Lithium bromide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The NOAEL for systemic toxicity is based on various findings at 45 mg/kg/bw regarding in vivo changes on body weight and water consumption, but also pronounced morphological changes in liver and kidneys. Some variations in one or the other sex on adrenals and thyroid glands (P generation however not in the F1 or F2 generation) were noted. A value of 15 mg/kg bw was derived as the systemic NOAEL.
The NOAEL for reproductive toxicity and foetal toxicity is considered to be 45 mg/kg bw/day as no obvious reproductive changes were observed in both generations.
Based on read-across the NOAEL for lithium bromide is 35 mg/kg bw/day for the parental generation (systemic toxicity) and 106 mg/kg bw/day for the P, F1 and F2 generation (reproductive and foetal toxicity).

Link to relevant study records

Reference

Reference 1

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-01-12 to 2012-01-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

January 22nd, 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

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 Netherlands)
- Age at study initiation: 8 - 10 weeks
- Weight at study initiation: (P) Males: 211 - 254 g; Females: 158 - 190 g;
- Fasting period before study: no
- Housing:
Pre mating: Rats were housed in groups of two per sex in sterilized standard suspended polysulfone cages (size: L 425 x B 266 x H 175 mm) with stainless steel top grill having facilities for holding pellet food and drinking water in a polycarbonate bottle with stainless steel sipper tubes. The last animal in all groups of P-Generation and G1, G3 and G4 groups were housed individually. The last 3 animals of G2 group in F1-Generation were housed individually.
Mating and Post mating: During mating, two animals were housed in a ratio of 1:1 (one male: one female). After confirming Day ‘0’ pregnancy by examining for presence of sperm in the vaginal smear/vaginal plug, males were transferred to their original cages. The females were housed individually in polysulfone cages (size: L 425 x B 266 x H 175 mm) during gestation and lactation period until weaning and sacrifice. The sterilized nesting material (paper shreds) was provided on GD 20 or near term. The steam sterilized corn cob was used as bedding material throughout the experiment. The cage along with bedding material was changed at least once a week.
- Diet (e.g. ad libitum): ssniff rats/mice pellet food - maintenance manufactured by ssniff Spezialdiäten GmbH. Ferdinand-Gabriel-Weg 16, D-59494 Söest, Germany was provided ad libitum to the animals.
- Water (e.g. ad libitum): Deep bore well water passed through activated charcoal filter and exposed to UV rays in Aquaguard online water filter-cum-purifier manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India, was provided ad libitum to animals in polycarbonate bottles with stainless steel sipper tubes.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 42 - 68
- Air changes (per hr): 12 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: Milli-Q water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Required quantity of the test item was weighed and mixed in Milli-Q water to attain desired concentrations of 0.5, 1.5 and 4.5 mg/mL for the low, mid and high dose groups respectively. Vehicle control group animals were administered Milli-Q water only. Dose formulations were prepared once every 8 days as stock solution for each dose. The prepared stock solution was mixed by inversion before taking for daily use. Homogenity of the dose formulation was maintained by constant stirring using magnetic stirrer. The prepared stock solution was stored in the experimental room. Following procedure was adopted when 1000 mL of dose solution was prepared;
The quantities of 0.5, 1.5 and 4.5 g of test item was weighed and mixed in Milli-Q water to attain desired concentrations of 0.5, 1.5 and 4.5 mg/mL for the low, mid and high dose groups respectively. The weight of the test item and the volume of the test item prepared varied depending upon the requirement.

VEHICLE
- Concentration in vehicle: 0.5, 1.5, 4.5 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Details on mating procedure:

- M/F ratio per cage: 1:1
- Length of cohabitation: 2 weeks
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): The females were housed individually in polysulfone cages (size: L 425 x B 266 x H 175 mm) during gestation and lactation period until weaning and sacrifice. The sterilized nesting material (paper shreds) was provided on GD 20 or near term. The steam sterilized corn cob was used as bedding material throughout the experiment. The cage along with bedding material was changed at least once a week.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For active ingredient concentration analysis, samples of test formulation were taken from all doses including vehicle control, prepared on Day 1 and once in 3 months intervals thereafter during the treatment period. The collected samples were sent to Analytical R&D Department of Advinus Therapeutics Limited, Bangalore for concentration analysis. The method of analysis was the same as the validated method done under Advinus Study No. G7467.
The test item concentrations in gavage prepared for dosing on 18.01.2011, 12.04.2011, 12.07.2011 and 20.09.2011 with nominal concentrations of the test item in gavage samples 0 mg/mL, 0.5 mg/mL, 1.5 mg/mL and 4.5 mg/mL indicated that the test item concentrations in solution were within the permissible limits of ± 15 %.from the nominal concentrations.

Duration of treatment / exposure:

(P) Treatment commenced from the age of 9 weeks and continued throughout the treatment period until F1 litters were weaned. Parents and pups not selected for F1 generation were sacrificed.
(F1) Treatment commenced for F1 generation from the time of weaning and continued until F2 were weaned and sacrificed.

Frequency of treatment:

The test item was administered to rats of the specific groups once in the afternoon hours on Day 1 for both males and females of P-Generation. From Day 2 onwards the test item was administered once daily in the morning hours.
Male rats: The test item was administered to the specific group of rats once daily at approximately the same time each day (varied by ± 2 hours) for at least 10 weeks prior to the mating period. Treatment was continued during mating and up to and including the day before sacrifice which was done after the completion of the mating process.
Female rats: As in males, females received the test item once daily at approximately the same time each day (varied by ± 2 hours) for at least 10 weeks prior to mating. Treatment was continued through mating, pregnancy and up to the weaning of F1 offspring, after which, parental females were sacrificed. F1-generation offspring were treated from weaning till they were sacrificed after obtaining F2 weanlings.
Vehicle control group (G1) animals were administered Milli-Q water only throughout the study.

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
- Age at mating of the mated animals in the study: 13 weeks

Remarks:

Doses / Concentrations:
0
Basis:
nominal conc.
mg/mL

Remarks:

Doses / Concentrations:
0.5
Basis:
nominal conc.
mg/mL

Remarks:

Doses / Concentrations:
1.5
Basis:
nominal conc.
mg/mL

Remarks:

Doses / Concentrations:
4.5
Basis:
nominal conc.
mg/mL

No. of animals per sex per dose:

25 animals per sex and dose for the parental and F1 generation.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
Dose selection was based on different studies regarding repeated oral exposure as well as oral exposure of pregnant rats with lithium carbonate (Ibrahim at al., 1990; Fritz et al., 1988; Marathe & Thomas et al., 1986; Hansen et al. , 2010). Further a dose range finding study was conducted treating male and female rats for 28 consecutive days orally by gavage (Study No. G7468).
Based on the available literature and experimental data provided, the dose levels of 5, 15 and 45 mg/kg bw/ day were selected for this two generation toxicity study in consultation with the Sponsor. In addition to the test doses, a vehicle control was included. Animals in the vehicle control were handled in a manner similar to the treatment groups except for test item administration.

Positive control:

No positive controls

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights of parental rats were recorded initially and at weekly intervals thereafter in males and in the pre mating period in females for both generations. All dams were weighed on presumed gestation days 0, 7, 14 and 20 and on lactation days 1, 4, 7, 14 and 21 and weights were recorded.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. After day 0 of pregnancy the food intake was recorded on gestation days (GD) 7, 14, 20 and on lactation days (LD) 4, 7, 14, 21.

WATER CONSUMPTION: Yes
- Time schedule for examinations: thrice a week for males and females. After Day 0 pregnancy, the water intake was recorded on presumed GDs 4, 7, 10, 14, 18 and 20 and on LDs 4, 7, 11,14, 16, 18 and 21.

Food and water consumption for both sexes was not measured during the cohabitation period.

Oestrous cyclicity (parental animals):

The oestrous cycle length and pattern was evaluated by vaginal smears examination for all females during a minimum of 2 weeks prior to mating and during mating. The oestrous cycle length was calculated for all females as the period between two successive diestrus stages.

Sperm parameters (parental animals):

For all the P and F1 males at termination, sperm from the right vas deferens was collected for evaluation of sperm motility. The sperm smears for the sperm morphology were prepared for all animals but evaluation was performed for the randomly selected 10 animals per group only. Likewise the right testis and corresponding epididymis were collected from all males for enumeration of homogenisation detergent resistant testicular spermatids and cauda epididymal sperm reserves, respectively. The sperm count was restricted to the selected animals. As there were no treatment-related effects observed in the sperm morphology, testicular spermatid count and epididymal sperm count, the examination was not extended to the remaining animals in control and high dose as well as all the animals in the lower dose groups. The frozen testes and epididymides samples were discarded.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were sacrificed after completion of the mating process.
- Maternal animals: All surviving animals were sacrificed after the last litter of each generation was weaned.

GROSS NECROPSY
- Gross necropsy consisted of the following tissues and organs for P and F1 parental animals: Adrenal glands, Brain, Epididymides, Gross lesions, Kidneys, Testes, Liver, Ovaries, Pituitary, Prostate, Sciatic nerves, Seminal vesicles and coagulating glands and their fluid, Spinal cord (cervical, thoracic and lumbar), Spleen, Thyroid with parathyroids, Uterus (with oviducts and cervix), Vagina
- The following tissues were collected from one randomly selected pup per sex per litter (F1 and F2 offspring): Brain, Coagulating glands, Epididymides, Gross lesions, Kidneys, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Uterus and vagina

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of the following tissues that were collected from one randomly selected pup per sex per litter (F1 and F2 offspring): Brain, Coagulating glands, Epididymides, Gross lesions, Kidneys, Ovaries, Prostate, Seminal vesicles, Spleen, Testes, Thymus, Uterus and vagina

HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated above were prepared for microscopic examination and weighed, respectively.

Statistics:

The statistical analysis of the experimental data was carried out using the validated package in Excel and using licensed copies of SYSTAT Statistical package ver.12.0. All quantitative variables like body weight, feed intake, spermatology parameters, organ weights and organ weight ratios data were tested for homogeneity of variances (Levene’s test) within the group before performing One-way analysis of variance (ANOVA). When the data are found to be non-optimal (non-normal or heteroschedastic), ANOVA was done using suitable transformation. Comparison of means between treatment groups and vehicle control group was done using Dunnett’s test when the overall treatment ‘F’ test is found to be significant. For the characters namely pre-implantation loss (%), post implantation loss (%), number of corpora lutea, implantations and pre-coital interval (days) was analysed after suitable transformation (Arc sine, √ x + ½) of the data. One-way analysis of variance (ANOVA) was carried out for the transformed data. Dunnett’s pair-wise comparison of the treated means with the control mean was done when the group differences are found significant. Z test was performed for testing the differences in proportions for the characters namely mating and fertility indices. Since the parametric tests proposed above are expected to be applicable and more efficient, the non parametric test (Kruskal Wallis followed by Mann Whitney U test) was used only for the non normal data measured in the nominal and ordinal scales wherever necessary. 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 vehicle control group

Reproductive indices:

The following indices were determined:
Male mating index, female mating index, male fertility index, female fertility index and the fecundity index.

Offspring viability indices:

The following indices were determined:
live birth index, 24 hour survival index, 4th day survival index, 7th day survival index, 14th day survival index and 21st day survival index.

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no clinical signs and mortalities observed in control, 5 and 15 and 45 mg/kg bw/day doses. However, incidences of hair thinning with hair re-growth were randomly observed in all the groups. These were considered incidental as these are common findings in rodents.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Males: The mean body weights were comparable with the control group during weeks 1 to 10 of treatment at 45 mg/kg bw/day dose group. However, the treatment significantly increased the mean body weights (5.9 to 7.1 %) during weeks 11 to 14 and net weight gains (16.6 %) at the end of 14 weeks of treatment when compared to vehicle control group. The weekly mean body weights and net weight gains were unaffected by the treatment at 5 and 15 mg/kg bw/day doses when compared to the vehicle control group.
Significantly higher food intake was observed during weeks 2 to 10 (8.2 to 12.7 %) at 45 mg/kg bw/day dose when compared to the vehicle control. The food intake was unaffected by the treatment at 5 and 15 mg/kg bw/day doses when compared to the vehicle control group.

Females: The mean body weights were unaffected by the treatment at 45 mg/kg bw/day dose. However, the net weight gains were apparently higher (9.7 %) when compared to the vehicle control but statistically not significant at the end of 10 weeks of treatment. The weekly mean body weights and net weight gains were unaffected by treatment at 5 and 15 mg/kg bw/day doses when compared to the vehicle control group.
Significantly higher food intake was observed during weeks 4 – 6 (7.9 to 9.3%) at 45 mg/kg bw/day when compared to the vehicle control. The food intake was not altered by the treatment at 5 and 15 mg/kg bw/day doses.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The calculated mean oestrous cycle length was 4.07, 3.90, 3.85 and 3.95 days in vehicle control, 5, 15 and 45 mg/kg bw/day doses, respectively. The mean oestrous cycle length in the treated groups was not significantly different from the vehicle control group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There were no intergroup differences in the percentage of total motility, percentage of progressive sperm motility and sperm morphology evaluated. Cauda epididymal sperm counts and testicular spermatid counts data were comparable between control and 45 mg/kg bw/day dose group. The minimal weight decrease observed in the cauda epididymides weight at 45 mg/kg bw/day did not show any changes in the sperm counts and hence considered as not toxicologically relevant. The percentage of progressive motile sperms in 15 and 45 mg/kg bw/day dose groups were higher. These minimal changes however were considered as not toxicologically relevant.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Pre-coital Time:
The mean pre-coital interval was apparently higher but statistically not significant and was considered incidental, as the interval was within the normal biological range. Further these finding was within the historical range.

Gestation Length:
There were no treatment-related effects on the gestation length (average days to litter) at 5 and 15 mg/kg bw/day doses. At 45 mg/kg bw/day dose, the gestation length (average days to litter) was significantly longer when compared to concurrent vehicle control. This was however considered incidental as the increase was within the historical range.

Fertility Indices:
No treatment-related changes were observed in the fertility indices of sires and dams. The incidences of higher male and female fertility indices observed at 45 mg/kg bw/day were considered toxicologically insignificant as the significance was due to the slightly lower control value. No treatment-related changes were observed in the uterine/implantation data except slightly higher post-implantation loss at 45 mg/kg bw/day dose, which subsequently led to lower mean litter size. The post-implantation loss observed was due to the lower number of pups in four dams (Rk5876, Fk5879, Rk5881, and Rk5889) only.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In liver, a significant increase in the absolute and relative weights was observed at 45 mg/kg bw/day in males. This change corresponded to the microscopic finding of higher incidences of increased hepatocyte cytoplasmic rarefaction in the livers at 45 mg/kg bw/day. In adrenals, a significant increase in the absolute and relative weights (combined and individual weights) was observed at 45 mg/kg bw/day in males. This increase corresponded to the microscopic finding of increased cortical cell vacuolation observed at 45 mg/kg bw/day in males. The increase in the absolute but not relative thyroid weight with no histopathological correlations was not considered treatment related. The increase in the thyroid weight in 45 mg/kg bw/day dose group males was considered test item related. This was not associated with any microscopic change in thyroid at 45 mg/kg bw/day. Some variations in other organ weights were noted but none were considered to be test item related.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no test item-related gross findings in males or females.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Microscopically, higher incidence of increased cytoplasmic rarefaction was observed in the liver at 45 mg/kg bw/day dose group in males. In females, higher incidences of focal basophilic hepatocytes and hepatocellular hypertrophy were observed at 45 mg/kg bw/day. Hepatocellular hypertrophy was of minimal severity and not observed in the lower dose groups. The basophilic hepatocytes involved approximately 10 to 15 hepatocytes with focal distribution. The relation of this lesion to test item administration is not clear as only low incidences were observed with minimal severity and focal distribution. In kidneys, higher incidences with minimal severity of dilated tubules were observed in 45 mg/kg dose groups of both males and females (11/25 and 3/25, respectively). In addition, a slightly more severe (mild) dilatation was observed also in males and females (10/25 and 13/25, respectively) Increased incidences were also observed at 15 mg/kg bw/day in males (P: 11/25, F1:6/25) and females (P: 3/25, F1:8/25). Adrenals showed higher incidences of cortical vacuolation in males at 45 mg/kg bw/day. In thyroid glands, increased colloid was observed in the follicular lumen at 45 mg/kg bw/day in females. Microscopic examination of reproductive organs did not reveal any test item related changes.

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Key result

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Effect level:

45 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings:

effects observed, treatment-related

VIABILITY (OFFSPRING)
At the doses tested there were no treatment-related effects on the number of pregnancies of the parental generation, number littered and number of live litters. The mean litter and mean viable litter size was not altered by the treatment at 5 and 15 mg/kg bw/day doses. At the high dose, the mean litter size and mean viable litter size were significantly lower when compared to vehicle control, however the findings fell well within historical range (HD range for mean litter size is 8.3 to 12.3), whereas the control values (mean values of 12.2 and 12.0, respectively) were at the higher boundaries of the historical control range. Therefore this finding was not considered to be toxicologically relevant. There were no external abnormalities in live or dead pups in any of the groups tested. No treatment-related changes were observed in the survival data of pups up to lactation day 21 at all the doses tested. An incidence of higher number of pup deaths up to Day 4 was observed at 45 mg/kg bw/day dose. This variation was due to the fact that 8 pups were dead in one dam (Rk5879) of no good health. With this one dam the overall rate was within the historical control range of 2-16.

CLINICAL SIGNS (OFFSPRING)
There were no clinical signs observed in control, 5 and 15 and 45 mg/kg bw/day doses. However, incidences of hair thinning with hair re-growth were randomly observed in all the groups. These were considered incidental as these are common findings in rodents. One male rat (Rk5986) died during blood collection in the 45 mg/kg bw/day dose group prior to sacrifice because of overdose of anaesthesia. One female rat died on GD 24 (Rk6042) due to dystocia in the 15 mg/kg bw/day dose group. The cause of death could not be ascertained as there were no gross and microscopic changes observed in this animal, so the findings were not considered test item related.

BODY WEIGHT (OFFSPRING)
There were no intergroup differences in the terminal body weights in both males and females.

SEXUAL MATURATION (OFFSPRING)
Microscopic examination of reproductive organs did not reveal any test item related changes. No test item related microscopic findings were observed in both male and female pups of F2 litters. There were no significant differences in the follicle counts between vehicle control and 45mg/kg bw/day dose group females.

ORGAN WEIGHTS (OFFSPRING)
In males test item related increase in the liver weight (absolute and relative) was observed at 45 mg/kg bw/day. This change corresponded to the microscopic finding of higher incidences of increased hepatocyte cytoplasmic rarefaction in the livers at 45 mg/kg bw/day. There were no statistically significant inter group differences in the organ weights of females.

GROSS PATHOLOGY (OFFSPRING)
There was a higher incidence of unilateral pelvis dilatation of kidneys at 45 mg/kg bw/day males. In females, there were no test item related gross pathology findings.

HISTOPATHOLOGY (OFFSPRING)
Microscopically, higher incidences of increased cytoplasmic rarefaction were observed in the liver at 45 mg/kg bw/day in males. In females, incidences of basophilic hepatocytes were observed at 45 mg/kg bw/day. The basophilic hepatocytes involved approximately 10 to 15 hepatocytes with focal distribution. The relation of this lesion to test item administration is not clear as only low incidences were observed with minimal severity and focal distribution. In kidneys, higher incidences with minimal severity of dilated tubules were observed in males and females at 15 mg/kg (6/25 and 8/25 respectively) and 45 mg/kg (19/25 and 16/25 respectively) and were considered as test item related. In addition, slightly more severe (mild) dilation was only seen in 2/25 males of 45 mg/kg group. The test item related microscopic changes observed in adrenals of males and thyroid of females in P generation were not evident in F1 generation parental animals.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

45 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

The NOAEL for systemic toxicity is based on various findings at 45 mg/kg/bw regarding in vivo changes on body weight and water consumption, but also pronounced morphological changes in liver and kidneys. Some variations in one or the other sex on adrenals and thyroid glands (P generation however not in the F1 or F2 generation) were noted. A value of 15 mg/kg bw was derived as the systemic NOAEL.
The NOAEL for reproductive toxicity and foetal toxicity is considered to be 45 mg/kg bw/day as no obvious reproductive changes were observed in both generations.

Executive summary:

This two generation reproduction toxicity study in rats with lithium carbonate was performed according to OECD Guideline No. 416 (2001). The test item was dissolved in Milli-Q water and administered orally to Wistar rats at dose levels of 5, 15 and 45 mg/kg bw/day. Similarly, concurrent vehicle control group animals were administered Milli-Q water (vehicle) alone.

A stability and homogeneity study (Advinus Study No.: G7467) for lithium carbonate was carried out at concentrations of 0.1 and 100.0 mg/mL in Milli-Q water. The results indicated that the test item was homogeneous in the vehicle and stable for up to 8 days at both the concentration levels when stored at room temperature. The dose formulations were analyzed for test item concentrations on Day 1 and once in every 3 months during the treatment period. The results indicated that the mean concentrations were within the 10% permissible variation against the nominal concentrations of 0.5, 1.5 and 4.5 mg/mL.

Each group consisted of 25 male and 25 female rats. Animals from all groups were observed for clinical signs, behaviour, physical abnormalities and changes in body weight, food and water consumption during various phases of the experiment. The oestrous cycle length and pattern was evaluated by vaginal smears examination for all females during 2 weeks prior to mating. After a minimum of 10 weeks of treatment, females were cohabitated with males in a 1:1 (one male to one female) ratio. The number, weight, survivability and mortality of pups were observed during the lactation period. Physical signs of postnatal development were observed daily until the criterion was met. Vaginal opening and preputial separation were also observed in pups selected for the F1-generation.

The animals were subjected to detailed necropsy at sacrifice and study plan specified organs were weighed. Andrological assessment like sperm motility was evaluated for all groups, whereas the sperm morphology, enumeration of homogenisation resistant testicular spermatids and caudaepididymal sperm counts were carried out only in control and high dose groups.

Histopathological examination of parents was initially carried out for the preserved organs including gross lesions from control and high dose group animals. Further, based on the microscopic changes observed in the high dose, liver, kidneys and adrenals from males and liver, kidneys and thyroid from females of P generation and liver, kidneys and thyroid from males, liver and kidneys from females of F1 generation were considered as target organs and were examined in lower dose groups. The reproductive organs of non-pregnant females were also examined in the low and mid dose groups.

The left testis was collected in modified Davidson’s fixative and one 4-5 µm thick section was prepared and stained with PAS and Haematoxylin for microscopic examination.

The post lactational ovaries were examined for qualitative depletion of primordial follicles. A quantitative evaluation of primordial and primary follicles was done in F1 females. Ovarian follicle count was carried out for the control and high dose groups and all the not littered females of F1 generation suspected of reduced fertility.

For F1 and F2 weanlings, histopathological examination of the organs of reproductive system and kidneys as potential primary target was carried out for the available one randomly selected pup/sex/litter in all the groups. All gross lesions were also examined for the pups with external abnormalities or clinical signs.

At 5 mg/kg bw/day had no effects on general health, body weights, food and water intake, oestrus cyclicity, preciotal time, gestation length, pups survivability, mating, fertility, fecundity or sperm parameters in both generations. There were no treatment-related changes with regard to any absolute or relative organ weights including reproductive organs and other gross or microscopic findings of parents, offspring or weanlings in both the generations.

At 15 mg/kg bw/day, treatment significantly increased the water intake periodically in males of both generations. There were no effects on general health, body weights, food intake, oestrous cyclicity, pre-coital time, gestation length, pups survivability, mating, fertility, and fecundity or sperm parameters in both the generations. There were no treatment-related changes in reproductive and other organ weights and gross findings of parents or weanlings in both the generations. Microscopically, slightly dilated tubules of kidneys were seen in both generation males and females, however they were considered to be an adaptation to the pharmacology of lithium carbonate (vasopressin-downregulation) and therefore not considered as a toxicological effect.

At 45 mg/kg bw/day, treatment-related findings included increased body weights and net body weight gains in males of P generation and increased water intake in both P and F1 generations in males. Apparently higher net body weight gains were observed in both P and F1 generations premating females. There were no treatment-related changes in reproductive organ weights and gross findings of parents or weanlings in both the generations. There were also no relevant treatment-related changes in oestrous cyclicity, pre-coital time, gestation length, pups survivability, mating, fertility, and fecundity or sperm parameters in both the generations when dose response and historical control ranges were taken into account. Postmortem examination in P generation demonstrated a higher body weight in males, a significant increase in the absolute and relative liver weight in males and in the relative liver weight in females. Furthermore a marginal increase in absolute and relative adrenal weight and an increase in absolute but not in relative weight of thyroid in males only was noted.

In F1 generation, the terminal body weight was not affected. A significant increase in the absolute and relative liver weight was observed in males only.

Microscopically, increased cytoplasmic rarefaction of hepatocytes in liver in males was observed, whereas in females, hepatocellular hypertrophy and focal basophilic hepatocytes were observed. Increased colloids in thyroid follicles of females were also observed. However, these changes were not present in the F1 parental rats. In F1 generation, the terminal body weight was not affected. A significant increase in the liver weight was observed in males. Microscopically, increased cytoplasmic rarefaction of hepatocytes in liver in males was observed. In females, focal basophilic hepatocytes were observed in the liver. Finally, pronounced and severely dilated tubules of kidneys were observed in both generations. Taking into account the steep dose response curve of lithium carbonate, the changes and histopathological findings in kidneys and liver as well as the variations noted with regard to adrenals and thyroids, are considered as an early onset of lithium carbonate systemic toxicity.

Evaluation of pups showed that in both generations, the mean weight of male, female and total pups per litter at all the doses tested were unaffected by treatment and that there were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes were observed in the survival data of pups up to lactation day 21 at all the doses tested. No relevant effects were seen for postnatal developmental observations in F1 and F2 pups such as pinna detachment, incisor eruption, ear opening, and eye opening. The mean age and body weights at acquisition of balano-preputial separation and vaginal opening in F1 were not affected by treatment when compared to vehicle control group. Finally, no test item related microscopic findings were observed in both male and female pups of F1 and F2 litters.

In view of the results observed:

- The “No Observed Adverse Effect Level (NOAEL)” for systemic toxicity in parental rats is considered to be 15 mg/kg bw/day. The effects observed at 45 mg/kg are considered to be of toxicological relevance. At this dose, not only various in vivo changes on body weight and water consumption but also pronounced morphological changes in liver and kidneys and some variations noted in one or the other sex on adrenals and thyroid glands ( in P generation), however, not in the F1 or F2 generation were noted.

- The “No Observed Adverse Effect Level (NOAEL)” for reproductive toxicity and foetal toxicity is considered to be 45 mg/kg bw/day as no clear substances related and biologically relevant effects on reproductive parameters were observed in the P, F1 and F2 generations. (Advinus, 2012)

Reference 2

Endpoint:

two-generation reproductive toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

HYPOTHESIS FOR THE ANALOGUE APPROACH
Lithium bromide completely dissociates in water forming lithium cation and the corresponding bomide anion. Thus, lithium salts with different anion moieties and bromide compounds were found to be suitable candidates for read-across. (Eco)toxicological properties were extrapolated to different endpoints by using the lowest effect concentration.
For further information, please refer to the read-across justification in IUCLID, section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

35 mg/kg bw/day (nominal)

Based on:

other: recalculated for LiBr

Sex:

male/female

Basis for effect level:

other: systemic toxicity

Remarks on result:

other: Based on Li2CO3

Key result

Dose descriptor:

NOAEL

Effect level:

106 mg/kg bw/day (nominal)

Based on:

other: recalculated for LiBr

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Based on Li2CO3

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Effect level:

35 mg/kg bw/day (nominal)

Based on:

other: recalculated for LiBr

Sex:

male/female

Basis for effect level:

body weight and weight gain

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: Based on Li2CO3

Key result

Dose descriptor:

NOAEL

Effect level:

106 mg/kg bw/day (nominal)

Based on:

other: recalculated for LiBr

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: Based on Li2CO3

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

106

Based on:

other: recalculated for LiBr

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Based on Li2CO3

Key result

Critical effects observed:

no

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

106 mg/kg bw/day (nominal)

Based on:

other: recalculated for LiBr

Sex:

male/female

Remarks on result:

other: Based on Li2CO3

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Reference 3

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

not available

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: The study report is not detailed enough and documentation is not available, nevertheless, the study was performed according to an OECD guideline and is scientifically acceptable.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

water

Details on mating procedure:

Not available in the short report.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Males, 42 days
Females, from 14 days before mating to day 4 of lactation

Frequency of treatment:

Daily

Details on study schedule:

Not available in the short report.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

Males, 13; females, 13 per dose group

Control animals:

yes, concurrent vehicle

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Pairing days until copulation tended to be prolonged in the 20 mg/kg and more-treated groups.

Key result

Dose descriptor:

NOEL

Generation:

F1

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects of the treatment on pup body weights and their morphology were observed.

Key result

Reproductive effects observed:

not specified

Conclusions:

Based on the results of the study the NOEL determined for reproductive/developmental toxicity was 5 mg/kg/day in both sexes of animals. The NOEL for the offspring was considered to be 80 mg/kg/day.

Executive summary:

A combined repeated dose and reproductive/developmental toxicity screening test was conducted in rats according to OECD test guideline 422 with the test substance lithium bromide (55.6 % purity). 13 males and 13 females were adminstered with the test substance in concentrations of 0 (vehicle - water), 5, 20, 80 mg/kg bw/day) by gavage for 42 days to males and to females from 14 days before mating to day 4 of lactation. No abnormalities were found regarding the estrous cycle. Some animals in the 20 mg/kg and more-treated groups did not copulate on the first estrous day during the mating period, and the numbers of vaginal estrus and pairing days until copulation tended to be increased in these groups. However, statistical differences were not found in these parameters from those in the control group. Indices of copulation index and fertility were not affected by the lithium bromide treatment. The treatment did not cause abnormal delivery conditions, and gestation length and gestation index were comparable to those in the controls. In addition, no effects of the treatment were observed on parameters indicating viability of embryos/fetuses until delivery, such as numbers of corpora lutea and implantations, the implantation index, number of newborn and the delivery index, and on those indicating postnatal viability, such as number of live newborn, indices of birth and live birth, number of live pups on day 4 of lactation, sex ratios on days 0 and 4 of lactation and viability index on day 4 of lactation. No effects of the treatment on pup body weights and their morphology were observed. A NOEL value of 5 mg/kg bw/day for reproductive/developmental toxicity was determined in both sexes of animals, because the pairing days until copulation tended to be prolonged in the 20 mg/kg and more-treated groups. The NOEL for the offspring was considered to be 80 mg/kg/day for the offspring. A NOEL value of 5 mg/kg bw/day in both sexes was determined for repeat dose toxicity of lithium bromide. (Hatano Institute, date not indicated)

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

106 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

GLP and guideline compliant 2-generation study with highest reliability.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A study according to OECD 416 was initiated by the Lead Registrant and a Member Registrant for non-European regulatory purposes. A summary of the findings is written below.

Key study

As no reproductive toxicity study is available for lithium bromide, read-across with a structural similar compound (lithium carbonate) was performed.

A two generation reproduction toxicity study in rats with lithium carbonate was performed according to OECD Guideline No 416 (2001). The test item was dissolved in Milli-Q water and administered orally to Wistar rats at dose levels of 5, 15 and 45 mg/kg bw/day. Similarly, concurrent vehicle control group animals were administered Milli-Q water (vehicle) alone.

A stability and homogeneity study (Advinus Study No.: G7467) for lithium carbonate was carried out at concentrations of 0.1 and 100.0 mg/mL in Milli-Q water. The results indicated that the test item was homogeneous in the vehicle and stable for up to 8 days at both the concentration levels when stored at room temperature. The dose formulations were analyzed for test item concentrations on Day 1 and once in every 3 months during the treatment period. The results indicated that the mean concentrations were within the 10 % permissible variation against the nominal concentrations of 0.5, 1.5 and 4.5 mg/mL.

Each group consisted of 25 male and 25 female rats. Animals from all groups were observed for clinical signs, behaviour, physical abnormalities and changes in body weight, food and water consumption during various phases of the experiment. The oestrous cycle length and pattern was evaluated by vaginal smears examination for all females during 2 weeks prior to mating. After a minimum of 10 weeks of treatment, females were cohabitated with males in a 1:1 (one male to one female) ratio. The number, weight, survivability and mortality of pups were observed during the lactation period. Physical signs of postnatal development were observed daily until the criterion was met. Vaginal opening and preputial separation were also observed in pups selected for the F1-generation.

The animals were subjected to detailed necropsy at sacrifice and study plan specified organs were weighed. Andrological assessment like sperm motility was evaluated for all groups, whereas the sperm morphology, enumeration of homogenisation resistant testicular spermatids and caudaepididymal sperm counts were carried out only in control and high dose groups.

Histopathological examination of parents was initially carried out for the preserved organs including gross lesions from control and high dose group animals. Further, based on the microscopic changes observed in the high dose, liver, kidneys and adrenals from males and liver, kidneys and thyroid from females of P generation and liver, kidneys and thyroid from males, liver and kidneys from females of F1 generation were considered as target organs and were examined in lower dose groups. The reproductive organs of non-pregnant females were also examined in the low and mid dose groups.

The left testis was collected in modified Davidson’s fixative and one 4-5 µm thick section was prepared and stained with PAS and haematoxylin for microscopic examination.

The post lactational ovaries were examined for qualitative depletion of primordial follicles. A quantitative evaluation of primordial and primary follicles was done in F1 females. Ovarian follicle count was carried out for the control and high dose groups and all the not littered females of F1 generation suspected of reduced fertility.

For F1 and F2 weanlings, histopathological examination of the organs of reproductive system and kidneys as potential primary target was carried out for the available one randomly selected pup/sex/litter in all the groups. All gross lesions were also examined for the pups with external abnormalities or clinical signs.

At 5 mg/kg bw/day had no effects on general health, body weights, food and water intake, oestrus cyclicity, pre-coital time, gestation length, pups survivability, mating, fertility, fecundity or sperm parameters in both generations. There were no treatment-related changes with regard to any absolute or relative organ weights including reproductive organs and other gross or microscopic findings of parents, offspring or weanlings in both the generations.

At 15 mg/kg bw/day, treatment significantly increased the water intake periodically in males of both generations. There were no effects on general health, body weights, food intake, oestrous cyclicity, pre-coital time, gestation length, pups survivability, mating, fertility, and fecundity or sperm parameters in both the generations. There were no treatment-related changes in reproductive and other organ weights and gross findings of parents or weanlings in both the generations. Microscopically, slightly dilated tubules of kidneys were seen in both generation males and females, however they were considered to be an adaptation to the pharmacology of lithium carbonate (vasopressin-downregulation) and therefore not considered as a toxicological effect.

At 45 mg/kg bw/day, treatment-related findings included increased body weights and net body weight gains in males of P generation and increased water intake in both P and F1 generations in males. Apparently higher net body weight gains were observed in both P and F1 generations premating females. There were no treatment-related changes in reproductive organ weights and gross findings of parents or weanlings in both the generations. There were also no relevant treatment-related changes in oestrous cyclicity, pre-coital time, gestation length, pups survivability, mating, fertility, and fecundity or sperm parameters in both the generations when dose response and historical control ranges were taken into account. Postmortem examination in P generation demonstrated a higher body weight in males, a significant increase in the absolute and relative liver weight in males and in the relative liver weight in females. Furthermore a marginal increase in absolute and relative adrenal weight and an increase in absolute but not in relative weight of thyroid in males only was noted.

In F1 generation, the terminal body weight was not affected. A significant increase in the absolute and relative liver weight was observed in males only.

Microscopically, increased cytoplasmic rarefaction of hepatocytes in liver in males was observed, whereas in females, hepatocellular hypertrophy and focal basophilic hepatocytes were observed. Increased colloids in thyroid follicles of females were also observed. However, these changes were not present in the F1 parental rats. In F1 generation, the terminal body weight was not affected. A significant increase in the liver weight was observed in males. Microscopically, increased cytoplasmic rarefaction of hepatocytes in liver in males was observed. In females, focal basophilic hepatocytes were observed in the liver. Finally, pronounced and severely dilated tubules of kidneys were observed in both generations. Taking into account the steep dose response curve of lithium carbonate, the changes and histopathological findings in kidneys and liver as well as the variations noted with regard to adrenals and thyroids, are considered as an early onset of lithium carbonate systemic toxicity.

Evaluation of pups showed that in both generations, the mean weight of male, female and total pups per litter at all the doses tested were unaffected by treatment and there were no external abnormalities in live or dead pups in any of the groups. No treatment-related changes were observed in the survival data of pups up to lactation day 21 at all the doses tested. No relevant effects were seen for postnatal developmental observations in F1 and F2 pups such as pinna detachment, incisor eruption, ear opening, and eye opening. The mean age and body weights at acquisition of balano-preputial separation and vaginal opening in F1 were not affected by treatment when compared to vehicle control group. Finally, no test item related microscopic findings were observed in both male and female pups of F1 and F2 litters.

In view of the results observed:

- The “No Observed Adverse Effect Level (NOAEL)” for systemic toxicity in parental rats is considered to be 15 mg/kg bw/day. The effects observed at 45 mg/kg are considered to be of toxicological relevance. At this dose, not only various in vivo changes on body weight and water consumption but also pronounced morphological changes in liver and kidneys and some variations noted in one or the other sex on adrenals and thyroid glands ( in P generation), however, not in the F1 or F2 generation were noted.

- The “No Observed Adverse Effect Level (NOAEL)” for reproductive toxicity and foetal toxicity is considered to be 45 mg/kg bw/day as no clear substances related and biologically relevant effects on reproductive parameters were observed in the P, F1 and F2 generations. (Advinus, 2012)

Based on read-across approach, the calculated NOAEL values for lithium bromide were 35 mg/kg bw/day for parental systemic toxicity and 106 mg/kg bw/day for the reproductive and foetal toxicity in the F1 and F2 generation.

Supporting study

A combined repeated dose and reproductive/developmental toxicity screening test was conducted in rats according to OECD test guideline 422 with the test substance lithium bromide (55.6 % purity).

13 males and 13 females were administered with the test substance in concentrations of 0 (vehicle - water), 5, 20, 80 mg/kg bw/day by gavage for 42 days to males and to females from 14 days before mating to day 4 of lactation. No abnormalities were found regarding the estrous cycle. Some animals in the 20 mg/kg and more-treated groups did not copulate on the first estrous day during the mating period, and the numbers of vaginal estrus and pairing days until copulation tended to be increased in these groups. However, statistical differences were not found in these parameters from those in the control group. Indices of copulation index and fertility were not affected by the lithium bromide treatment. The treatment did not cause abnormal delivery conditions, and gestation length and gestation index were comparable to those in the controls. In addition, no effects of the treatment were observed on parameters indicating viability of embryos/fetuses until delivery, such as numbers of corpora lutea and implantations, the implantation index, number of newborn and the delivery index, and on those indicating postnatal viability, such as number of live newborn, indices of birth and live birth, number of live pups on day 4 of lactation, sex ratios on days 0 and 4 of lactation and viability index on day 4 of lactation. No effects of the treatment on pup body weights and their morphology were observed. A NOEL value of 5 mg/kg bw/day for reproductive/developmental toxicity was determined in both sexes of animals, because the pairing days until copulation tended to be prolonged in the 20 mg/kg and more-treated groups. The NOEL for the offspring was considered to be 80 mg/kg/day for the offspring. A NOEL value of 5 mg/kg bw/day in both sexes was determined for repeated dose toxicity of lithium bromide.

This result has to be seen as very limited due to the fact that only an abstract of this report is available. Further only a NOEL value was derived from the study and without the full report it is not possible to derive a NOAEL. Therefore this study is not comparable to the key study with lithium carbonate. (Hatano Institute, no date indicated)

Effects on developmental toxicity

Description of key information

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. These values are equivalent to a NOAEL for lithium bromide of 71 mg/kg bw/day for dams and a NOAEL of 212 mg/kg bw/day for the fetuses according to a read-across approach.

Link to relevant study records

Reference

Reference 1

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

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

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

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 b.w. 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

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)

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.

Details on maternal toxic effects:

Maternal toxic effects:yes

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

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Dose descriptor:

NOEL

Effect level:

90 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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 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 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.

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 materno-toxic dose level of 90 mg Lithium Carbonate/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

HYPOTHESIS FOR THE ANALOGUE APPROACH
Lithium bromide completely dissociates in water forming lithium cation and the corresponding bomide anion. Thus, lithium salts with different anion moieties and bromide compounds were found to be suitable candidates for read-across. (Eco)toxicological properties were extrapolated to different endpoints by using the lowest effect concentration.
For further information, please refer to the read-across justification in IUCLID, section 13.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

71 mg/kg bw/day

Based on:

other: recalculated for LiBr

Basis for effect level:

other: maternal toxicity

Remarks on result:

other: Based on Li2CO3

Key result

Dose descriptor:

NOEL

Effect level:

212 mg/kg bw/day

Based on:

other: recalculated for LiBr

Basis for effect level:

other: embryotoxicity

Key result

Abnormalities:

not specified

Key result

Developmental effects observed:

not specified

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

212 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and guideline compliant study with highest reliability.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

Guideline-conform developmental studies are not available

A study according to OECD 414 with lithium carbonate was conducted by the Lead Registrant and a Member Registrant for non-European regulatory purposes. This study is valuable for read-across to evaluate the potential toxicity of lithium and lithium compounds with respect to reproduction and fertility. The findings are summarised below.

Prenatal developmental toxicity study of Lithium carbonate in rats by oral administration (LPT, 24635, 2010)

With regard to the developmental toxicity, the most reliable and key study is the prenatal developmental toxicity study performed in Wistar 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.

Conversion of the obtained NO(A)ELs results in

- NOAEL (maternal toxicity) of 71 mg lithium bromide/kg bw/day

- NOAEL (embryotoxicity) of 212 mg lithium bromide/kg bw/day

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on the studies on reproductive and prenatal developmental toxicity of lithium carbonate in rats according to OECD 416, OECD 414 (read-across approach) and the results of a combined repeated dose and reproductive/developmental toxicity screening test on teratogenic and reproductive effects of lithium bromide in rats, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.