopically. - GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Labs
- Age at study initiation: 5 weeks (m) and 12 weeks (f) @ start of dosing
- Weight at study initiation: (P) Males: 165 +/- 9 g; Females: 141 +/- 6 g
- Fasting period before study: no
- Housing: stainless steel wire mesh bottom cages; polypropylene cages with hardwood chips as bedding for females after mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- according to "Guide for the Care and Use of Laboratory Animals"
- Air changes (per hr): 15-18
- Photoperiod (hrs dark / hrs light): 12 / 12 hours - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: adjusted
- Amount of vehicle: 2.5 mL/kg bw
- Purity: distilled
- Gavage solutions were prepared fresh weekly and dispensed as needed for daily dosing. The test article and gavage solutions were stored at room temperature in light-protective containers. - Details on mating procedure:
- - M/F ratio per cage: 1 / 2
- Length of cohabitation: 10 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After 10 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
- Any other deviations from standard protocol: no - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The stability of Metformin in gavage solutions was verified prior to study initiation. Solutions containing 48, 120 and 240 mg/ml Metformin were prepared and the test article concentration of each determined on the day of preparation and 7 and 14 days thereafter. In addition, the Metformin content in gavage solutions were verified prior to use.
UV Spectophotometry
Perkin Elmer Lambda 5 UV VIS spectrophotometer
Calibration with linear standard curve (R=0.99994)
Results (Mean concentrations of 3 measurements):
----------------------------------------------------------------
Dose Group Target Concentration Analytical Concentration
(mg/mL) (mg/mL)
----------------------------------------------------------------
A 0 -
B 48 48.7
C 120 122.8
D 240 241.2
----------------------------------------------------------------
Conclusion: Metformin concentration in gavage solutions was stable up to 14 days when stored at room temperature in light protective containers. Metformin concentrations were within 4% of the target level for all gavage solutions. - Duration of treatment / exposure:
- Twelve males and 24 females were given daily oral doses of 120, 300 or 600 mg/kg body weigth, beginning nine and two weeks, respectively before mating until lactation day 21 for the females.
- Frequency of treatment:
- daily
- Dose / conc.:
- 120 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- Dose / conc.:
- 600 mg/kg bw/day
- No. of animals per sex per dose:
- 12 (m), 24 (f)
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of males were recorded weekly. Individual body weights of females were recorded weekly prior
to insemination, on gestation days 0, 6, 14 and 19 and on lactation days 0 (as soon as possible after delivery), 7, 14 and 21. - Oestrous cyclicity (parental animals):
- not examined
- Sperm parameters (parental animals):
- not examined
- Litter observations:
- viability: daily
general appearance : daily
physical development: daily
Offspring body weights were determined on lactation days 0, 7, 14 and 21.
The sex ratio of each litter was determined on days 0, 4, 14 and 21 of lactation. - Postmortem examinations (parental animals):
- The dams and their offspring were subjected to gross necropsy on day 21 postpartum or, for dams not delivering litters, at least 23 days after the final day of cohabitation.
All lesions or physical anomalies were recorded.
The genito urinary tract of the dams was carefully examined for identification and quantification of implantation scars.
The sex of each pup was confirmed by gonadal inspection.
Males were euthanized at conclusion of the cohabitation period and examined grossly.
The testes of all males were weighed and examined microscopically.
The following data was recorded for females sacrifice at midgestation:
- number of corpora lutea
- number and distribution of viable fetuses
- number and distribution of resorptions
- number and distribution of all implantations - Postmortem examinations (offspring):
- offspring were subjected to gross necropsy on day 21 postpartum
- Statistics:
- Standard statistical methods have been applied for data processing.
- Reproductive indices:
- Fertility index, Litter Size, Litter Sex Ratio
- Offspring viability indices:
- Lacation index, Viability Index
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No findings attributable to Metformin treatment were noted.
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Description (incidence):
- No findings attributable to Metformin treatment were noted.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No significant differences in absolute body weight or body weight gain were noted among groups for either sex.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- No deviation from normal morphology was observed during histopathological evaluation of the testes.
- Histopathological findings: neoplastic:
- no effects observed
- Description (incidence and severity):
- No neoplastic ledions have been observed
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Cortical red spots, probably related to routine postmortem congestion; were noted in the kidneys of one to three males per group. The presence of a skin sore, which was noted during daily observations, was confirmed for a single mid-dose male. Slight,. unilateral enlargement of the testes was observed in one high-dose male. Microscopically, the enlarged gonad appeared normal and therefore, no biological importance is given to the gross observation.
Incidental findings, which occurred in one to two females per group (when present) included discoloration of the kidneys or lungs, hydropelvis, skin sore, firm subcutaneous tissue mass, "fluid-filled" gravid uterus and peritoneal (splenic) adhesions. - Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Description (incidence and severity):
- No statistically significant differences in the number of corpora lutea per pregnant female were noted among groups.
No statistically significant differences in the total number of implantation scars per dam occurred among groups.
No statistically significant differences in the number of resorptions per dam were noted among groups
No statistically significant differences in the number of viable, non viable or total fetuses present per dam were noted between the control and any test groups.
Uterine distribution of fetuses was comparable for all groups.
No statistically significant differences in the gestation index were demonstrated between the control and any test group.
The average length of gestation was comparable for all groups.
No statistically significant differences in the total number of offspring, number of offspring cast live or number of stillbirths per litter were noted between the control and Metformin-treated groups. Likewise, the percent of total pups per litter cast live or dead were similar for all groups. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 600 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No evidence for any Metformin-related effect upon general appearance or behavior of offspring was observed at any dose level studied.
- Mortality / viability:
- no mortality observed
- Description (incidence and severity):
- No mortality was observed during this study.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- No statistically significant differences in mean litter pup weight were noted between the control and Metformin treated groups.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- not examined
- Nipple retention in male pups:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No gross lesions related to Metformin treatment were noted for offsprings at any dose level studied.
- Histopathological findings:
- not examined
- Other effects:
- no effects observed
- Description (incidence and severity):
- No statistically significant differences in litter sex ratio were noted among groups on lactation days 4, 14 and 21. The mean average litter viability and lactation indices
ranged from 98.1-99.4 and 97.4-100.0, respectively for all groups. - Key result
- Dose descriptor:
- NOEL
- Generation:
- F1
- Effect level:
- 600 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- Under the conditions of this study, Metformin did not have any untoward effects upon fertility or reproductive performance at dosage levels of 120, 300 or 600 mg/kg body weight.
- Executive summary:
The potential effect of Metformin on fertility and reproductive performance was evaluated in Sprague-Dawley rats. Twelve males and 24 females were given daily oral doses of 120, 300 or 600 mg/kg body weight of the drug, beginning nine and two weeks, respectively before mating. Daily dose administration continued to study termination. A control group, also consisting of 12 males and 24 females, was given distilled-deionized water under the requisite experimental conditions.
The animals were observed daily, and body weight recorded weekly prior to and during cohabitation.
The dams were observed daily for physical and behavioral abnormalities during gestation, parturition and lactation. Individual body weights were recorded on gestation days 0, 6, 14 and 19. Approximately one-half of the pregnant females were euthanized on gestation day 14 and subjected to
gross necropsy. The number of implantations, viable fetuses, resorptions and corpora luteawererecorded. Remaining females were allowed to deliver. Dam body weights were recorded on lactation days 0, 7, 14 and 21. All litters were observed dailytodetermine the viability, general appearance and
physical development of the offspring.
Offspring body weights were determined on lactation days 0, 7, 14 and 21. The sex ratio of each litter was determined on days 0, 4, 14, and 21of lactation. The dams and their offspring were subjected to
gross necropsy on day 21 postpartum or, for dams not deliveringlitters, at least 23 days after the final day of cohabitation.
All lesions or physical anomalies were recorded. The genitourinary tract of the dams was carefully examined for identification and quantification of implantation scars. The sex of each pup was confirmed by gonadal inspection. Males were euthanized at conclusion of the cohabitation period and examined grossly.
The testes of all males were weighed and examined microscopically.
Daily, oral Metformin treatment at dosage levels of 120, 300 and 600 mg/kg body weight beginning nine and two weeks prior to mating for males and females, respectively did not have any adverse effect upon mating, fertility or reproductive performance in Sprague-Dawley rats. Continued Metformin treatment of the dams for 21 days postpartum had no untoward effect upon littersize,growth, development or viability at any dose level studied.
Furthermore, no treatment-related gross anomalies were noted in males, females or offspring at the designated dosage levels.
- Endpoint:
- reproductive toxicity, other
- Remarks:
- Segment III study
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1984-04-24 to 1984-11-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: ICH S5 Detection of Toxicity to Reproduction for Medicinal Products
- Qualifier:
- according to guideline
- Guideline:
- other: FDA, Segment III Study
- Principles of method if other than guideline:
- - Principle of test:
- Short description of test conditions: Pregnant rats were treated daily with the test item (3 dose levels) beginning on gestation day 15 until study termination at 21 days postpartum
- Parameters analysed / observed:
dams: physical or behavioral abnormalities during gestation, parturition and lactation,
body weights
gross necropsy
genito-urinary tract for implantation scars
pups/litters: viability,
general appearance and physical development,
body weights,
sex ratio,
gross necropsy - GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 100 days at start of dosing
- Weight at study initiation:
- Fasting period before study: no
- Housing: individual during acclimatisation and cohabited during mating.
- Housing: Stainless steel wire mesh bottom cages; polyprepylene cages with hardwood chips as bedding for females after mating .
- Diet: NIH open formula 07, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature
- Humidity (%): according to Guide for the Care and Use of Laboratory Animals
- Air changes (per hr): 15 - 18
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: adjusted
- Amount of vehicle: 2.5 mL/kg bw
- Purity: distilled
- Gavage solutions were prepared fresh weekly and dispensed as needed for daily dosing. The test article and gavage solutions were stored at room temperature in light-protective containers. - Details on mating procedure:
- The animals were randomly mated, one male to one female, within each treatment group. Vaginal lavage was conducted daily on all females during the mating period.The smears were microscopically examined and insemination confirmed by the presence of sperm therein. If insemination was not confirmed after ten days, the female was cohabited with a second male of proven fertility.
Males were euthanized and discarded, without necropsy , at conclusion of the mating period.
Inseminated fernales (dams) were transferred to nesting boxes for the gestation, parturition and lactation phases of the study.
The dams were observed daily and body weights periodically determined during gestation. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Perkin-Elmer Lambda 5 UV-VIS spectrophotometer
nominal concentration (mg/mL) analytical concentration (mg/mL)
48 48.7 +/- 1.0
120 121.8 +/- 3.4
240 240.8 +/- 6.5 - Duration of treatment / exposure:
- Daily dose administration (oral) was initiated for each dam on gestation day 15 and continued until study termination (lactation day 21). Dams and their litters were observed daily during the lactation phase of the study.
Specifically, general appearance of dams and offspring, body weight of dams and offspring, sex ratio and viability of offspring and deveiopment of the offspring were evaluated. - Frequency of treatment:
- Daily
- Dose / conc.:
- 120 mg/kg bw/day (nominal)
- Dose / conc.:
- 300 mg/kg bw/day (nominal)
- Dose / conc.:
- 600 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 20 pregnant females per dose
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before dosing
BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded once weekly during the mating phase. Individual body weights of females were recorded on gestation days 0, 6, 14 and 19 and on lactation days 0 (as soon as possible after delivery), 7, 14 and 21. Individual pup weights were determined on lactation days 0, 7, 14 and 21.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21 (of lactation)
- Organs examined: All dams and pups were euthanized (C02 gas) and subjected to gross necropsy on lactation day 21. The external body surface and orifices, internal body cavities and their organs and the carcass were examined.
All pups, except those cannabilized, were dissected and examined for gross anomalies including cleft lip/ palate, talpes, gastroschisis, exencephalacia, meningocele, accessory digits, spinal bifida, ablephera or other obvious malformations. The sex of each was confirrned by gonadal inspection. - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
PARAMETERS EXAMINED
The following parameters were examined in offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities
In addition to routine daily observation, the pups were further evaluated for achievement of the following criteria:
surface righting reflex (five seconds) pinna unfolding (both)
hair growth (first coat)
incisor eruption (upper and lower recorded separately)
eye opening (both)
Each litter was examined daily and the day upon which all pups achieved each criteria was recorded.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
Referenceopen allclose all
Effect on fertility: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 600 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
- Quality of whole database:
- The study is of high quality and useful for the assessment of hazard to fertility and reproduction in humans.
Effect on fertility: via inhalation route
- Endpoint conclusion:
- no study available
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
- Quality of whole database:
- All three studies are well described and were performed under GLP regulation.
Effects on developmental toxicity
Description of key information
Three developmental toxicity studies are available for metformin hydrochloride: Two teratogenicity studies (rat, rabbit) and one perinatal study in rats. The studies are well described, performed under GLP regulation and of high quality. They are well suited for assessing the risks for humans.
No teratogenicity or embryotoxicity toxicity was observed in pregnant animals (rats and rabbits) treated up to the highest dose of each study. The highest dose tested in the two rat studies (Teratogenicity study, perinatal study) was 600 mg/kg bw/day, respectively, which is the NOAEL for maternal and developmental toxicity in both studies.
In the rabbit, metformin produces a maternal toxicity starting at the lowest studied dose (50 mg/kg/day). Considerable mortality and clinical symptoms are noted. These observation might be explained with a specific sensitivity of this species and/or a metformin specific metabolism in this species. However, no developmental toxicity was observed in the offspring of rabbits and the developmental NOAEL was 140 mg/kg bw/day, i.e. the highest dose tested.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1984-04-24 to 1984-11-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- Principles of method if other than guideline:
- The daily oral treatment of pregnant rats was from day 6 through day 15 of gestation covering the period of organogenesis (as described in OECD 414, 2001 and earlier).
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Labs
- Age at study initiation: 20 weeks at start of mating
- Weight at study initiation: Females: 271 +/- 17 g
- Fasting period before study: no
- Housing: stainless steel wire mesh bottom cages; polypropylene cages with hardwood chips as bedding for females after mating
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- according to "Guide for the Care and Use of Laboratory Animals"
- Air changes (per hr): 15-18
- Photoperiod (hrs dark / hrs light): 12 / 12 hours - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: adjusted
- Amount of vehicle: 2.5 mL/kg bw
- Purity: distilled
- Gavage solutions were prepared fresh weekly and dispensed as needed for daily dosing. The test article and gavage solutions were stored at room temperature in light-protective containers. - Analytical verification of doses or concentrations:
- yes
- Remarks:
- UV Spectrophotometry
- Details on analytical verification of doses or concentrations:
- The stability of Metformin in gavage solutions was verified prior to study initiation. Solutions containing 48, 120 and 240 mg/ml Metformin were prepared and the test article concentration of each determined on the day of preparation and 7 and 14 days thereafter. In addition, the Metformin content in gavage solutions were verified prior to use.
UV Spectophotometry
Perkin Elmer Lambda 5 UV VIS spectrophotometer
Calibration with linear standard curve (R=0.99994)
Results (Mean concentrations of 3 measurements):
----------------------------------------------------------------
Dose Group Target Concentration Analytical Concentration
(mg/mL) (mg/mL)
----------------------------------------------------------------
A 0 -
B 48 47.2
C 120 120.0
D 240 238.0
----------------------------------------------------------------
Conclusion: Metformin concentration in gavage solutions was stable up to 14 days when stored at room temperature in light protective containers. Metformin concentrations were within 4% of the target level for all gavage solutions. - Details on mating procedure:
- Mating procedures were initiated after conclusion of the acclimation phase. The animals were approximately 105 days of age at this time.
Two females were randomly assigned to and cohabited with each male. Vaginal smears were obtained daily and insemination confirmed by the presence of sperm therein. The day on which insemination was confirmed was designated as day 0 of gestation. One hundred twenty five inseminated females were randomly assigned to five experimental groups (25 dams/group).
Each dam was assigned an identification number and treated as follows:
-----------------------------------------------------
Group Treatment Dose (mg/kg)
-----------------------------------------------------
A Vehicle -
B Acetylsalicylic acid 250
C Test Material 120
D Test Material 300
E Test Material 600
-----------------------------------------------------
No male was allowed to impregnate more than one female per group. - Duration of treatment / exposure:
- days 6 - 15 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- 11 days
- Dose / conc.:
- 120 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- Dose / conc.:
- 600 mg/kg bw/day
- No. of animals per sex per dose:
- 25 dams per group,
5 groups: 1 control, 3 treatment groups, 1 positive control - Control animals:
- yes, concurrent vehicle
- other: positive control: Acetylsalicylic acid (dose: 250 mg/kg bw/day)
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality; once daily for detailed observation
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
BODY WEIGHT: Yes
- Time schedule for examinations: on days 0, 6, 11, 15 and 20 of gestation
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #20
- Organs examined:
Special attention was given to examination of the urogenital system. The uterus and ovaries were excised, the uterus weighed and its contents examined. The following data were recorded for each dam:
Number of corpora lutea
Number and position of resorption and implantation sites
Number and position of live and dead fetuses
Sex, body weight and crown to rump length of each live fetus - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- Each fetus was visually examined for external anomalies. One third of the fetuses from each litter were randomly selected for and fixed in Bouin's solution and examined-for visceral anomalies using the Wilson free hand slicing technique (Wilson, 1965). All fetuses having external anomalies were also processed and examined in this manner.
The remaining fetuses were eviscerated, fixed in 70% isopropyl alcohol, macerated in a 2% aqueous potassium hydroxide solution, stained with Alizarin Red S (Dawson, 1926), cleared in glycerine and examined under low magnification for skeletal abnormalities and variations. These fetuses have been preserved in 100% glycerin. - Statistics:
- Continuous data were analyzed using analysis of variance {Snedecor and Cochran, 1967).
Differences between the vehicle control and test groups were determined using the Least Significant Differences Test.
Discrete data were evaluated using Fisher's Exact Test (Siegel, 1956).
Nonparametric litter data were analyzed using the Kruskal-Wallis Test.
Differences between the vehicle control and test groups were identified using the Mann-Whitney U Test (Siegel, 1956).
Significance was judged at the level p < 0.05 for all tests. - Clinical signs:
- no effects observed
- Description (incidence and severity):
- No finding occurred in any test group at an incidence significantly different from that of the vehicle control group.
- Dermal irritation (if dermal study):
- not examined
- Description (incidence and severity):
- not relevant
- Mortality:
- no mortality observed
- Description (incidence):
- No mortality occurred in any test group and consequently, all dams were sacrificed on gestation day 20.
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- Absolute body weight and body weight gain of dams exposed to 120, 300 and 600 mg/kg bw/day were comparable to control values at all times.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- no effects observed
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No finding occurred in any test group at an incidence significantly different (p >/= 0.05) from that of the vehicle control group.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Description (incidence and severity):
- No significant differences were evident between the vehicle control and any test item-treated group for any of the following variables:
Number of corpora lutea, total implantation sites, total resorption sites, percent resorption sites (as compared to implantation sites), or incidence of dams having two or more resorption sites. - Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Description (incidence and severity):
- No significant differences in the total number of fetuses, number of live fetuses, number of dead fetuses or number of stunted fetuses (runts) per litter were evident between the vehicle control and any Metformin HCl-treated group.
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Other effects:
- no effects observed
- Details on maternal toxic effects:
- Maternal toxic effects: no effects
Details on maternal toxic effects: No findings occured in any test group. - Key result
- Dose descriptor:
- NOEL
- Effect level:
- 600 mg/kg bw/day
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- No significant differences in male to fetal weight or fetal crown to rump length were evident between vehicle control and any Metformin-treated group.
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- No significant differences in male to female ratio were evident between vehicle control and any Metformin-treated group.
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- not examined
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects:no effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- The test material exposure produced no evidence of any teratogenic effect. No signs of maternal toxicity, treatment related embryo lethality or fetotoxicity were evident at any dosage level studied. The NOEL of the study was 600 mg/kg bw/day for developmental and maternal toxicity.
- Executive summary:
The potential teratogenicity of Metformin hydrochloride was evaluated in Sprague-Dawley rats. Dosages of 0, 120, 300 and 600 mg/kg body weight Metformin were administered daily by oral gavage to groups of 25 inseminated females on days 6 through 15 of gestation.
Acetylsalicylic acid (positive control) was administered in the same manner to a separate group of 25 inseminated females at a level of 250 mg/kg body weight.
All dams were sacrificed and subjected to uterine section on day 20 of gestation. Fetuses were excised and subjected to external and visceral or skeletal examination.
Acetylsalicylic acid exposure induced various teratogenic effects indicating that the test system could detect potential teratogenic agents.
Metformin exposure produced no evidence of any teratogenic effect. No signs of maternal toxicity, treatment-related embryo lethality or fetotoxicity were evident at any dosage level studied. External, visceral and skeletal examination of fetuses from Metformin-treated dams revealed no findings beyond those seen in vehicle control treated fetuses. The NOEL of the study was 600 mg/kg bw/day for developmental and maternal toxicity.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1991-03-15 to 1991-09-13
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Deviations:
- no
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: U.S. Food and Drug Administration (Segment II: Developmental toxicity (Embyo-foetal toxicity and teratogenic potential).
- Principles of method if other than guideline:
- The daily oral treatment of pregnant rabbits was from day 6 through day 18 of gestation covering the period of organogenesis (as described in OECD 414, 2001 and earlier).
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hazleton Research Products, Inc., Denver, Pennsylvania
- Age at study initiation: approx. 5 months
- Weight at study initiation: 2.85 to 4.04 kg
- Fasting period before study: no
- Housing: individual
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°F): 60 - 70
- Humidity (%): 40 - 60 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- yes
- Remarks:
- HPLC-UV
- Details on analytical verification of doses or concentrations:
- System:
Shimadzu LC-6A HPLC pump
Shimadzu SPD-6A UV detector (218 nm)
VYDAC C18 HPLC column
Mobile phase:15% Acetonitril / 85% pentanesulfonic buffer pH 2.8
The results of the the chemical analysis confirmed the stability of the test item in the vehicle and the correct preparation of the dosing formulations during the course of the study. - Details on mating procedure:
- The female rabbits were artificially inseminated with spermatozoa from untreated proven breeder male rabbits. The bucks had been obtained from the same source and were of the same strain as the female rabbits. Because the male rabbits were used only for the purpose of breeding the female rabbits and were not administered the test article, they were not considered to be part of the Test System.
The selected female rabbits were intravenously administered 20 USP Units of HCG(b)/kg approximately three hours before insemination. An estimated 0.25 mL of semen that had been diluted with normal saline to a concentration of 6.0 x 10^6 spermatozoa / 0.25 mL saline was used to artificially inseminate each rabbit.
The day artificial insemination was performed was designated as day o of presumed gestation. Each rabbit was artificially inseminated once on one of four consecutive days (one-fourth of the total number of rabbits assigned to each dosage group was inseminated each day).
The spermatozoa used to inseminate the female rabbits were obtained from four proven male breeders. - Duration of treatment / exposure:
- on days 6 through 18 of presumend gestation
- Frequency of treatment:
- daily
- Duration of test:
- 29 days
- Dose / conc.:
- 50 mg/kg bw/day
- Dose / conc.:
- 100 mg/kg bw/day
- Dose / conc.:
- 140 mg/kg bw/day
- No. of animals per sex per dose:
- 20 (f) per group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- The purpose of this study was to evaluate the developmental toxicity (embryo-fetal toxicity and teratogenic potential) of metformin hydrochloride administered orally via stomach tube to female New Zealand White (NZW) rabbits.
The standards of the U.S. Food and Drug Administration (FDA), were used as the basis for study design and compliance with Good Laboratory Practices (GLPs).
The first insemination occurred on March 18, 1991, and the last Caesarean-sectioning was performed on April 19, 1991.
Each dosage group was randomly assigned 20 presumed pregnant New Zealand White (NZW) rabbits.
The test article, metformin hydrochloride, was administered orally via stomach tube once daily to artificially inseminated rabbits on days 6 through 18 of presumed gestation. Dosages of 0 (Vehicle), 50, 100 and 140 mg/kg bw/day were given to the rabbits as solutions prepared at concentrations of 0 (Vehicle) , 25. 50 and 70 mg/mL, respectively.
The vehicle was reverse osmosis membrane processed deionized water (R.O. deionized water).
The dosage volume was 2 mL/kg, adjusted daily based on the individual body weights recorded immediately before intubation.
The rabbits were examined daily for clinical observations, abortions, premature deliveries and deaths. Body weights were recorded on day 0 of presumed gestation, and then daily on days 6 through 29 of presumed gestation.
Feed consumption values were recorded on days 0 through 29 of presumed gestation. Rabbits that were found dead, aborted or prematurely delivered were necropsied on the day the event occurred.
The procedures used were the same as those described below for rabbits sacrificed on day 29 of presumed gestation.
On day 29 of presumed gestation, surviving rabbits were sacrificed, and a gross necropsy of the thoracic and abdominal viscera was performed. Each uterus was excised and examined for number and placement of implantations, early and late resorptions and live and dead fetuses.
The number of corpora lutea in each ovary was recorded. Each foetus was weighed and examined for sex and gross external, soft tissue and skeletal alterations. - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of the rabbits were recorded four times before insemination, and then on day 0 and days 6 through 29 of presumed gestation.
FOOD CONSUMPTION : Yes
- Feed consumption values were recorded daily during the acclimation and study periods.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
- Organs examined: The thoracic and abdominal cavities of each rabbit were examined for gross lesions. Gross lesions (with the exception of parovarian cysts, which are considered to be common spontaneous lesions in rabbits) were retained in neutral buffered 10% formalin. All other tissues were discarded. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of live and dead fetuses: Yes - Fetal examinations:
- - External examinations: Yes: [all per litter ]
- Soft tissue examinations: Yes: [all per litter ]
- Skeletal examinations: Yes: [all per litter ]
Each Caesarean-delivered fetus was placed in an individual container, weighed, examined for gross external alterations and individually identified with a tag noting litter number, study number and uterine placement. Live fetuses were sacrificed by an intraperitoneal injection of pentobarbital sodium. All fetuses were examined internally to identify sex and visceral alterations: the brain was free hand cross-sectioned (a single cross-section was made between the parietal and the frontal bones and examined for hydrocephaly. Fetal gross lesions were preserved in neutral buffered 10% formalin. The fetuses were eviscerated, stained with alizarin red S and evaluated for skeletal alterations. All skeletal preparations were stored in 80% glycerin with thymol crystals added to retard fungal growth. Delivered pups, aborted conceptuses, fetuses of females that aborted and late resorptions were examined to the extent possible. Representative photographs were taken of selected fetal alterations. - Statistics:
- Standard statistical methods have been applied for data processing:
Parametric: Bartlett's Test, Dunett's test
Nonparametric: Kruskal-Wallis Test, Fisher's Exact Test - Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- decreased motor activity, ataxia, impaired righting reflex, tonic extensor convulsions, body jerks, mucoid faeces and a red substance in the cage pan
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- One low dosage group and four (P<0.01) high dosage group animals died on days 10, 14, 15 or 18 of gestation. The death of the low dose animal was due to an intubation accident.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Weight losses occurred in the 100 and 140 mg/kg/day dosage groups on days 15 to 19 of gestation. Body weight gains for the entire dosage period (calculated as days 6 to 19 of gestation) did not significantly differ among the four dosage groups.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Absolute and relative maternal feed consumption values were reduced in the 100 and 140 mg/kg bw/day dosage groups for the entire dosage period (calculated as days 6 to 19 of gestation) . These reductions in feed consumption values were significant in the 140 mg/kg bw/day dosage group.
- Food efficiency:
- effects observed, treatment-related
- Description (incidence and severity):
- reduced
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Gross pathological findings:
- no effects observed
- Description (incidence and severity):
- No gross lesions were considered effects of the test article because: 1) the lesions that occurred were single events; 2) the incidences were not dosage-dependent; and 3) the lesions are common in this strain of rabbit.
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- not examined
- Histopathological findings: neoplastic:
- not examined
- Number of abortions:
- effects observed, treatment-related
- Description (incidence and severity):
- One middle and two high dosage group animals aborted on days 25 or 26 of gestation.
One control group animal aborted on day 20 of gestation. - Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- effects observed, treatment-related
- Description (incidence and severity):
- premature delivery @140 mg/kg bw
- Changes in number of pregnant:
- no effects observed
- Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 50 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- body weight and weight gain
- clinical signs
- food consumption and compound intake
- mortality
- number of abortions
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Fetal body weight changes:
- no effects observed
- Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- No fetal gross external, soft tissue or skeletal alterations that occurred in this study were considered effects of administration of the test article to the animals at dosages as high as 140 mg/kg bw/day.
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 140 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Based on these data, the maternal no-observable-effect-level (NOEL) for metformin hydrochloride in pregnant rabbits is less than 50 mg/kg/day.
Dosages of 50 mg/kg/day and higher caused deaths and clinical observations; the 100 and 140 mg/kg/day dosages also caused abortions, body weight losses and reduced feed consumption values; premature delivery was also caused by the 140 mg/kg/day dosage.
The developmental NOEL for metformin hydrochloride is 140 mg/kg; day. No adverse effects on embryo-fetal viability, growth or morphology occurred up to the highest dosage tested. - Executive summary:
Purpose
The purpose of this study was to evaluate the developmental toxicity (embryo-fetal toxicity and teratogenic potential) of metformin hydrochloride administered orally via stomach tube to female New Zealand White (NZW) rabbits. The standards of the U.S. Food and Drug Administration (FDA), were used as the basis for study design and compliance with Good Laboratory Practices (GLPs). The first insemination occurred on March 18, 1991, and the last Caesarean-sectioning was performed on April 19, 1991.
Methods
Each dosage group was randomly assigned 20 presumed pregnant New Zealand White (NZW) rabbits. The test article, metformin hydrochloride, was administered orally via stomach tube once daily to artificially inseminated rabbits on days 6 through 18 of presumed gestation. Dosages of 0 (Vehicle), 50, 100 and 140 mg/kg bw/day were given to the rabbits as solutions prepared at concentrations of 0 (Vehicle) , 25. 50 and 70 mg/mL, respectively. The vehicle was reverse osmosis membrane processed deionized water (R.O. deionized water). The dosage volume was 2 mL/kg, adjusted daily based on the individual body weights recorded immediately before intubation. The rabbits were examined daily for clinical observations, abortions, premature deliveries and deaths. Body weights were recorded on day 0 of presumed gestation, and then daily on days 6 through 29 of presumed gestation. Feed consumption values were recorded on days 0 through 29 of presumed gestation. Rabbits that were found dead, aborted or prematurely delivered were necropsied on the day the event occurred. The procedures used were the same as those described below for rabbits sacrificed on day 29 of presumed gestation. On day 29 of presumed gestation, surviving rabbits were sacrificed, and a gross necropsy of the thoracic and abdominal viscera was performed. Each uterus was excised and examined for number and placement of implantations, early and late resorptions and live and dead foetuses. The number of corpora lutea in each ovary was recorded. Each foetus was weighed and examined for sex and gross external, soft tissue and skeletal alterations.
Results
One middle and two high dosage group animals aborted on days 25 or 26 of gestation, one low dosage group and four (P<0.01) high dosage group animals died on days 10, 14, 15 or 18 of gestation; one of these high dosage group animals aborted before it died. One high dosage group animal prematurely delivered its litter.
These deaths, abortions and the premature delivery in the low, middle and high dosage groups were considered effects of the test article because these does also had other effects of the test article (clinical observations, weight loss and reduced feed consumption}.
One control group animal aborted. One low dosage group animal died as the result of an intubation accident. These events were considered unrelated to the test article because the incidences were within the ranges observed historically.
Dried faeces occurred in increased numbers of 100 and 140 mg/kg/day dosage group does and was considered an effect of the test article. Other clinical observations that occurred in animals that died or aborted and were considered possible effects of the test article included decreased motor activity, ataxia, impaired righting reflex, tonic extensor convulsions, body jerks, mucoid faeces and a red substance in the cage pan.
Weight losses occurred in the 100 and 140 mg/kg/day dosage groups on days 15 to 19 of gestation. Body weight gains for the entire dosage period (calculated as days 6 to 19 of gestation) did not significantly differ among the four dosage groups.
During the postdosage period (calculated as days 19 to 29 of gestation), body weight gains were increased in the test material groups, as compared with the control group value. Significant increases (P<0.05) in body weight gains occurred in the 100 and 140 mg/kg/day dosage groups on days 24 to 29 of gestation. Body weights were comparable throughout the study in the four dosage groups.
Absolute (g/day) and relative (g/kg/day) maternal feed consumption values were reduced in the 100 and 140 mg/kg/day dosage groups for the entire dosage period (calculated as days 6 to 19 of gestation). These reductions in feed consumption values were significant (P<0.05 to P<0.01) in the 140 mg/kg/day dosage group. Within the dosage period, absolute and relative feed consumption values were significantly reduced (P<0.05 to P<0.01) on days 9 to 12 and 12 to 15 of gestation and relative feed consumption values were significantly reduced (P<0.05) on days 6 to 9 and 15 to 19 of gestation in the 140 mg/kg/day dosage group. During the post dosage period, absolute and relative feed consumption values were slightly increased in the 50, 100 and 140 mg/kg/day dosage groups, as compared with the control group values.
No Caesarean-sectioning or litter observations were affected by dosages of metformin hydrochloride as high as 140 mg/kg/day. The litter averages for corpora lutea, implantations, litter sizes, live foetuses, early and late resorptions and the number of animals with any resorptions with viable foetuses were comparable, and did not significantly differ. The litter averages for percent male foetuses, fetal body weights and percent resorbed conceptuses were also comparable in the four dosage groups and did not significantly differ.
No fetal gross external, soft tissue or skeletal malformations or variations that occurred in this study were considered effects of administration of the test article to the does at dosages as high as 140 mg/kg/day.
Conclusions
Based on these data, the maternal no-observable-effect-level (NOEL) for metformin hydrochloride in pregnant rabbits is less than 50 mg/kg/day.
Dosages of 50 mg/kg/day and higher caused deaths and clinical observations; the 100 and 140 mg/kg/day dosages also caused abortions, body weight losses and reduced -feed consumption -values;- premature delivery was also caused by the 140 mg/kg/day dosage.
The developmental NOEL for metformin hydrochloride is 140 mg/kg/day. No adverse effects on embryo-fetal viability, growth or morphology occurred at the highest dosage tested. Therefore, metformin hydrochloride should not be classified as a developmental toxicant.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 1984-04-24 to 1984-11-16
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: FDA, Segment III Study
- GLP compliance:
- yes
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc.
- Age at study initiation: 100 days at start of dosing
- Weight at study initiation:
- Fasting period before study: no
- Housing: individual during acclimatisation and cohabited during mating.
- Housing: Stainless steel wire mesh bottom cages; polyprepylene cages with hardwood chips as bedding for females after mating .
- Diet: NIH open formula 07, ad libitum
- Water: Tap water, ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature (°C): room temperature
- Humidity (%): according to Guide for the Care and Use of Laboratory Animals
- Air changes (per hr): 15 - 18
- Photoperiod (hrs dark / hrs light): 12 / 12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: adjusted
- Amount of vehicle: 2.5 mL/kg bw
- Purity: distilled
- Gavage solutions were prepared fresh weekly and dispensed as needed for daily dosing. The test article and gavage solutions were stored at room temperature in light-protective containers. - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Perkin-Elmer Lambda 5 UV-VIS spectrophotometer
nominal concentration (mg/mL) analytical concentration (mg/mL)
48 48.7 +/- 1.0
120 121.8 +/- 3.4
240 240.8 +/- 6.5 - Details on mating procedure:
- The animals were randomly mated, one male to one fe male, within each treatment group.
Vaginal lavage was conducted daily on all females during the mating period.
The smears were microscopically examined and insemination confirmed by the presence of sperm therein .
If insemination was not confirmed after ten days, the female was cohabited with a second male of proven fertility.
Males were euthanized and discarded, without necropsy , at conclusion of the mating period.
Inseminated fernales (dams) were transferred to nesting boxes for the gestation, parturition and lactation phases of the study.
The dams were observed daily and body weights periodically determined during gestation. - Duration of treatment / exposure:
- Daily dose administration (oral) was initiated for each dam on gestation day 15 and continued until study termination (lactation day 21). Dams and their litters were observed daily during the lactation phase of the study.
Specifically, general appearance of dams and offspring, body weight of darns and offspring, sex ratio and viability of off spring and deveiopment of the offspring were evaluated. - Frequency of treatment:
- Daily
- Duration of test:
- gestation day 15 to day 21 of lactation
- Dose / conc.:
- 120 mg/kg bw/day
- Dose / conc.:
- 300 mg/kg bw/day
- Dose / conc.:
- 600 mg/kg bw/day
- No. of animals per sex per dose:
- 20 pregnant females per dose
- Control animals:
- yes, concurrent vehicle
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before dosing
BODY WEIGHT: Yes
- Time schedule for examinations:
Individual body weights were recorded once weekly during the mating phase. Individual body weights of females were recorded on gestation days 0, 6, 14 and 19 and on lactation days 0 (as soon as possible after delivery), 7, 14 and 21. Individual pup weights were determined on lactation days 0, 7, 14 and 21.
Offspring Development:
- Time schedule for examinations: daily
In addition to routine daily observation, the pups were further evaluated for achievement of the following criteria:
surface righting reflex (five seconds) pinna unfolding (both)
hair growth (first coat)
incisor eruption (upper and lower recorded separately)
eye opening (both)
Each litter was examined daily and the day upon which all pups achieved each criteria was recorded.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day #21 (of lactation)
- Organs examined: All dams and pups were euthanized (C02 gas) and subjected to gross necropsy on lactation day 21. The external body surface and orifices, internal body cavities and their organs and the carcass were examined.
All pups, except those cannabilized, were dissected and examined for gross anomalies including cleft lip/ palate, talpes, gastroschisis, exencephalacia, meningocele, accessory digits, spinal bifida, ablephera or other obvious malformations. The sex of each was confirrned by gonadal inspection.
OTHER: - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes / No / No data
Examinations included:
- Gravid uterus weight: Yes / No / No data
- Number of corpora lutea: Yes / No / No data
- Number of implantations: Yes / No / No data
- Number of early resorptions: Yes / No / No data
- Number of late resorptions: Yes / No / No data
- Other: - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter - Statistics:
- Standard statistical methods have been applied for data processing.
- Clinical signs:
- no effects observed
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- no effects observed
- Water consumption and compound intake (if drinking water study):
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed - Changes in number of pregnant:
- no effects observed
- Other effects:
- no effects observed
- Details on maternal toxic effects:
- Maternal toxic effects:no effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 600 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 600 mg/kg bw/day
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Key result
- Abnormalities:
- no effects observed
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed - Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- no effects observed
- Details on embryotoxic / teratogenic effects:
- Embryotoxicity / teratogenicity:no effects
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 600 mg/kg bw/day
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- Under the conditions of this study, Metformin did not produce any untoward effect upon pregnant Sprague-Dawley rats during the third trimester of gestation, parturition or lactation at dose levels of 120, 300 and 600 mg/kg body weight. Furthermore, the drug had no adverse effect upon litter size, offspring viability, litter sex ratio, offspring development or offspring body weight at any dose level studied. No treatment-related physical anomalies were noted during gross necropsy of the dams and their litters.
Referenceopen allclose all
Metformin was orally given to twenty pregnant Sprague-Dawley rats at dosage levels of 120, 300 or 600 mg/kg body weight. Daily dose administration was begun on gestation day 15 and continued through study termination at 21 days post partum. A control group, also containing 20 pregnant animals, was administered distilled-deionized water under the requisite experimental conditions.
The dams were observed daily for physical or behavioral abnormalities during gestation, parturition and lactation. Individual body weights were recorded on gestation days 0, 6, 14 and 19 and lactation days 0, 7, 14 and 21 for all dams. All litters were observed daily to determine the viability, general appearance and physical development of the offspring.
Offspring body weights were determined on lactation days 0, 7, 14 and 21. The sex ratio of each litter was determined on days 0, 4, 14 and 21 of lactation. All dams and offspring were subjected to gross necropsy on day 21 postpartum or at the time of death. All lesions or physical anomalies were recorded. The genitourinary tract of all dams was carefully examined for identification and quantification of implantation scars. The sex of each pup was confirmed at necropsy by gonadal inspection.
Under the conditions of this study, Metformin did not produce any untoward effect upon pregnant Sprague-Dawley rats during the third trimester of gestation, parturition or lactation at dose levels of 120, 300 and 600 mg/kg body weight. Furthermore, the drug had no adverse effect upon litter size, offspring viability, litter sex ratio, offspring development or offspring body weight at any dose level studied. No treatment-related physical anomalies were noted during gross necropsy of the dams and their litters.
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 140 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rabbit
- Quality of whole database:
- All three studies are well described and were performed under GLP regulation.
Effect on developmental toxicity: via inhalation route
- Endpoint conclusion:
- no study available
Effect on developmental toxicity: via dermal route
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Based on the data provided, metformin hydrochloride does not need to be classified for reproductive toxicity.
Additional information
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