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Toxicological information

Toxicity to reproduction

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test animals

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

Administration / exposure

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. The 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
Doses / concentrationsopen allclose all
Dose / conc.:
5 mg/kg bw/day
Dose / conc.:
15 mg/kg bw/day
Dose / conc.:
45 mg/kg bw/day
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

Examinations

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 v12.0. 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
There were no clinical signs and mortalities observed in control, 5, 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
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
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
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
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 this 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
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 correlation 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
There were no test item-related gross findings in males or females.

HISTOPATHOLOGY
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.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (nominal)
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

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Body weight and weight changes:
effects observed, non-treatment-related
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, non-treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
effects observed, non-treatment-related
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, non-treatment-related

Details on results (P1)

CLINICAL SIGNS AND MORTALITY
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 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 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.

BODY WEIGHT AND FOOD CONSUMPTION
Males:
The weekly mean body weights and net weight gains were unaffected by treatment at all the tested doses when compared to the vehicle control group.
The food intake was unaffected by the treatment at all the tested doses when compared to the vehicle control group. However, an incidence of significantly lower food intake during week 4 at 15 mg/kg bw/day dose observed was considered to be incidental because of its isolated occurrence.
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 (8.5 %) 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. Thus, the treatment slightly increased the body weight towards the end of treatment and net weight gains in males of P-generation at 45 mg/kg bw/day weight dose. In females, the net weight gains were apparently higher (statistically not significant) when compared to vehicle control in both P and F1 generations.
The food intake was not altered by the treatment at all the tested doses when compared to the vehicle control group. Thus, treatment had no effect on food consumption in either sex during the premating period at the 5 and 15 mg/kg bw/day doses. At 45 mg/kg bw/day, treatment significantly increased the food intake in males (8.2 to 12.7 %) and on few occasions in females (7.9 to 9.3 %) during the pre-mating period in P generation.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE
The calculated mean oestrous cycle length was 3.89, 4.43, 4.0 and 4.05 days in vehicle control, 5, 15 and 45 mg/kg bw/day doses, respectively. The oestrous cycle length was significantly higher at 5 mg/kg bw/day dose. This was considered as incidental as there was no dose dependency observed. Thus the treatment did not alter the oestrous cycle length between control and treatment groups in both P and F1 generations.

REPRODUCTIVE FUNCTION: SPERM MEASURES
There were no statistically significant inter group differences in the sperm motility (percentage of progressive motile sperms and total motility) and sperm morphology parameters evaluated. Cauda epididymal sperm counts and testicular spermatid count data were comparable between control and 45 mg/kg bw/day dose groups.

REPRODUCTIVE PERFORMANCE

Pre-coital Time:
The mean pre-coital time was apparently higher but statistically not significant in all the doses tested. This was considered incidental, as the normal biological range is 1-4 as per the available literature. Further this was within the historical range (HD for mean pre-coital time is 2.53 days with a range of lowest 1 to highest 12 days).

Gestation Length:
There were no treatment-related effects on the gestation length (average days to litter) at all the tested doses.

Fertility Indices:
No treatment-related changes were observed in the fertility indices of sires and dams at 5 and 15 mg/kg bw/day dose. At 45 mg/kg bw/day dose, the treatment significantly reduced the male and female fertility indices and was less than the Advinus historical control data (HD range: Male fertility index: 88 to 100%, Female fertility index: 80 to 100 %). However, literature showed historical ranges for male fertility index of 70 to 100 % and for female fertility index of 72 to 100 %, (Hood, 2nd Ed, Developmental and Reproductive Toxicology). In addition, there were no significant changes in the oestrous cyclicity, sperm parameters and reproductive organ weights and histology (including follicle count) in these animals. Hence, as it was only observed in F1 generation and in absence of other interrelated reproductive findings it was not considered a direct test article related reproductive effect but rather a possible indirect (stress related) effect.

ORGAN WEIGHTS
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
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
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. Microscopic examination of reproductive organs did not reveal any test item related changes. There were no significant differences in the follicle counts between vehicle control and 45 mg/kg bw/day dose group females.

Effect levels (P1)

open allclose all
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

Target system / organ toxicity (P1)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
no effects observed

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

VIABILITY AND DEVELOPMENTAL EXAMINATIONS
At the doses tested there were no treatment-related effects on the number of pregnancies of the parental generation, on the number littered and on the 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’s dead 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.
Pinna detachment:
The observation for pinna detachment was started on postnatal day (PND) 1. The first sign of detachment began on PND 2 in all groups and the process was completed on PND 4 in all groups. The pinna detachment was seen in a significantly higher percentage of pups on PND 2 and 3 at 45 mg/kg bw/day dose. This earlier onset was considered incidental.
Incisor Eruption:
The observation for incisor eruption was started on PND 7. The first sign of eruption of incisors was noticed on PND 9 in control, 5 and 15 mg.kg bw/day doses and on PND 8 in the 45 mg/kg bw/day dose. The eruption was completed in all pups on PND 12 at 5 and 15 mg/kg bw/day doses and was delayed by one day (PND 13) in control and 45 mg/kg bw/day dose. The incisor eruption was seen in a significantly higher percentage of pups on PND 11 and 12 at 5 mg/kg bw/day dose, PND 9, 11 and 12 at 15 mg/kg bw/day and on PND 9 at 45 mg/kg bw/day doses. This change was considered not toxicologically relevant as there was no dose dependency observed.
Ear Opening:
The observation for ear opening was started on PND 10 and continued until the criterion was met in all pups. The ear canal of both ears began to open on PND 12 in all the treated groups and was delayed by one day in the control group. The ear opening was completed on PND 15 in 5, 15 and 45 mg/kg/bw/day doses and was delayed by 1 Day in the control group. No significant changes were observed between the control and treated groups.
Eye Opening:
The observation for the total separation of the upper and lower eye lids and the complete opening of both eyes was started on PND 13 and the first sign of eye opening began on PND 14 in all groups. The eye opening was completed in all pups on PND 16 in all the treated groups and was delayed by one day in the control group. The eye opening was seen in a significantly higher percentage of pups on PND 14 and 15 at 45 mg/kg bw/day dose. This earlier onset was considered not toxicologically relevant.

BODY WEIGHT
Evaluation of pups showed that the mean weight of male, female and total pups per litter at all the doses tested were unaffected by treatment.

SEXUAL MATURATION
Balano-preputial separation is the complete retraction of prepuce from the penis when gentle pressure is applied to the animal’s prepuce. This was evaluated for 25 males in each group. These males were selected from parental generation at weaning and formed the parents of the F1-generation. The average age at acquisition of balano-preputial separation in the control 5, 15 and 45 mg/kg bw/day exposure dose groups were 43.64, 43.20, 43.92 and 44.16 days, respectively. The mean age and body weights at acquisition of balanopreputial separation were not affected by treatment when compared to vehicle control group.
It is the visible break in the membranous sheath covering the vaginal orifice which results in separation of vaginal edges. This was evaluated for 25 females in each group. These females were selected from the parental generation at weaning and formed the parents of the F1-generation. The average duration post weaning of vaginal opening (patency) in the control 5, 15 and 45 mg/kg bw/day exposure dose groups were 31.56, 31.72, 31.68 and 32.20 days, respectively. The mean age and body weights at acquisition of vaginal patency were not affected by treatment when compared to vehicle control group.

HISTOPATHOLOGY
No test item related microscopic findings were observed in both male and female pups of F1 litters.

Effect levels (F1)

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

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
no effects observed

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Details on results (F2)

VIABILITY AND DEVELOPMENTAL EXAMINATIONS
Test item at the doses tested had no treatment-related effects on the number of pregnancies, number littered and number of live litters. The mean litter size and mean viable litter size were not altered by treatment. 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.

Pinna detachment:
The observation for pinna detachment was started on postnatal day (PND) 1. The first sign of detachment began on PND 2 in all groups and the process was completed on PND 4 in all groups. The pinna detachment was seen in a significantly higher percentage of pups on PND 3 at 5 mg/kg bw/day and PND 2 and 3 at 45 mg/kg bw/day doses. This earlier onset was considered not toxicologically relevant.

Incisor Eruption:
The observation for incisor eruption was started on PND 7. The first sign of eruption of incisors was noticed on PND 8 in the 5 mg/kg bw/day dose and was delayed by one day in other groups. The eruption was completed in all pups on PND 13 in all the groups. The incisor eruption was seen in a significantly lower percentage of pups on PND 10 and 12 at 15 mg/kg bw/day dose and at a significantly higher percentage of pups on PND 9 to 11 at 45 mg/kg bw/day doses. This earlier onset was considered not toxicologically relevant as there was no dose dependency observed.

Ear Opening:
The observation for ear opening was started on PND 10 and continued until the criterion was met in all pups. The ear canal of both ears began to open on PND 12 in all the groups. The ear opening was completed on PND 15 in all the groups. The ear opening was seen in a significantly higher percentage of pups on PND 12 at 45 mg/kg bw/day dose group. This earlier onset was considered not toxicologically relevant due to lack of dose correlation.

Eye Opening:
The observation for the total separation of the upper and lower eye lids and the complete opening of both eyes was started on PND 13 and the first sign of eye opening began on PND 14 in all groups. The eye opening was completed in all pups on PND 17 in all groups. The eye opening was seen in a significantly higher percentage of pups on PND 15 and 16 at 5 mg/kg bw/day, PND 15 at 15 mg/kg bw/day and on PND 14 and 15 at 45 mg/kg bw/day doses. This earlier onset was considered not toxicologically relevant.

BODY WEIGHT
Evaluation of pups showed that the mean weight of male, female and total pups per litter at all the doses tested were unaffected by treatment.

HISTOPATHOLOGY
No test item related microscopic findings were observed in both male and female pups of F2 litters.

Effect levels (F2)

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

Target system / organ toxicity (F2)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Applicant's summary and conclusion

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.
Based on read-across approach, the calculated NOAEL values for lithium bromide were 10.5 mg/kg bw/day for parental systemic toxicity and 31.6 mg/kg bw/day for the reproductive and foetal toxicity in the F1 and F2 generation.
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 adrenalsand 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)