Lithium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2010-11-04 to 2011-01-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 October 2008

Deviations:

yes

Remarks:

See below

Principles of method if other than guideline:

The recovery groups were not included for the control and high dose groups. Further, the number of groups, number of animals, spectrum for haematology and clinical chemistry, organ weights, organ collection and preservation and histopathology deviate from OECD 407 prescription.

GLP compliance:

yes

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:

Wistar

Details on species / strain selection:

The rat is the standard laboratory rodent species used for toxicity assessment and also recommended by various regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxicology, Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore 560 058, India (Parent stock obtained from Harlan Netherland)
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: males: 180 - 221 g; females: 134 - 167 g
- Housing: groupwise two animals of same sex per cage in sterilized suspended polysulfone cages
- Diet: ad libitum (ssniff rats/mice pellet food)
- Water: ad libitum (Deep bore-well water passed through activated charcoal filter)
- Acclimation period: After physical examination for good health and the suitability for the study, the rats were acclimatized for five days before start of the treatment.

DETAILS OF FOOD AND WATER QUALITY: Analysis and contaminant analysis reports confirm that all parameters are within specification. Documents are included in the report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24
- Humidity (%): 57 – 67
- Air changes (per hr): 12 – 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2010-11-11 To: 2010-12-13

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route has been chosen because this route allows assessing the
maximum local and systemic toxicological profile of the test item, which may
be used for extrapolative risk assessment for the human.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Required quantity of test item was weighed and mixed in Milli-Q water to attain desired concentrations of 3, 6, 10 and 15 mg/mL for the low, mid, midintermediate and high dose groups respectively. Vehicle control group animals were administered the vehicle only. Dose formulation was prepared once in 8 days as stock solution for each dose. The prepared stock solution was mixed by invasion (3-5 times) before taking for daily use. Homogenity of the Dose formulation was maintained by constant stirring using magnetic stirrer except on days 1 and 2. The prepared stock solution was stored in the experimental room. On treatment Day 9, the high dose of 150 mg/kg bw/day was reduced to 120 mg/kg bw/day for the G5 group. For this, the required quantity of the test item was weighed and mixed in Milli-Q water to attain desired concentration of 12 mg/mL.

VEHICLE
- Concentration in vehicle: 3, 6, 10 and 15 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For active ingredient concentration analysis, samples of the test formulations
were taken from all doses including the vehicle control, prepared on Days 1 and
22 of the treatment period. The collected samples were sent to Analytical R&D Department of Advinus Therapeutics Limited, Bangalore for concentration analysis. Analysis was done as per the validated method (Study No.: G7467).
The stability and homogeneity of test item in the vehicle was carried out separately before initation of the study. Based on the results, the test item was found to be stable for 8 days in the vehicle at room temperature.
Stability and homogeneity of the test item in the vehicle at 0.1 mg/mL and
10 mg/mL concentrations were carried out before initation of the study as per
the method described in a separate study no. G7467.
The results of the homogeneity study indicated that the test item was found to be homogeneous in the vehicle. Based on stability results, the test item was found to be stable for 8 days in the vehicle at both the concentration levels when stored at ambient conditions.
The test item concentration in gavage samples prepared for dosing were 0, 3, 6, 10 and 15 mg/mL on 11.11.2010 and 0, 3, 6 and 10 mg/mL on 02.12.2010 were analysed. The results indicated that the test item concentration in solution prepared was within the permissible limits of ± 15 % from the nominal concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Remarks:

On treatment Day 9, the high dose of 150 mg/kg bwt/day was reduced to 120 mg/kg bw/day.

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

30 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Control: 3
Treatment groups: 6

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Repeated oral dosing with lithium carbonate at 0, 106, 478, 1067 and 2083 ppm via the diet in Sprague-Dawley rats for 21 days resulted in statistical decreases in absolute liver, kidney, spleen and heart weight at 2083 ppm (approximating 160 mg/kg bw/day). Relative organ weights were also altered: brain and heart weight were increased, whereas liver and spleen weight were increased. Blood lithium levels were 0.0, 0.06, 0.23, 0.36 and 0.81 mEq lithium/L* (Ibrahim et al., 1990). Previous studies in female pregnant rats with lithium carbonate from gestation day 6-15 resulted in embryotoxicity and skeletal abnormalities related to delayed skeletal maturation, but also in maternal toxicity as polyuria, body weight gain decrease and mortality in dams (Fritz, 1988; Marathe & Thomas, 1986). The dose of 50 mg/kg bw/day was considered as a NOAEL for maternal and developmental effects. There were however not data on lithium serum levels. Lithium carbonate was recently administered to female rats, first as dose range finding for a developmental study at dose levels of 15, 50 and 200 mg/kg bw/day orally by gavage from the 6th to 19th day of pregnancy (see IUCLID section 7.8.2). Administration of the high dosed animals was discontinued due to mortality and poor general condition. The no-observed-effect level (NOEL) was 50 mg/kg bw/day for the dams. There was no increase in the incidence of external malformations or variations. Based on the data obtained, the dose levels of 10, 30 and 90 mg/kg bw/day were selected for the main prenatal developmental toxicity study of lithium carbonate in rats from the 6th to 19th day of pregnancy (see IUCLID section 7.8.2). Under the test conditions, the NOEL was 30 mg/kg bwt/day for the dams. At 90 mg/kg bw/day, piloerection 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 above 90 mg/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. Toxicokinetic analysis revealed a clear dose-related systemic exposure to Lithium, with mean peak plasma levels of 1.66, 3.59 and 9.65 mg lithium/L (or 0.24, 0.52 and 1.40 mEq lithium/L).

* 1mEq/L = 6, 9 mg Li/L

Based on the available literature and experimental data provided, the dose levels of 30, 60, 100 and 150 mg/kg bw/day have been selected for this study.

- Fasting period before blood sampling for clinical biochemistry: fasted overnight

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, morbidity and mortaltiy was checked twice daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation of treatment on Day 1 and at weekly (± 1 day) intervals during treatment period. In addition, the detailed clinical examination was done on Day 12 to thoroughly examine the rats.
- Parameters examined: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self mutilation, walking backwards) were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of treatment and at week 1 of treatment. From week 2 onwards, the body weights were recorded twice weekly.

FOOD INTAKE:
- Food consumption for each animal calculated by using the food consumed at each measuring interval per cage and dividing by the number of days in the intervening period to determine the food intake/rat/day.


WATER INTAKE: Yes
- Time schedule for examinations: daily from Day 8

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters checked in table [No.1 under "Any other information on materials and methods incl. tables"] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters checked in table [No.2 under "Any other information on materials and methods incl. tables"] were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during 4th week (Day 26) of treatment period
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity, other: home cage observations, observations during removal of subject from home cage, open field observation

IMMUNOLOGY: No

Other examinations:

Toxikokinetics
The purpose of the toxicokinetic phase of the study was to determine the plasma exposures of test item in Wistar rats. The study was conducted at four dose levels. A total of 54 animals were divided into five groups: G2, G3, G4 and G5, each consisting of 6 males and 6 female animals and for G1 (vehicle control group) 3 males and 3 female rats were assigned. As per the sampling schedule, blood samples were collected from three rats per time point in each treatment group. No rat was bled for two consecutive time points. About 0.7 mL of blood was collected from the retro-orbital plexus under isoflurane anesthesia, at each sampling time points on Days 1 and 28. Blood samples were drawn on Day 1 at 0.5, 1, 2, 4, 8, 12, and 24 hrs for groups G2, G3, G4 and G5 and on Day 28 at pre - dose, 0.5, 1, 2, 4, 8, 12, and 24 hrs for groups G2, G3 and G4. The toxicokinetic sampling of the G5 group was discontinued due to preterminal death or sacrifice of the animals. For group G1, blood samples were drawn at 0 and 2 hours after dosing. Blood was collected within ± 2 to 5 minutes of the scheduled time for each time point.
Bioanalysis: Bioanalysis was performed using a validated method in the Sipra Labs Limited.
Toxicokinetic evaluation was performed within the Department of Drug Metabolism, Pharmacokinetics and Clinical Pharmacology, Advinus Therapeutics Limited using the non-compartmental analysis tool of validated WinNonlin® software (version 5.2). The toxicokinetic parameters that were calculated include area under the concentration - time curve until the last sampling point (AUC0-24h), peak plasma concentration (Cmax), time for the peak plasma concentration (Tmax) and half-life (T1/2). The elimination rate constant value (k) was obtained by linear regression of the log-linear terminal phase of the concentration-time profile using 3 declining concentrations in terminal phase with a correlation coefficient of >0.8. The terminal half-life value (T1/2) was calculated using the equation ln2/k. Dose-exposure relationship, accumulation on repeated administration was also be assessed. After last (24 hour) blood collection, the animals in the toxicokinetic groups were euthanized under isoflurane anaesthesia and following tissues were collected and frozen immediately for possible toxicokinetic analysis.
1. Brain
2. Muscle
3. Thyroid with parathyroid
4. Femur bone

Statistics:

Parameters such as body weight, net body weight gains, food consumption, organ weights and their ratios, gross and histopathology data were analysed using ProvantisTM built-in statistical tests.
The statistical analysis of the experimental data was carried out using the validated package in Excel and/or using licensed copies of SYSTAT Statistical package Ver.12.0. All quantitative variables like laboratory investigations (haematology, coagulation and clinical chemistry) will be tested for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test) within the group before performing a one-factor ANOVA modeling by treatment groups. Non-optimal (non-normal or heteroschedastic) data was transformed, before ANOVA is performed. Comparison of means between treatment groups and control group was done using Dunnett’s test if the overall treatment, ‘F’ test is found to be significant. All analysis and comparisons were evaluated at the 5 % (P<0.05) level. Statistically significant differences (P<0.05), indicated by the aforementioned tests are designated by the superscripts throughout the report as stated below:
+/-: Significantly higher (+)/lower (-) than the control group

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed in either sex at 30 and 60 mg/kg bw/day dose level during the 28-day treatment period. Toxic signs like dehydration and hypoactivity were observed in few animals of the 100 mg/kg bw/day dose levels. All the high dose (G5; 150 mg/kg bwt/day) animals were found normal and
active during the first 5 days treatment. On day 6 of treatment clinical signs
such as dehydration, tremors, weakness, hypoactivity and piloerection were
observed in most of the animals.

Mortality:

mortality observed, treatment-related

Description (incidence):

No mortalities were observed in the control and low doses in either sex. At 60 and 100 mg/kg bwt/day doses, one female each in the toxicokinetic groups died after 12th hour blood collection on Day 28. Twelve male and seven female rats at 150/120 mg/kg bwt/day died pre-terminally.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males:
At 150/120 mg/kg bwt/day dose, mean body weights and cumulative net body
weight gains on Day 8 were significantly lower.
At 100 mg/kg bwt/day dose, mean cumulative net body weight gains were
significantly lower during Days 8-11 and significantly higher during Days 11-15
and 25-28 of treatment period. Initially the body weight was decreased further it
was compensated by increased body weight so that the terminal body weight
was equal to controls.
At 30 and 60 mg/kg bwt/day doses, mean cumulative net body weight gains
were significantly higher during Days 8-11 and 22-25 of treatment period.The
absolute (G2-16% : G3- 8%) and percentage (G2-15.5% : G3- 6 %) of body
weight gains were apparently higher when compared to concurrent control but
statistically not significant.

Females
At 150/120 mg/kg bwt/day dose, mean body weights on Days 8 and 11 were
significantly lower. The cumulative net body weight gains were lower when
compared to the initial body weight.
The mean cumulative net body weight gains were significantly higher during
Days 1-8 in the 30, 60 and 100 mg/kg bwt/day doses. The absolute (G2+40 %:
G3+ 23 %: G4+32%) and percentage (G2+41 %: G3+ 23 %: G4+34 %) of body
weight gains were apparently higher when compared to concurrent control group
but statistically not significant.
Thus the treatment with Lithium carbonate slightly increased the body weights
at all the tested doses in males and females.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males:
The food consumption was significantly higher during Days 15- 22 and 22-28 in
the 30, 60 and 100 mg/kg bwt/day doses.

Females:
The food consumption was significantly higher during Days 8-15, 15-22 and
22 - 28 in the 100 mg/kg bwt/day dose and during Days 8-15 and 15-22 in the
30 mg/kg bwt/day dose and during Days 15-22 in the 60 mg/kg bwt/day dose.
Thus the treatment with Lithium carbonate increased the food intake at all the
tested doses in both sexes.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

At 150/120 mg/kg bwt/day dose, statistically significant decreased water
consumption was observed in males on Day 8-9, 9-10, and 10-11 before
termination. In females, statistically significant increased water consumption
was observed on Days 8-9 and 9-10.
The water consumption was significantly higher at the 100 mg/kg bwt/day dose
over the entire dose period (G4: 95-228 % males, 83-186 % females when
compared to control). At 30 and 60 mg/kg bwt/day doses, the water
consumption was apparently higher (G2: 2-46 % males, 4-26 % females, G3: 8-
28 % males, 13-46 % females when compared to control) but statistical
significant not achieved.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hematology evaluation showed an increase in the mean platelet volume at
100 mg/kg in both sexes (males-9.60, females-10.57) and at 60 mg/kg in males
(9.88). This was considered as incidental as the differences were minimal and
the values were similar to the in house historical control data (males-10.42,
females-10.61).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In both males and females at 100 mg/kg dose groups, minimal increase (less
than one fold) in the ALT level was considered as treatment related.

Endocrine findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Sensory, open field and homecage as well as handling observations did not show any treatment-related abnormalities in any of the doses tested in both sexes.

Motor activity: At 100 mg/kg bwt/day dose, the motor activity score indicated
signifcantly lower distance traveled, stereotypic time, bursts of stereotype
movement and horizontal counts and significantly higher resting period in
males. Significantly higher resting period was observed, and is considered an
indication of hypoactivity of the animals. In females no significant changes
observed
At 30 and 60 mg/kg bwt/day doses, no significant changes observed
Neuromuscular observation:
Landing hind limb footsplay: No significant changes were observed at all the
tested doses except for significantly higher hind limb foot splay in the 100
mg/kg bwt/day dose in females. The observation is considered as an incidental
finding as no changes were observed in other parameters.
Grip strength: At 100 mg/kg bwt/day dose, significantly lower hind limb grip
strength was observed in males. In females, no significant changes observed.
At 30 and 60 mg/kg bwt/day doses, no significant changes observed.

Physiological observation:
Body temperature: The physiological observation of body temperature was
unaffected in both sexes except for lower body temperature in the 30 mg/kg
bwt/day dosed males. The observation is considered as an incidental finding as
no changes were observed in other parameters.
Thus, the changes observed in motor activity and decreased grip strength at the
100 mg/kg bwt/day dose males was an indicative of hypoactivity of the animals
and considered treatment - related.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The terminal fasting body weight and organ weight parameters were performed
upto 100 mg/kg dose groups as all the rats at 150/120 mg/kg dose groups were
either pre-terminally found dead or moribund sacrificed on or before treatment
day 11.
Adrenal weight changes were observed in both sexes. At 100 mg/kg relative
weight increase was observed in males and both absolute and relative weights
were increased in females. At 60 mg/kg absolute and relative adrenal weight
changes were observed only in females.
In females, liver weight increase (absolute and relative) was observed at 100
mg/kg and in the lower dose groups (30 and 60 mg/kg) the increase was
observed only in the absolute weights. Lower weight of spleen at 100 mg/kg in
males was considered as treatment related associated with the decreased body
weight. This weight decrease could be associated with the stress due to
treatment.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The treatment did not reveal any gross pathological findings up to 100 mg/kg
bwt/day dose groups. At 150/120 mg/kg bwt/day dose group, small sized
thymus and spleen were observed in both sexes and small sized seminal vesicles
were observed in males. These changes could be associated with the in life
observations of body weight loss and decreased feed intake due to the presence
of exaggerated clinical signs. The single incidence of liver discolouration
observed in the dead male at 150/120 mg/kg bwt/day dose group was not
regarded as treatment related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histopathological examination was performed at 0, 30 and 60 mg/kg.
Microscopic examination revealed the following changes at 60 mg/kg bwt/day
dose group.
Liver: No treatment related microscopic changes were observed in
males. In females, minimal degree of hepatocellular hypertrophy of diffuse
distribution was observed at 60 mg/kg bwt/day (4/6) and this finding was
considered as a treatment related metabolic adaptive response.
Kidneys: Increased incidences of dilated tubules were observed in the
cortex region in both sexes of 60 mg/kg bwt/day dose groups (males-3/6,
females-4/6). This change was considered as treatment related.
Adrenals: In males, increased corticular cell vacuolation was observed
at 60 mg/kg bwt/day (3/6). Females did not show any significant
morphological alteration between the control and 60 mg/kg bwt/day dose
groups.
Thyroids, parathyroids, pituitary and brain did not show any treatment related
microscopic changes.
At the 30 mg/kg bwt/day dose no histopathological changes observed.

Histopathological findings: neoplastic:

no effects observed

Details on results:

Toxicokinetics
Following oral administration of lithium carbonate solution to rats, the maximum plasma concentration of lithium was measured at 0.5 hour across dose levels, except in males at 100 mg/kg bwt/day on Day 1 where it was 1h and in males dosed at 150 mg/kg bwt/day on Day 1 where the Tmax was observed at 4 h. The plasma exposure (AUClast) of lithium was comparable between genders, suggesting no gender difference. The male to female AUClast ratio ranged from 1.07 to 1.31 across dose levels on Day 1. The male to female Cmax ratio ranged from 0.98 to 1.06.The plasma exposure (AUClast and Cmax) of lithium increased linearly with increase in dose on Day 1 and Day 28 in both the genders. Overall a 5-fold increase in dose from 30 mg/kg bwt/day to 150 mg/kg bwt/day resulted in 3.5 and 3.4-fold increase of lithium AUClast in male and female rats, respectively on Day 1. A 2-fold increase in dose from 30 mg/kg bwt/day to 60 mg/kg bwt/day led to 1.32 and 1.43-fold increase in Cmax in males and females, respectively. Further 1.67-fold increase in dose from 60 mg/kg bwt/day to 100 mg/kg bwt/day led to 1.31 and 1.22 –fold increase in Cmax in males and females, respectively and 1.50-fold increase in dose from 100 mg/kg bwt/day to 150 mg/kg bwt/day led to 1.20 and 1.15-fold increase in Cmax in males and females, respectively. The Day 28 to Day 1 AUClast ratio ranged from 1.01 to 1.07 in males and 0.79 to 1.18 in female rats. The Day 28 to Day 1 Cmax ratio ranged from 1.16 to 1.54 in males and 1.10 to 1.44 in female rats. On repeat administration of lithium carbonate for 28 days there was no indication of accumulation. No quantifiable concentrations of lithium were observed in the plasma samples obtained from animals in the control group.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

30 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

no

System:

hepatobiliary

Organ:

liver

2

Key result

Critical effects observed:

no

System:

urinary

Organ:

kidney

Applicant's summary and conclusion

Conclusions:

Considering higher net body weight gains, food and water consumption at 30, 60 and 100 mg/kg doses, the microscopic changes of hepatocellular hypertrophy in liver of females, increased cortical vacuolation in adrenals of males and dilated tubules of kidneys in both sexes at 60 mg/kg dose “No Observed Effect Level (NOEL)” is less than 30 mg/kg following 28 days of repeated oral administration of Lithium carbonate to Wistar rats under the test conditions and doses employed. However, the changes in food and water consumption were not considered to be adverse, therefore the “No Observed Adverse Effect Level (NOAEL)” is considered to be 30 mg/kg following 28 days of repeated oral administration of Lithium carbonate to Wistar rats under the test conditions and doses employed.

Executive summary:

The objectives of this study was to assess the toxicity potential of test item Lithium carbonate when administered to Wistar rats orally through gavage for 28 consecutive days and also to provide information for selecting the dose levels to a subsequent two generation reproduction toxicity study. The test item was dissolved in Milli-Q water, administered orally by gavage at dose levels of 30, 60, 100 and 150 mg/kg bw/day to low (G2), mid (G3), mid intermediate (G4) and high (G5) dose group of rats, respectively at a dose volume of 10 mL/kg bw/day. Each group consisted of 6 male and 6 female rats. Additional groups of 6 male and 6 female rats per treatment group (G2TK, G3TK, G4TK and G5TK) were treated with Lithium carbonate for collecting blood for toxicokinetics (TK) at designated time points of 0 (Day 28 only), 0.5, 1, 2, 4, 8, 12 and 24 hours post dosing on Days 1 and 28. In the vehicle control group (G1TK), 3 rats /sex were sampled for TK at pre-dose (0 hour) and 2 hours after dosing on Days 1 and 28. During the course of the experiment, treatment at 150 mg/kg bw/day dose caused toxic signs viz., dehydration, weakness, tremors, hypoactivity and piloerection from treatment Day 6 onwards. Further, 2 female rats in the toxicokinetic group died pre-terminally on Day 8. Hence, the high dose was reduced from 150 mg/kg bw/day to 120 mg/kg bw/day on Day 9. Further 12 male and 5 female rats died from Days 9 to 11. Hence, the remaining surviving animals in this group were sacrificed on Day 11 to avoid unnecessary sufferings of animals. All rats were observed for clinical signs, detailed clinical examination, and functional (neurological) abnormalities, changes in body weight, food and water consumption. The rats were fasted overnight (water allowed) and subjected to a full detailed necropsy at the end of the treatment and study plan specified organs were weighed and examined histopathologically. Toxicokinetic group animals were euthanized and specified organs were collected and frozen immediately for possible toxicokinetic analysis.

Following were the results obtained:

 

Clinical signs: There were no clinical signs observed in either sex at 30 and 60 mg/kg bw/day dose level during the 28-day treatment period. Toxic signs like dehydration and hypoactivity were observed in few animals of the 100 mg/kg bw/day dose levels. All the high dose (G5; 150 mg/kg bw/day) animals were found normal and active during the first 5 days treatment. On day 6 of treatment clinical signs such as dehydration, tremors, weakness, hypoactivity and piloerection were observed in most of the animals.

 

Mortality: Twelve male and seven female rats at 150/120 mg/kg bw/day died pre-terminally. There was no mortality observed at 30, 60 and 100 mg/kg bw/day doses.

 

Neurological examination: No treatment-related neurological abnormalities /dysfunctions were observed at 30 and 60 mg/kg bw/day doses. At 100 mg/kg bw/day dose, the motor activity score indicated signficantly lower distance traveled, stereotypic time, bursts of stereotype movement and horizontal counts and significantly higher resting period in males. Significantly lower hind limb grip strength was also observed in males. In females no significant changes were observed up to 100 mg/kg bw/day.

 

Body weights and body weight gain: At 150/120 mg/kg bw/day dose, mean body weights were significantly lower in both sexes. The cumulative net body weight gains were lower when compared to the initial body weight. At 100 mg/kg bw/day dose, mean cumulative net body weight gains were significantly lower during Days 8-11 and significantly higher during Days 11-15 and 25-28 of treatment period in males. Initially the body weight was decreased further it was compensated by increased body weight so that the terminal body weight was equal to controls. In females, the mean cumulative net body weight gains were significantly higher during Days 1-8. The absolute and percentage of body weight gains were apparently higher when compared to concurrent control group but statistically not significant. At 30 and 60 mg/kg bw/day doses in males, mean cumulative net body weight gains were significantly higher during Days 8-11 and 22-25 of treatment period. The absolute and percentage of body weight gains were apparently higher when compared to concurrent control but statistically not significant. In females, the mean cumulative net body weight gains were significantly higher during Days 1-8. The absolute and percentage of body weight gains were apparently higher when compared to concurrent control group but statistically not significant.

 

Food consumption: The food consumption was significantly higher during Days 15- 22 and 22-28 in the 30, 60 and 100 mg/kg bw/day doses in males. In females, the food consumption was significantly higher during Days 8-15, 15-22 and 22 - 28 in the 100 mg/kg bw/day dose and during Days 8-15 and 15-22 in the 30 mg/kg bw/day dose and during Days 15-22 in the 60 mg/kg bw/day dose. Thus, the treatment with Lithium carbonate increased the food intake at all the tested doses in both sexes except for 150/120 mg/kg bw/day dose.

 

Water consumption: At 150/120 mg/kg bw/day dose, statistically significant water consumption was observed in males on Day 8-9, 9-10, and 10-11 before termination. In females, statistically significant increased water consumption was observed on Days 8-9 and 9-10. Water consumption was significantly higher at 100 mg/kg bw/day dose in both the sexes over the entire dose period. At 30 and 60 mg/kg bw/day doses, the water consumption was also slightly higher when compared to vehicle control in both the sexes, however not statistically significant.

 

Toxicokinetics: After oral administration of lithium carbonate solution to rats, the maximum plasma concentration of lithium was measured at 0.5 hour across dose levels, except in males at 100 mg/kg bw/day on Day 1 where it was 1h and in males dosed at 150 mg/kg bw/day on Day 1 where the Tmax was observed at 4 h. The plasma exposure (AUClast) of lithium was comparable between genders, suggesting no gender difference. The male to female AUClast ratio ranged from 1.07 to 1.31 across dose levels on Day 1. The male to female Cmax ratio ranged from 0.98 to 1.06. The plasma exposure (AUClast and Cmax) of lithium increased linearly with increase in dose on Day 1 and Day 28 in both the genders. Overall a 5-fold increase in dose from 30 mg/kg bw/day to 150 mg/kg bw/day resulted in 3.5 and 3.4- fold increase of lithium AUClast in male and female rats, respectively on Day 1. A 2-fold increase in dose from 30 mg/kg bw/day to 60 mg/kg bw/day led to 1.32 and 1.43-fold increase in Cmax in males and females, respectively. Further 1.67-fold increase in dose from 60 mg/kg bw/day to 100 mg/kg bw/day led to 1.31 and 1.22 –fold increase in Cmax in males and females, respectively and 1.50-fold increase in dose from 100 mg/kg bw/day to 150 mg/kg bw/day led to 1.20 and 1.15-fold increase in Cmax in males and females, respectively. The Day 28 to Day 1 AUClast ratio ranged from 1.01 to 1.07 in males and 0.79 to 1.18 in female rats. The Day 28 to Day 1 Cmax ratio ranged from 1.16 to 1.54 in males and 1.10 to 1.44 in female rats. On repeat administration of lithium carbonate for 28 days there was no indication of accumulation.

 

Clinical pathology investigation: There were no treatment-related changes in the hematology and coagulation parameters. Minimal increase in ALT activity was observed in both the sexes at 100 mg/kg bw/day dose (39 and 67% versus controls, respectively in males and females).

 

Terminal fasting body weights, Organ weights and organ weight ratios: Organ weight and organ weight ratios showed increased adrenal weights in both the sexes and liver weight in females at 100 mg/kg bw/day dose. In females, absolute and relative adrenal weight increase was also observed at
60 mg/kg and absolute liver weight increases were also seen at 30 and 60 mg/kg bw/day dose groups. Lower spleen weight was observed at 100 mg/kg bw/day in males, however not statistically significant.

 

Gross and histopathology: Grossly, at 150/120 mg/kg bw/day dose group, small sized thymus and spleen in both the sexes and small sized seminal vesicles in males were observed. Histopathological examination was performed at 0, 30 and 60 mg/kg. Microscopically, hepatocellular hypertrophy in liver of females, increased cortical vacuolation in adrenals of males and dilated tubules of kidneys in
both sexes at 60 mg/kg bw/day dose. These effects were not observed at the 30 mg/kg dosed animals.

 

Considering higher net body weight gains, food and water consumption at 30, 60 and 100 mg/kg doses, the microscopic changes of hepatocellular hypertrophy in liver of females, increased cortical vacuolation in adrenals of males and dilated tubules of kidneys in both sexes at 60 mg/kg dose, the “No Observed Effect Level (NOEL)” is less than 30 mg/kg following 28 days of repeated oral administration of Lithium carbonate to Wistar rats under the test conditions and doses employed. However, the changes in body weight, food and water consumption were not considered to be adverse, therefore the “No Observed Adverse Effect Level (NOAEL)” is considered to be 30 mg/kg following 28 days of repeated oral administration of Lithium carbonate to Wistar rats under the test conditions and doses employed.