Metformin hydrochloride

Toxicity to reproduction

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1984

Report date:

1984

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

26 May 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: FDA Segment I study

Test guideline 3

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

Test material

Test material information

Constituent 1

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:

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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

120 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

600 mg/kg bw/day

No. of animals per sex per dose:

12 (m), 24 (f)

Control animals:

yes, concurrent vehicle

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Key result

Critical effects observed:

no

Results: F1 generation

General toxicity (F1)

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.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result

Critical effects observed:

no

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

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.