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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1984
Report date:
1984

Materials and methods

Test guideline
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

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:
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

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.
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
Doses / concentrationsopen allclose all
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)

Examinations

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.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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.

Effect levels (maternal animals)

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

Maternal abnormalities

Key result
Abnormalities:
no effects observed

Results (fetuses)

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

Effect levels (fetuses)

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

Fetal abnormalities

Key result
Abnormalities:
no effects observed

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

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.