Lithium amide

Developmental toxicity / teratogenicity

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

2001

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

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

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 degree C
- Humidity (%): 55 +/- 15 %
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light

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 b.w. 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 ata 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 AllessaChemie 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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

90 mg/kg bw/day

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:

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

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Examinations: macroscopic examination of the internal organs and placentae of the dams

OTHER: Toxicokinetics
On the last administration day, three spare animals/group (groups 1 to 4) were used for blood sampling for plasma determination and were not laparotomised. In order to obtain at least 50 μL K3EDTA plasma per animal and sampling time, sufficient whole blood was collected from the retrobulbar venous plexus under light ether anaesthesia at the following sampling times:
Groups 2 to 4: 0 (predose), 1, 2, 4, 8 and 24 h p.a.
Group 1: 0 (predose), 24 h p.a.
Total number of samples: 60 plasma samples

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:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Yes: all per litter

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

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

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:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

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

Details on maternal toxic effects:

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.

Reproduction data
No test item-related influence on the prenatal fetal development was detected at either 10, 30 or 90 mg lithium carbonate/kg b.w./day with respect to the number of corpora lutea, implantation sites, resorptions and live fetuses or the values calculated for the pre- and post-implantation losses. The number of implantation sites compared to the number of Corpora lutea was significantly reduced (at p ≤ 0.01 or p ≤ 0.05) in all test item-treated groups compared to the control. This finding was considered as incidental as the preimplantation loss occurred before start of test item-treatment.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

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:

effects observed, non-treatment-related

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

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 sex distribution, fetal and placental weights, and number of live fetuses at birth 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.

Effect levels (fetuses)

Key result

Dose descriptor:

NOEL

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: embryotoxicity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Toxicokinetics

The toxicokinetic analysis based on lithium plasma levels revealed a clear dose related 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 bw/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 ithium 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 present test conditions, the no-observed-effect level (NOEL) was 30 mg lithium carbonate/kg bw/day for the dams. The NOEL for the fetuses was >= 90 mg lithium carbonate/kg bw/day.

Executive summary:

An 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 maternel toxic dose level of 90 mg lithium carbonate/kg bw/day.