Lithium bis(oxalato)borate

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-19 to 2012-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-03-19 to 2012-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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)

Version / remarks:

adopted March 22, 1996

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: USA EPA Health Effects Test Guide OPPTS 870.3650, July 2000

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: Toxi-Coop Zrt., Cserkesz u. 90, H-1103 Budapest, Hungary
- Age at study initiation: 85 – 90 days
- Body weight range at study initiation: 313 to 365 g (males); 184 to 218 g (females)
- Fasting period before study: no
- Housing in wire-mesh cages:
before mating: 2 animals of the same sex/cage
during mating: 1 male and 1 female / cage
pregnant females: individually
males after mating: 2 animals/cage
- Diet: SSNIFF SM R/M-Z+H pelleted breeding and maintenance diet, ad libitum
- Water: regularly monitored tap water, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 8 to 12 per hour
- Photoperiod: 12 hours light (6:00 - 18:00) and 12 hours dark

IN-LIFE DATES: 2012-03-21 to 2012-05-08 (necropsy of last animals)

Route of administration:

oral: gavage

Vehicle:

other: dehydrated sunflower oil

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water and hydrolyses in water, so dehydrated sunflower oil was used as vehicle. As determined in pre-tests, sunflower oil was suitable for the formulation analysis for the test item.
- Concentration in vehicle: 2 mg/mL, 6 mg/mL or 20 mg/mL
- Amount of vehicle: 5 mL dose preparation/kg body weight
- Lot/batch no.: 19/5
- Purity: Natural product, not specified on CoA (pharmaceutical grade Ph.Hg.VIII)

PREPARATION OF DOSING SOLUTIONS:
Sunflower oil was dehydrated by adding 1 g Na2SO4/10 mL sunflower oil. The supernatant was used for formulation after full sedimentation. Formulations were prepared daily in the formulation laboratory of the test facility immediately before administration. The test item was administered as a suspension in the vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Samples of test item dosage formulation were taken twice during the study for an analytical determination of concentration, homogeneity and stability. A flame-photometric method was validated for the determination of lithium bis(oxalato)borate (LiBOB) concentration in dosage formulations. The sufficient stability in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation using IR spectroscopy at the Department of Analytical Chemistry of Eötvös Loránd University Pázmány Péter sétány 1/A, H-1117 Budapest before the commencement of the study.

The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirm that the formulations were homogeneous and stable from the time of preparation until completion of dosing.

Duration of treatment / exposure:

- Males: for 14 days before mating, continued during the mating period (up to 2 weeks) until sacrifice (i.e. at least 4 weeks in total)
- Females: for 14 days before mating, continued during the mating period (up to 2 weeks), during pregnancy, during lactation and until day 10 post-partum at maximum

Frequency of treatment:

Once daily (7 days / week)

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle

Dose / conc.:

30 mg/kg bw/day (nominal)

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- The Dose selection rationale was based on the results of a 14-day oral gavage dose-range finding (DRF) study with lithium bis(oxalato)borate in the rat (Study no. 559.400.3242) performed at the same test facility and using the same species and strain at 0, 50, 100 or 250 mg / kg bw / day. Due to fatalities at the high dose, the dosage was reduced from 250 to 150 mg / kg bw / day after seven days of administration. The No Effect Level in the DRF study was established at 50 mg / kg bw / day.

Positive control:

No positive control

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, at the beginning and end of each working day
- Parameters monitored: fatalities or signs of moribundity, pertinent behavioral changes, signs of difficult or prolonged parturition and all signs were recorded including onset, degree and duration of signs

CLINICAL SIGNS
- Time schedule: once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing

DETAILED CLINICAL OBSERVATIONS: Yes, outside the home cage in a standard arena (modified Irwin screen)
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: on the first day of treatment (day 1), then once a week until sacrifice
Females: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 0 and 4 of lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 rats / sex / dose level (selected at random)
- Parameters examined: erythrocytes, reticulocytes, haemoglobin concentration, relative volume of erythrocytes, mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, thrombocyte counts, leucocyte counts, differential white cell count: neutrophils, eosinophils, basophils, lymphocytes, monocytes
- Coagulation: prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Animals fasted: Yes
- How many animals: 5 rats / sex / dose level (selected at random)
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, cholesterol, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once in the last week of the treatment period
- Dose groups that were examined: 5 rats / sex / dose level (selected at random)
- Battery of functions tested: sensory activity, e.g. auditory, visual and proprioceptive / grip strength / motor activity (FOB: modified Irwin screen in standard arena)

Sacrifice and pathology:

SACRIFICE
- Male animals: after the end of the mating period (on day 47 or 48)
- Maternal animals: on day 11 post-partum at maximum; Non-pregnant females were treated up to and including the day before necropsy (for 43 days at maximum)

GROSS NECROPSY on all adult animals
- Including external and internal examinations of the cranial, thoracic, and abdominal cavities, appearance of the tissues and organs. Any abnormalities were recorded, comprising details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The numbers of implantation sites and of corpora lutea were recorded.

ORGAN WEIGHTS (absolute and relative organ weight to body and brain)
- adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed from five males and five females randomly selected from each dose group
- All males: epididymides, testes, brain
- Paired organs weighed individually

PRESERVATION OF TISSUES
- Five male and five female animals were randomly selected from each group and the following tissues were preserved: adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (with lachrymal gland and Harderian glands), female mammary gland, gonads (testes with epididymides; ovaries, uterus with vagina), heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular and mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum, spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder
- All animals: uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions

HISTOPATHOLOGY: Yes
- All animals in the control and high dose groups and non-pregnant females with cohabiting males: ovaries (follicular, luteal, and interstitial compartments, epithelial capsule, ovarian stroma), uterus, vagina, pituitary, testes and epididymides
- Randomly selected animals (5 males / 5 females) in the control and high dose groups and dead animals: all preserved organs and tissues
- Randomly selected animals (5 males / 5 females) of the low and medium dose groups: additional histological examination of kidneys due to test item related histopathological changes in kidneys of rats from the high dose groups.

Statistics:

Body weights, food consumption and reproductive data:
- Bartlett’s homogeneity of variance test
- One-way ANOVA and Dunnett test (if normally distributed and variances homogeneous)
- Bartlett’s Duncan Multiple Range test (if ANOVA positive) to assess the significance of inter-group differences
- Kruskal-Wallis analysis of variance (If variances inhomogeneous), inter-group comparisons with Mann-Whitney U-test
- Chi2 test, where applicable

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation, piloerection, activity decrease, decreased body tone

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

high dose females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Details on results:

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One high dose (100 mg/kg bw/day) male rat and four female rats (one from the control group and three from the high dose group) died during the study. The death of one male animal and two pregnant females at 100 mg/kg bw/day was described as a consequence of an application error. One control female and one high dose treated dam died after prolonged parturition. A test item related effect was excluded as cause for all five fatalities.

- Test item related salivation with variable frequency within a group but in a dose related manner regarding the degree, incidence and onset was observed in males that received 30 or 100 mg/kg bw/day. Slight, moderate or marked salivation occurred in males (10/11) at 100 mg/kg bw/day from day 6 up to the end of the observation period. Slight or moderate salivation appeared in three male animals (3/12) at 30 mg/kg bw/day from day 25 up to the end of treatment period (1/3) or for five days (1/3), or for one day (1/3). Test item related clinical signs were noted for some surviving females towards the delivery: piloerection (3/8), activity decrease (2/8), decreased body tone (2/8) and on some postpartal days (piloerection (2/8), decreased activity (1/8) and decreased body tone (1/8). All animals recovered up to postpartal day 4.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the gestation period, a test item related depression of the body weight development compared to their control group was detected in females receiving 100 mg/kg bw/day. The mean body weight on gestational day 21 and the summarized body weight gain (between gestation days 0 and 21) were also significantly lower at 100 mg/kg bw/day than in the control group. There were no significant differences in the mean body weight and body weight gain of female animals between control and dosed groups (100, 30 or 10 mg/kg bw/day) during the pre-mating period. The body weight and body weight gain of the male animals were unaffected during the entire observation period. Statistical significances noted for the mean body weight gain of all male groups receiving test item during week 3 (mating days 13 – 20) was due to the relatively low mean body weight gain of their control group, and was therefore not considered to be toxicologically relevant.

A test item related reduction of the mean daily food consumption was observed in female animals at 100 mg/kg bw/day during the gestation period and also between lactation days 0 and 4. This finding coincided with a reduction of body weight gain. Other than the above, statistically significant differences of the mean daily food consumption between the control group females and female rats from all test item treated groups were not observed. Mean daily food consumption of male rats was statistically different (significance: p < 0.05) to the control group data at the medium dose (30 mg/kg bw/day) from day 41 to 47, but as the measured mean food consumption was normal for this strain and age and the significance was due to the relatively small standard deviation, this was considered to be a statistical artifact without biological relevance. Test item related effects on the mean food consumption of male rats were not detected.

FUNCTIONAL OBSERVATIONS
No test item related changes were detected. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups. The limb tone was not examinable in one control male animal due to an unusual (crooked) limb position. A more pronounced startle reactions noted for one male (1/5) and one female (1/5) animal at 30 and 10 mg/kg bw/day dosages, respectively, were considered to be an individual variation without any toxicological significance.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significantly higher mean weights of kidneys (absolute and relative to body and brain weights) were indicative of a test item influence both in male and female animals at 100 mg/kg bw/day and were coinciding with changes in clinical chemistry parameters, necropsy and histopathology observations. Other statistically significant changes in high dose females where a lower mean thymus weight (absolute and relative to body and brain weights) and a higher mean spleen weight relative to body weight. The toxicological significance of these observed organ weight changes were considered negligible, as they were well within the range of the historical control data of the Test Facility. Other statistically significant changes from several mean organ weight data were considered to be not related to the administration of the test item: lower mean testes weight relative to body weight at 30 mg/kg bw/day; lower fasted body weight, absolute weight of brain and heart, and lower brain weight at 30 and 10 mg/kg bw/day in female animals. They were considered to be of little or no biological significance as there was no dose-response and the means were well within the normal range of historical control data of the Test Facility.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no necropsy findings from the five fatalities that would indicate a test item related effect as cause of death. Foamy content in the lungs and trachea, and dark red content in the intestines were observed in one male of the high dose group (100 mg/kg bw/day) that died on day 4. Enlarged adrenal glands, smaller than normal spleen and brownish liquid content in the uterine horns and vagina were noted for a dam dosed with 100 mg/kg bw/day that was necropsied prematurely on postpartal day 2 (Day 39). Reddish mottled lungs (1/2), spotted surface of kidneys (1/2), thin wall of stomach at the cardia (1/2), yellowish mucous content in the intestines (1/2) and hyperemic duodenum (2/2), as well as brownish liquid content (1/2) and embryonic death (1/2) in the uterine horns of two pregnant animals (2/11) at 100 mg/kg bw/day were found during necropsy on gestational days 24 and 21 (Day 38 and 37, respectively). Visceral organs were found to be pale in one dam of the control group (1/10) that died prematurely.

In male and female animals that survived as scheduled, several test item related renal changes were grossly visible in both high dose groups (100 mg/kg bw/day). The kidneys of some males and females of the high dose groups were found to be pale (2/11 male and 4/8 dams) and enlarged (1/11 male and 3/8 dams). Congenital absence of the left kidney and uterine horn in combination with enlargement of the right kidney were observed in one dam from the low dose group (10 mg/kg bw/day). Hydrometra was noted for one non-pregnant female rat (1/2) in the control group and one low dose animal. Hydrometra is a frequent observation in experimental rats and was considered to be of no toxicological relevance, due to the lack of histopathological changes.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the rats that died prior to their scheduled deaths, passive hyperemia (1/1 male) as well as alveolar emphysema and acute hemorrhages (in all five animals) were detected. These pulmonary alterations were considered to be the probable cause of death in connection with a suspected shock. In addition vacuolization of tubular epithel cells (1/1 male, 1/1 dam, 1/2 not delivered pregnant), segmental tubular basophilia (1/1 male, 1/1 dam, 2/2 not delivered pregnant) and in some cases mineral deposits (1/1 male, 1/1 dam, 1/2 not delivered pregnants) were detected in the kidneys in the 100 mg/kg bw/day treated animals. No histological signs of possible endometritis were detected in the dead female animals. No morphological evidence of other lesions of possible toxic origin were observed in the other investigated organs of dead animals.

Histological examination of male and female rats 100 mg/kg bw/day that survived as scheduled revealed focal or multifocal tubular basophilia accompanied with slight inter-tubular lymphocytic infiltration in the cortical region of the kidneys, affecting the proximal convoluted tubules (5/5 male, 5/5 dams) and mineralization (mineral deposits) in the lumina of descending proximal tubules (5/5 male, 4/5 dams) as signs of test item related renal lesions. The finding coincided with effects described from clinical chemistry, necropsy and organ weights. The tubular basophilia accompanied with slight inter-tubular lymphocytic infiltration in the cortical region affecting the proximal convoluted tubules reflected a decreased functional activity of these tubules. Tubular dilatation and atrophy of epithelial cells were observed and described as a secondary change. The mineralization (mineral deposits) in the lumina of descending proximal tubules was mainly present at the border of cortical-medullary zone. The above mentioned lesions were not detectable in the kidneys of male and female animals from the middle and low dose groups and were therefore considered to be related to the administration of the test item.

Focal alveolar emphysema in the lungs occurred sporadically to a minimal or mild degree (3/5 control male, 1/5 control female dam; 2/5 male and 1/5 dam at 100 mg/kg bw/day). These findings were considered to be a consequence of hypoxia, dyspnoea and circulatory disturbance developed during the scheduled death by exsanguination. One sided lymphatic pyelitis was observed in a single animal at 10 mg/kg bw/day (1/5 dam) as an individual disease. The hyperplasia of bronchus associated lymphoid tissue (BALT) in a control (1/5 of male) and 100 mg/kg bw/day treated animals (1/1 male fatality and 1/5 dam) was considered to be a physiological adaptation. Histology of the reproductive organs (testes, epididymides, ovaries, uterus, vagina and pituitary) revealed no differences between the test item treated animals and the control groups. The ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases. The uterus, cervix and vagina had a normal structure in phase with the sexual cycle. In some animals (5/12 control: 2/2 non-pregnant females and 3/9 surviving dams), dilatation of the uterus was observed, but in absence of inflammation or other pathological lesions, that was considered to be a physiological phenomenon connected to the normal sexual cycle. Pituitary histology was normal in animals treated with the test item and in their control groups. No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed in males or females. The structure and the cell morphology of the endocrine glands were similar between the control groups and the treated animals of both sexes.

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

histopathology: non-neoplastic

Conclusions:

The NOAEL for systemic toxicity is based on various findings at 100 mg/kg/bw regarding in vivo changes on clinical signs, body weight, clinical parameters and changes in organ pathology. A value of 30 mg/kg bw was derived as the systemic NOAEL for parental animals.

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test was to provide initial information concerning the toxic potential of lithium bis(oxalato)borate. The test was performed according to OECD guideline 422. The test item was administered orally (by gavage) at 0, 10, 30 or 100 mg/kg bw/day, corresponding to concentrations of 0, 2, 6 and 20 mg/mL at a 5 mL/kg bw dose volume to four dose groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) once a day. All animals of the parent (P) generation received test item or vehicle prior to mating (for 14 days) and throughout the mating phase. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. Five dams and the cohabiting males were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, and organ weight. All parental animals were subjected to gross pathology one day after the last treatment. Selected organs of parental animals were weighed. Full histopathology was performed on the five selected animals of both, the control and high dose groups. Histopathology on reproductive organs and pituitary was performed on all animals of the control and high dose groups. The reproductive organs and pituitary of non-pregnant female animals and cohabiting males of the low and mid dose groups were also processed and evaluated histologically. The kidneys of animals at the low and medium doses were processed as well and evaluated histologically due to histopathology findings in kidneys of the high dose animals. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. The results are listed below:

Hematology

Statistically higher percentage of the neutrophil granulocytes and reduced percentage of lymphocytes were seen in females of the high dose groups. Other than that, hematology examinations did not reveal test item related changes in the examined hematological parameters in male or female animals at any dose level (100, 30 or 10 mg/kg bw/day).

Clinical chemistry

A slightly higher mean concentration of creatinine, urea and inorganic phosphorous referred to a test item influence on renal function in male and female animals dosed with 100 mg/kg bw/day. Changes in elevated mean concentration of bile acids might be indicative a test item effect on hepatic function in female animals dosed with 100 mg/kg bw/day.

Necropsy

Test item related renal changes were observed in male (2/11) and female (4/8) animals dosed with 100 mg/kg bw/day: the kidneys were found to be pale and enlarged. Other than this, there were no test item related findings in animals that survived until the scheduled termination of the in life phase.

Organ weight

The kidney weights (absolute and relative to body and brain weights) were significantly elevated in male and female animals at 100 mg/kg bw/day with respect to controls.

Histopathology

Histopathology investigations revealed test item related renal lesions in males and females of the high dose group (100 mg/kg bw/day). Segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration, mineral deposits in the lumina of descending proximal tubules and segmental vacuolization of tubular epithelial cells were observed.

Reproduction

There were no differences between the control and test item treated groups in the delivery data of dams. The reproductive ability of male and female animals was unaffected by the treatment with Lithium-bis(oxalato)borate at 100, 30 or 10 mg/kg bw/day.

Offspring

A test item effect on the offspring development was observed in the slightly higher extra uterine mortality (number and percent) between postnatal days 0 and 4, and in the less litter weight and litter weight gain and mean pup’s weight on postnatal day 0 in 100 mg/kg bw/day group. The higher mortality of pups was probably a consequence of maternal toxicity.

Under the conditions of the present study, Lithium-bis(oxalato)borate caused salivation (male), clinical signs and changes in body weight and food consumption (female) towards end of gestation and changes in clinical chemistry parameters (higher creatinine, urea and inorganic phosphorous, in male and female animals, elevated bile acid level in female animals), and changes in organ pathology in male and female animals (enlarged and pale kidneys, higher kidney weights and segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration, mineral deposits in the lumina of descending proximal tubules and segmental vacuolization of tubular epithelial cells) following an oral administration at 100 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 30 mg/kg bw/day, salivation was observed in some male animals. At 10 mg/kg bw/day, there was no test item related effect.

Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:

NO(A)EL for male rats: 30 mg/kg bw/day

NO(A)EL for female rats: 30 mg/kg bw/day

(TOXICOOP, 2012)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted March 22, 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: USA EPA Health Effects Test Guide OPPTS 870.3650, July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Lithium bis(oxalato)borate

EC Number:

456-990-3

EC Name:

Lithium bis(oxalato)borate

Cas Number:

244761-29-3

Molecular formula:

C4BLiO8 (Hill Empirical Formula) C4BO8.Li (CAS Empirical Formula)

IUPAC Name:

boron(3+) lithium(1+) dioxalate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90, H-1103 Budapest, Hungary
- Age at study initiation: 85 – 90 days
- Body weight range at study initiation: 313 to 365 g (males); 184 to 218 g (females)
- Fasting period before study: no
- Housing in wire-mesh cages:
before mating: 2 animals of the same sex/cage
during mating: 1 male and 1 female / cage
pregnant females: individually
males after mating: 2 animals/cage
- Diet: SSNIFF SM R/M-Z+H pelleted breeding and maintenance diet, ad libitum
- Water: regularly monitored tap water, ad libitum
- Acclimation period: 20 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70 %
- Air changes: 8 to 12 per hour
- Photoperiod: 12 hours light (6:00 - 18:00) and 12 hours dark

IN-LIFE DATES: 2012-03-21 to 2012-05-08 (necropsy of last animals)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: dehydrated sunflower oil

Details on exposure:

VEHICLE
- Justification for use and choice of vehicle: The test item is not soluble in water and hydrolyses in water, so dehydrated sunflower oil was used as vehicle. As determined in pre-tests, sunflower oil was suitable for the formulation analysis for the test item.
- Concentration in vehicle: 2 mg/mL, 6 mg/mL or 20 mg/mL
- Amount of vehicle: 5 mL dose preparation/kg body weight
- Lot/batch no.: 19/5
- Purity: Natural product, not specified on CoA (pharmaceutical grade Ph.Hg.VIII)

PREPARATION OF DOSING SOLUTIONS:
Sunflower oil was dehydrated by adding 1 g Na2SO4/10 mL sunflower oil. The supernatant was used for formulation after full sedimentation. Formulations were prepared daily in the formulation laboratory of the test facility immediately before administration. The test item was administered as a suspension in the vehicle.

Details on mating procedure:

- M/F ratio per cage: 1 / 1
- Length of cohabitation: Each female was placed with the same male until mating was assumed or 14 days had elapsed
- Proof of pregnancy: detection of vaginal plug or 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
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front. Samples of test item dosage formulation were taken twice during the study for an analytical determination of concentration, homogeneity and stability. A flame-photometric method was validated for the determination of lithium bis(oxalato)borate (LiBOB) concentration in dosage formulations. The sufficient stability in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation using IR spectroscopy at the Department of Analytical Chemistry of Eötvös Loránd University Pázmány Péter sétány 1/A, H-1117 Budapest before the commencement of the study.

The analytical results indicated that the doses were accurately formulated during the toxicity study. The results also confirm that the formulations were homogeneous and stable from the time of preparation until completion of dosing.

Duration of treatment / exposure:

- Males: for 14 days before mating, continued during the mating period (up to 2 weeks) until sacrifice (i.e. at least 4 weeks in total)
- Females: for 14 days before mating, continued during the mating period (up to 2 weeks), during pregnancy, during lactation and until day 10 post-partum at maximum

Frequency of treatment:

Once daily (7 days / week)

Details on study schedule:

- Animal age at mating in the study: 14 weeks

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle

Doses / concentrations 2

Dose / conc.:

30 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

No. of animals per sex per dose:

12 males and 12 females

Control animals:

yes, concurrent vehicle

Details on study design:

- The Dose selection rationale was based on the results of a 14-day oral gavage dose-range finding (DRF) study with lithium bis(oxalato)borate in the rat (Study no. 559.400.3242) performed at the same test facility and using the same species and strain at 0, 50, 100 or 250 mg / kg bw / day. Due to fatalities at the high dose, the dosage was reduced from 250 to 150 mg / kg bw / day after seven days of administration. The No Effect Level in the DRF study was established at 50 mg / kg bw / day.

Positive control:

No positive control

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day, at the beginning and end of each working day
- Parameters monitored: fatalities or signs of moribundity, pertinent behavioral changes, signs of difficult or prolonged parturition and all signs were recorded including onset, degree and duration of signs

CLINICAL SIGNS
- Time schedule: once a day, after treatment at approximately the same time, considering the peak period of anticipated effects after dosing

DETAILED CLINICAL OBSERVATIONS: Yes, outside the home cage in a standard arena (modified Irwin screen)
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: on the first day of treatment (day 1), then once a week until sacrifice
Females: on the first day of treatment (day 1), then once a week until mated (or until sacrifice) and on days 0, 7, 14 and 20 post-coitum and days 0 and 4 of lactation

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

WATER CONSUMPTION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 rats / sex / dose level (selected at random)
- Parameters examined: erythrocytes, reticulocytes, haemoglobin concentration, relative volume of erythrocytes, mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, thrombocyte counts, leucocyte counts, differential white cell count: neutrophils, eosinophils, basophils, lymphocytes, monocytes
- Coagulation: prothrombin time, activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of sacrifice
- Animals fasted: Yes
- How many animals: 5 rats / sex / dose level (selected at random)
- Parameters examined: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, cholesterol, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, gamma glutamyltransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once in the last week of the treatment period
- Dose groups that were examined: 5 rats / sex / dose level (selected at random)
- Battery of functions tested: sensory activity, e.g. auditory, visual and proprioceptive / grip strength / motor activity (FOB: modified Irwin screen in standard arena)

Sperm parameters (parental animals):

Parameters examined in male parental generation:
- testis / epididymis histopathology (with special emphasis on stages of spermatogenesis in the male gonads and interstitial testicular cell structure)

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
− Litter weight on postnatal days 0 and 4
− Mean body weight gain per litter between postnatal days 0-4
− Number of live births per litter, and number of viable pups per litter on postnatal days 0 and 4
− Survival Index of pups on postnatal day 4
− Sex ratio % (on postnatal days 0 and 4)

GROSS EXAMINATION OF DEAD PUPS:
- yes: attempts to determine cause of death of stillborns or pups found dead later

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: after the end of the mating period (on day 47 or 48)
- Maternal animals: on day 11 post-partum at maximum; Non-pregnant females were treated up to and including the day before necropsy (for 43 days at maximum)

GROSS NECROPSY on all adult animals
- Including external and internal examinations of the cranial, thoracic, and abdominal cavities, appearance of the tissues and organs. Any abnormalities were recorded, comprising details of the location, color, shape and size. Special attention was paid to the organs of the reproductive system. The numbers of implantation sites and of corpora lutea were recorded.

ORGAN WEIGHTS (absolute and relative organ weight to body and brain)
- adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed from five males and five females randomly selected from each dose group
- All males: epididymides, testes, brain
- Paired organs weighed individually

PRESERVATION OF TISSUES
- Five male and five female animals were randomly selected from each group and the following tissues were preserved: adrenals, aorta, bone marrow (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (with lachrymal gland and Harderian glands), female mammary gland, gonads (testes with epididymides; ovaries, uterus with vagina), heart, kidneys, large intestines (cecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular and mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum, spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder
- All animals: uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries, pituitary and all organs showing macroscopic lesions

HISTOPATHOLOGY: Yes
- All animals in the control and high dose groups and non-pregnant females with cohabiting males: ovaries (follicular, luteal, and interstitial compartments, epithelial capsule, ovarian stroma), uterus, vagina, pituitary, testes and epididymides
- Randomly selected animals (5 males / 5 females) in the control and high dose groups and dead animals: all preserved organs and tissues
- Randomly selected animals (5 males / 5 females) of the low and medium dose groups: additional histological examination of kidneys due to test item related histopathological changes in kidneys of rats from the high dose groups.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring were sacrificed at day 4 post-partum.
- These animals were subjected to postmortem examinations as follows:

GROSS NECROPSY
- macroscopic: pups were examined externally for gross external abnormalities

Statistics:

Body weights, food consumption and reproductive data:
- Bartlett’s homogeneity of variance test
- One-way ANOVA and Dunnett test (if normally distributed and variances homogeneous)
- Bartlett’s Duncan Multiple Range test (if ANOVA positive) to assess the significance of inter-group differences
- Kruskal-Wallis analysis of variance (If variances inhomogeneous), inter-group comparisons with Mann-Whitney U-test
- Chi2 test, where applicable

Reproductive indices:

- Mating index (measure of animals’ ability to mate)
- Fertility Index (measure of male's ability to produce sperm that can fertilize eggs and measure of female’s ability to become pregnant)
- Gestation Index (measure of pregnancy that provides at least one live pup)

Mortality indices:
- Pups (incl. sex ratios)
- Pre-implantation
- Post-implantation
- Intrauterine
- Postnatal

Offspring viability indices:

preimplantation loss, postimplantation loss, pup weight / litter, sex ratio

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation, piloerection, activity decrease, decreased body tone

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

high dose females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
One high dose (100 mg/kg bw/day) male rat and four female rats (one from the control group and three from the high dose group) died during the study. The death of one male animal and two pregnant females at 100 mg/kg bw/day was described as a consequence of an application error. One control female and one high dose treated dam died after prolonged parturition. A test item related effect was excluded as cause for all five fatalities.

- Test item related salivation with variable frequency within a group but in a dose related manner regarding the degree, incidence and onset was observed in males that received 30 or 100 mg/kg bw/day. Slight, moderate or marked salivation occurred in males (10/11) at 100 mg/kg bw/day from day 6 up to the end of the observation period. Slight or moderate salivation appeared in three male animals (3/12) at 30 mg/kg bw/day from day 25 up to the end of treatment period (1/3) or for five days (1/3), or for one day (1/3). Test item related clinical signs were noted for some surviving females towards the delivery: piloerection (3/8), activity decrease (2/8), decreased body tone (2/8) and on some postpartal days (piloerection (2/8), decreased activity (1/8) and decreased body tone (1/8). All animals recovered up to postpartal day 4.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
During the gestation period, a test item related depression of the body weight development compared to their control group was detected in females receiving 100 mg/kg bw/day. The mean body weight on gestational day 21 and the summarized body weight gain (between gestation days 0 and 21) were also significantly lower at 100 mg/kg bw/day than in the control group. There were no significant differences in the mean body weight and body weight gain of female animals between control and dosed groups (100, 30 or 10 mg/kg bw/day) during the pre-mating period. The body weight and body weight gain of the male animals were unaffected during the entire observation period. Statistical significances noted for the mean body weight gain of all male groups receiving test item during week 3 (mating days 13 – 20) was due to the relatively low mean body weight gain of their control group, and was therefore not considered to be toxicologically relevant.

A test item related reduction of the mean daily food consumption was observed in female animals at 100 mg/kg bw/day during the gestation period and also between lactation days 0 and 4. This finding coincided with a reduction of body weight gain. Other than the above, statistically significant differences of the mean daily food consumption between the control group females and female rats from all test item treated groups were not observed. Mean daily food consumption of male rats was statistically different (significance: p < 0.05) to the control group data at the medium dose (30 mg/kg bw/day) from day 41 to 47, but as the measured mean food consumption was normal for this strain and age and the significance was due to the relatively small standard deviation, this was considered to be a statistical artifact without biological relevance. Test item related effects on the mean food consumption of male rats were not detected.

FUNCTIONAL OBSERVATIONS
No test item related changes were detected. The behavior, physical condition and reactions to different type of stimuli of animals selected for examination were considered to be normal in all groups. The limb tone was not examinable in one control male animal due to an unusual (crooked) limb position. A more pronounced startle reactions noted for one male (1/5) and one female (1/5) animal at 30 and 10 mg/kg bw/day dosages, respectively, were considered to be an individual variation without any toxicological significance.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
The evaluated testes and epididymides were histologically normal and characteristic for the sexually mature organism in all investigated male animals of the control groups and the treated groups. The various spermatogenic cells (the spermatogonia, the spermatocytes, the spermatids and spermatozoa) representing different phases in the development and differentiation of the spermatozoons and the interstitial cells were the same in quantity and morphologically in the testes of all animals investigated. The histological results of epididymides and pituitary were normal in all cases.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No significant differences between the control and test item treated male and female animals were detected in the examined parameters of reproductive performance. The percentage of fertile male animals was higher and the percentage of infertile male animals was slightly lower with respect to controls in all test item treated groups, but lacked statistical significance. The copulatory and fertility indices were comparable between all groups. There were no significant differences between the control and test item treated groups in the mean of number and percentage of sperm positive (mated) female animals or the copulatory, fertility and gestational indices. The number and percentage of non-pregnant and pregnant animals, dams delivered and number of pregnant animals with live born pups and the mean pre-coital interval and number of conceiving days were similar to the control group data for all test item treated groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
Significantly higher mean weights of kidneys (absolute and relative to body and brain weights) were indicative of a test item influence both in male and female animals at 100 mg/kg bw/day and were coinciding with changes in clinical chemistry parameters, necropsy and histopathology observations. Other statistically significant changes in high dose females where a lower mean thymus weight (absolute and relative to body and brain weights) and a higher mean spleen weight relative to body weight. The toxicological significance of these observed organ weight changes were considered negligible, as they were well within the range of the historical control data of the Test Facility. Other statistically significant changes from several mean organ weight data were considered to be not related to the administration of the test item: lower mean testes weight relative to body weight at 30 mg/kg bw/day; lower fasted body weight, absolute weight of brain and heart, and lower brain weight at 30 and 10 mg/kg bw/day in female animals. They were considered to be of little or no biological significance as there was no dose-response and the means were well within the normal range of historical control data of the Test Facility.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no necropsy findings from the five fatalities that would indicate a test item related effect as cause of death. Foamy content in the lungs and trachea, and dark red content in the intestines were observed in one male of the high dose group (100 mg/kg bw/day) that died on day 4. Enlarged adrenal glands, smaller than normal spleen and brownish liquid content in the uterine horns and vagina were noted for a dam dosed with 100 mg/kg bw/day that was necropsied prematurely on postpartal day 2 (Day 39). Reddish mottled lungs (1/2), spotted surface of kidneys (1/2), thin wall of stomach at the cardia (1/2), yellowish mucous content in the intestines (1/2) and hyperemic duodenum (2/2), as well as brownish liquid content (1/2) and embryonic death (1/2) in the uterine horns of two pregnant animals (2/11) at 100 mg/kg bw/day were found during necropsy on gestational days 24 and 21 (Day 38 and 37, respectively). Visceral organs were found to be pale in one dam of the control group (1/10) that died prematurely.

In male and female animals that survived as scheduled, several test item related renal changes were grossly visible in both high dose groups (100 mg/kg bw/day). The kidneys of some males and females of the high dose groups were found to be pale (2/11 male and 4/8 dams) and enlarged (1/11 male and 3/8 dams). Congenital absence of the left kidney and uterine horn in combination with enlargement of the right kidney were observed in one dam from the low dose group (10 mg/kg bw/day). Hydrometra was noted for one non-pregnant female rat (1/2) in the control group and one low dose animal. Hydrometra is a frequent observation in experimental rats and was considered to be of no toxicological relevance, due to the lack of histopathological changes.

HISTOPATHOLOGY (PARENTAL ANIMALS)
In the rats that died prior to their scheduled deaths, passive hyperemia (1/1 male) as well as alveolar emphysema and acute hemorrhages (in all five animals) were detected. These pulmonary alterations were considered to be the probable cause of death in connection with a suspected shock. In addition vacuolization of tubular epithel cells (1/1 male, 1/1 dam, 1/2 not delivered pregnant), segmental tubular basophilia (1/1 male, 1/1 dam, 2/2 not delivered pregnant) and in some cases mineral deposits (1/1 male, 1/1 dam, 1/2 not delivered pregnants) were detected in the kidneys in the 100 mg/kg bw/day treated animals. No histological signs of possible endometritis were detected in the dead female animals. No morphological evidence of other lesions of possible toxic origin were observed in the other investigated organs of dead animals.

Histological examination of male and female rats 100 mg/kg bw/day that survived as scheduled revealed focal or multifocal tubular basophilia accompanied with slight inter-tubular lymphocytic infiltration in the cortical region of the kidneys, affecting the proximal convoluted tubules (5/5 male, 5/5 dams) and mineralization (mineral deposits) in the lumina of descending proximal tubules (5/5 male, 4/5 dams) as signs of test item related renal lesions. The finding coincided with effects described from clinical chemistry, necropsy and organ weights. The tubular basophilia accompanied with slight inter-tubular lymphocytic infiltration in the cortical region affecting the proximal convoluted tubules reflected a decreased functional activity of these tubules. Tubular dilatation and atrophy of epithelial cells were observed and described as a secondary change. The mineralization (mineral deposits) in the lumina of descending proximal tubules was mainly present at the border of cortical-medullary zone. The above mentioned lesions were not detectable in the kidneys of male and female animals from the middle and low dose groups and were therefore considered to be related to the administration of the test item.

Focal alveolar emphysema in the lungs occurred sporadically to a minimal or mild degree (3/5 control male, 1/5 control female dam; 2/5 male and 1/5 dam at 100 mg/kg bw/day). These findings were considered to be a consequence of hypoxia, dyspnoea and circulatory disturbance developed during the scheduled death by exsanguination. One sided lymphatic pyelitis was observed in a single animal at 10 mg/kg bw/day (1/5 dam) as an individual disease. The hyperplasia of bronchus associated lymphoid tissue (BALT) in a control (1/5 of male) and 100 mg/kg bw/day treated animals (1/1 male fatality and 1/5 dam) was considered to be a physiological adaptation. Histology of the reproductive organs (testes, epididymides, ovaries, uterus, vagina and pituitary) revealed no differences between the test item treated animals and the control groups. The ovaries had a normal structure characteristic of the species, age and phase of the active sexual cycle in all cases of control and treated groups. The cortex contained primary, secondary and tertiary follicles and corpora lutea, indicating the active maturation of oocytes and ovulation. The epithelial capsule and ovarian stroma were normal in all cases. The uterus, cervix and vagina had a normal structure in phase with the sexual cycle. In some animals (5/12 control: 2/2 non-pregnant females and 3/9 surviving dams), dilatation of the uterus was observed, but in absence of inflammation or other pathological lesions, that was considered to be a physiological phenomenon connected to the normal sexual cycle. Pituitary histology was normal in animals treated with the test item and in their control groups. No morphological evidence of acute or subacute injury (degeneration, inflammation, necrosis etc.) of the liver, small and large intestines, cardiovascular system, the immune system, the hematopoietic system, the skeleton, the male and female reproductive system or the central or peripheral nervous system was observed in males or females. The structure and the cell morphology of the endocrine glands were similar between the control groups and the treated animals of both sexes.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

30 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; body weight; food consumption, some clinical chemistry parameters; histopathology

2

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

> 100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effects at all dosages applied

Results: F1 generation

General toxicity (F1)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at dosage with maternal toxicity

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

at dosage with maternal toxicity

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at dosage with maternal toxicity

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Details on results (F1)

VIABILITY (OFFSPRING)
In the high dose group, the number of dead male offspring on postnatal day 0 and the number of dead male and female offspring between days 0 and 4 were slightly but significantly higher than in the other groups, including the control. Statistical significances indicated only slight differences, considered to be caused by maternal toxicity.

CLINICAL SIGNS (OFFSPRING)
In the high dose group, the number and percentage of cold and not fed pups were slightly higher than in the control group. An indirect effect of the test item through maternal toxicity was assumed, as high mortality occurred in litters of dams where clinical signs where detected and for which inadequate nursing was noted.

BODY WEIGHT (OFFSPRING)
At the dosage with maternal toxicity (high dose: 100 mg/kg/bw/day), the mean litter weight and litter weight gain and also offspring’s mean weight at birth was found to be reduced when compared to the vehicle control group.

SEX RATIO (OFFSPRING)
There were no significant differences in the ratio or in the litter means of genders between control and test item treated groups on postnatal days 0 or 4.

GROSS PATHOLOGY (OFFSPRING)
No test item related macroscopic alterations were found in offspring subjected to gross pathological examination. There were no signs of test item related effects and no skeletal or visceral malformations were observed in offspring during the macroscopic examination.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

F1

Effect level:

30 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: overall effects: - body weight (litter and pup’s weights slightly depressed)

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The NOAEL for systemic toxicity is based on various findings at 100 mg/kg/bw regarding in vivo changes on clinical signs, body weight, clinical parameters and changes in organ pathology. A value of 30 mg/kg bw was derived as the systemic NOAEL for parental animals and the offspring.
The NOAEL for reproductive performance is considered to be 100 mg/kg bw/day as no obvious reproductive changes were observed in the parental animals..

Executive summary:

The purpose of this Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test was to provide initial information concerning the toxic potential of lithium bis(oxalato)borate and on its possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, pregnancy, parturition as well as on development of the F1 offspring from conception to day 4 post-partum associated with oral administration to rats at repeated doses. The test was performed according to OECD guideline 422. The test item was administered orally (by gavage) at 0, 10, 30 or 100 mg/kg bw/day, corresponding to concentrations of 0, 2, 6 and 20 mg/mL at a 5 mL/kg bw dose volume to four dose groups of Hsd.Brl.Han:Wist rats (n=12/sex/group) once a day. All animals of the parent (P) generation received test item or vehicle prior to mating (for 14 days) and throughout the mating phase. Test item or vehicle was administered to male animals post mating up to the day before the necropsy. For females with live pups, test item was administered through the gestation period and up to lactation days 3 – 11, i.e. up to the day before the necropsy. The dams were allowed to litter and rear their young up to termination on day 4 postpartum. Pups were weighed and observed for possible abnormalities and were euthanized on postnatal day 4. Observations included mortality, clinical signs, body weight, food consumption, evidence for mating, pregnancy and parturition, as well as the development of pups. Five dams and the cohabiting males were selected from each group for further toxicity examinations such as functional observations, hematology, clinical chemistry, gross necropsy, and organ weight. All parental animals were subjected to gross pathology one day after the last treatment. Selected organs of parental animals were weighed. Full histopathology was performed on the five selected animals of both, the control and high dose groups. Histopathology on reproductive organs and pituitary was performed on all animals of the control and high dose groups. The reproductive organs and pituitary of non-pregnant female animals and cohabiting males of the low and mid dose groups were also processed and evaluated histologically. The kidneys of animals at the low and medium doses were processed as well and evaluated histologically due to histopathology findings in kidneys of the high dose animals. The results were interpreted comparing treatment groups with respect to controls, which were treated concurrently with vehicle (sunflower oil) only. The results are listed below:

Hematology

Statistically higher percentage of the neutrophil granulocytes and reduced percentage of lymphocytes were seen in females of the high dose groups. Other than that, hematology examinations did not reveal test item related changes in the examined hematological parameters in male or female animals at any dose level (100, 30 or 10 mg/kg bw/day).

Clinical chemistry

A slightly higher mean concentration of creatinine, urea and inorganic phosphorous referred to a test item influence on renal function in male and female animals dosed with 100 mg/kg bw/day. Changes in elevated mean concentration of bile acids might be indicative a test item effect on hepatic function in female animals dosed with 100 mg/kg bw/day.

Necropsy

Test item related renal changes were observed in male (2/11) and female (4/8) animals dosed with 100 mg/kg bw/day: the kidneys were found to be pale and enlarged. Other than this, there were no test item related findings in animals that survived until the scheduled termination of the in life phase.

Organ weight

The kidney weights (absolute and relative to body and brain weights) were significantly elevated in male and female animals at 100 mg/kg bw/day with respect to controls.

Histopathology

Histopathology investigations revealed test item related renal lesions in males and females of the high dose group (100 mg/kg bw/day). Segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration, mineral deposits in the lumina of descending proximal tubules and segmental vacuolization of tubular epithelial cells were observed.

Reproduction

There were no differences between the control and test item treated groups in the delivery data of dams. The reproductive ability of male and female animals was unaffected by the treatment with Lithium-bis(oxalato)borate at 100, 30 or 10 mg/kg bw/day.

Offspring

A test item effect on the offspring development was observed in the slightly higher extra uterine mortality (number and percent) between postnatal days 0 and 4, and in the less litter weight and litter weight gain and mean pup’s weight on postnatal day 0 in 100 mg/kg bw/day group. The higher mortality of pups was probably a consequence of maternal toxicity.

Under the conditions of the present study, Lithium-bis(oxalato)borate caused salivation (male), clinical signs and changes in body weight and food consumption (female) towards end of gestation and changes in clinical chemistry parameters (higher creatinine, urea and inorganic phosphorous, in male and female animals, elevated bile acid level in female animals), and changes in organ pathology in male and female animals (enlarged and pale kidneys, higher kidney weights and segmental tubular basophilia accompanied with slight intertubular lymphocytic infiltration, mineral deposits in the lumina of descending proximal tubules and segmental vacuolization of tubular epithelial cells) following an oral administration at 100 mg/kg bw/day to Hsd.Brl.Han:Wistar rats during the Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test. At 30 mg/kg bw/day, salivation was observed in some male animals. At 10 mg/kg bw/day, there was no test item related effect. Male and female reproductive performance (gonad function, mating behavior, conception, pregnancy, parturition) and dam’s delivery data was not affected by the test item at any dose level. At 100 mg/kg bw/day, the extra uterine mortality of offspring was slightly higher in percentage and mean with respect to control and the offspring’s body weight development (for litter and pup’s weights) was slightly depressed.

Based on these observations the No Observed (Adverse) Effect Levels (NO(A)EL) were determined as follows:

NO(A)EL for male rats: 30 mg/kg bw/day

NO(A)EL for female rats: 30 mg/kg bw/day

NO(A)EL for reproductive performance of the male and female rats: 100 mg/kg bw/day

NO(A)EL for F1 Offspring: 30 mg/kg bw/day (TOXICOOP, 2012)