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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:
2010-02-18 to 2010-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
2001
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:
2004
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Lithium carbonate
EC Number:
209-062-5
EC Name:
Lithium carbonate
Cas Number:
554-13-2
Molecular formula:
CH2O3.2Li
IUPAC Name:
dilithium carbonate
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Lithium ccarbonate pharm. 801 fibredrum
- Physical state: White, crystalline powder
- Analytical purity: 99.6 %

Test animals

Species:
rat
Strain:
other: Crl: CD(SD)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Research, Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 185 - 234 g
- Housing: MAKROLON cages (type III) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 15 cm
- Diet: Commercial ssniff® R/Z V1324, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous hydroxypropyl methyl cellulose gel (Methocel)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle 0.5 % aqueous hydroxypropyl methyl cellulose gel (Methocel)1 to the appropriate concentrations and was administered orally at a constant volume of 5 mL/kg bw once daily from the 6th to the 19th day of pregnancy. The dose of the test item was adjusted to the animal's body weight daily. The control animals received the vehicle at a constant volume of 5 mL/kg bw orally once daily in the same way. The test item mixtures were freshly prepared every day approx. 1h before use.

Applied volume: 5 mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the test item formulations, samples of approx. 10 mL were taken at the following times:
At the beginning of the administration period: Analysis of concentration/homogeneity.
At start of administration, during (middle) administration and before administration to the last animal of each group (3 samples/dose level group).
Total number of samples: 9

At termination of the administration period at time point when the majority of animals was dosed: Analysis of concentration/homogeneity.
At start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 sample/dose level group).
Total number of samples: 9

Thus, the sum of all samples is 18.

The samples were labelled with the study number, species, type of sample, test item, concentration, sampling time and date and were stored immediately after withdrawal at -20 degree C or colder until dispatch.

The formulation samples were analysed for Lithium levels according to GLP by the Test Site Allessa Chemie GmbH. The Phase Plan “Bestimmung des Lithiumgehaltes in Trägergemisch mittels ICP-OES (Teil-Prüfplan VP-Nummer 005/2010)” and any amendments to this Phase Plan are part of the LPT Study Plan 24635.

The analysis of the test item-carrier mixtures for Lithium levels revealed that the formulations used for the administrations in groups 2 to 4 were correctly prepared. The measured actual concentrations ranged from 96.45% to 103.64% of the nominal values. The results were within the expected range of the theoretical concentrations.
Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. The female breeding partners were randomly chosen. Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm. If findings were negative, mating was repeated with the same partner. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
From the 6th to the 19th day of pregnancy.
Frequency of treatment:
Once daily from the 6th to the 19th day of pregnancy.
Duration of test:
20 days after mating
No. of animals per sex per dose:
25
Control animals:
yes
Details on study design:
Summary on animals examined: 21 dams per dose group

Evaluated litters: 20 per dose group

Non pregnant dams: 1 in dose groups 0, 30 and 90 mg/kg bw/ day, i.e. 3 in total

Dams without viable fetuses: 1 (dose group 30 mg/kg bw/day)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: No data

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. day 0-3, 3- 6, 6-9, 9-12, 12-15, 15-18 and 18-20).

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes


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:
Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter
weight). Fetuses were inspected externally for damages, especially for malformations. The fetuses were sacrificed by an ether atmosphere.
Statistics:
For all numerical values, homogeneity of variances was tested using the BARTLETT chi-square test. When the variances were homogeneous, the DUNNETT test (p <= 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out, limit of significance was p <= 0.01. For the comparison of classification measurements (for example malformation-, resorption-, retardation- and variation rate) the FISHER's exact test (n < 100) or chi2-test with YATES' correction for continuity (n >= 100) (p <= 0.05 and p <= 0.01) was employed.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Slight but significant reductions was noted for the net weight change and the food intake.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOEL
Effect level:
30 mg/kg bw/day
Based on:
other: test material: lithium carbonate
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Effect levels (fetuses)

Key result
Dose descriptor:
NOEL
Effect level:
90 mg/kg bw/day
Based on:
other: test material: lithium carbonate
Basis for effect level:
other: embryotoxicity

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Influence on the dam

Mortality: None of the dams treated with 10, 30 or 90 mg Lithium Carbonate/kg b.w./day died prematurely during the course of the study.

Clinical signs: Pilo-erection was noted in four high-dosed dams treated with 90 mg Lithium Carbonate/kg b.w./day on two to four days, starting on gestation day 17 or 19 and lasting until laparotomy on gestation day 20. The drinking water intake of all high-dosed dams treated with 90 mg Lithium Carbonate/kg b.w./day was increased starting on gestation day 17, 18 or 19 and lasting until laparotomy on gestation day 20.

Body weight and body weight gain: Marginal reductions were noted for the mean body weights of the high-dosed dams (90 mg Lithium Carbonate/kg b.w./day) during the last gestation days. The increase in the mean body weight from the start value (day 0 of pregnancy) was 66.9% at the time point of laparotomy (control: 74.4%). Significant reductions (at p ≤ 0.01) were noted for the net weight change of the high-dosed dams from day 6 of gestation to laparotomy on gestation day 20 (carcass weight minus day 6 body weight).

Food consumption: Slight but statistically significant reductions (at p <= 0.01 or p <= 0.05) were determined for the relative food consumption of the high-dosed dams (90 mg Lithium Carbonate/kg b.w./day) on gestation days 7, 9, 11 to 13 and 19 (up to 18.3% below the control value).

Drinking water consumption: Increased intake of drinking water was noted in all high dosed females treated with 90 mg Lithium Carbonate/kg b.w./day on one to four days, starting on gestation day 17 (qualitative observation by visual appraisal).

Necropsy findings: No test item-related pathological findings were noted.

Uterus and carcass weights: The gravid uterus weight and the carcass weight were not influenced by the exposure to the test item.

Influence on the fetus

No test item-related influence was noted on the prenatal fetal development at 10, 30 or 90 mg Lithium Carbonate/kg b.w./day with respect to the number of corpora lutea, implantation sites, resorptions, sex distribution, fetal and placental weights, number of live fetuses at birth and the values calculated for the pre- and postimplantation loss when compared to the control. No dead fetuses or runts were noted at laparotomy.

Malformations: No malformations were noted in the fetuses during external/ internal examination, skeletal examination (according to DAWSON) or soft tissue evaluation (according to WILSON).

Variations: No test item-related variations were noted in the fetuses during external / internal examination, skeletal examination (according to DAWSON) or soft tissue evaluation (according to WILSON). Retardations No test item-related influence was noted for the incidence of skeletal retardations.

Toxicokinetics: The toxicokinetic analysis based on Lithium plasma levels revealed a clear doserelated systemic exposure to Lithium. Mean peak plasma levels of 1.66, 3.59 and 9.65 mg Li/L plasma, respectively, were observed at 10, 30 or 90 mg Lithium Carbonate/ kg b.w./day on gestation day 19. The plasma concentrations declined with a mean elimination half-life for Lithium between 8.4 to 12.0 hours. Toxicokinetics demonstrated dose proportional increases of Lithium plasma concentrations between 10 and 90 mg Lithium Carbonate/kg b.w./day. Peak time and half-life and increased with dose levels.

Analysis of test item formulation (performed by the Test Site AllessaChemie GmbH, Germany): The analysis of the test item-carrier mixtures for Lithium levels revealed that the formulations used for the administrations in groups 2 to 4 were correctly prepared. The measured actual concentrations ranged from 96.45% to 103.64% of the nominal values. The results were within the expected range of the theoretical concentrations.

Applicant's summary and conclusion

Conclusions:
Under the test conditions of prenatal developmental toxicity study with lithium carbonate, the no-observed-effect level (NOEL) for the fetuses was above 90 mg lithium carbonate/kg bw/day. Thus, the NO(A)EL for developmental toxicity is 90 mg lithium carbonate /kg bw. The no-observed-effect level (NOEL) was 30 mg lithium carbonate/kg bw/day for the dams (maternal toxicity). Thus, the NO(A)EL for maternal toxicity is 30 mg lithium carbonate/kg bw/day.
Executive summary:

A prenatal developmental toxicity study was performed in rats (strain: Crl CD (SD)) according to OECD Guideline 414 and EU method B.31. In this rat embryotoxicity study, the test item lithium carbonate was administered to female rats at concentrations of 10, 30 or 90 mg/kg bw/day orally by gavage from the 6th to 19th day of pregnancy. Under the present test conditions, the no-observed-effect level (NOEL) was 30 mg lithium carbonate/kg bw/day for the dams (maternal NOEL). At 90 mg lithium carbonate/kg bw/day, pilo-erection was noted in a few dams. Furthermore, slight but significant reductions were noted for the net weight change and the food intake. The NOEL for the fetuses was >= 90 mg lithium carbonate/kg bw/day. There was no test item-related increase in the incidence of fetal malformations, external/ internal, skeletal or soft tissue variations or skeletal retardations. The toxicokinetic analysis revealed a clear dose-related systemic exposure to lithium. In conclusion, no embryotoxic properties of the test item were noted during external/ internal, skeletal and soft tissue examinations. No test item-related increase was noted in the incidence of malformations, variations or retardations, not even at the materno-toxic dose level of 90 mg lithium carbonate/kg bw/day.