Lithium bis(oxalato)borate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 NOV 2020 - 12 AUG 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other 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:

2021

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 25th June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

[EPA 712–C–98–199], August 1998

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: US FDA Toxicological Principles for the Safety Assessment of Food Ingredients, Redbook 2000, IV.C. 4. a. Subchronic Toxicity Studies with Rodents (2003).

Version / remarks:

2003

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

Details on species / strain selection:

Rat, Han: WIST of Wistar origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt., Cserkesz u. 90, 1103 Budapest
- Females (if applicable) nulliparous and non-pregnant: yes, nulliparous and non-pregnant animals
- Age at study initiation: young adult rats, 8- 9 weeks old
- Weight at study initiation: 257-284 g for male animals, 160 – 177 g for female animals
- Housing: Group caging; max. 2 or 3 animals of the same sex/ cage; Type III polypropylene/polycarbonate with certified laboratory wood bedding; The cages and bedding were changed twice a week.
- Diet (e.g. ad libitum): ssniff® SM R/M-Z+H complete diet, ad libitum except overnight food deprivation before the blood sampling.
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY:
The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used, which is shown in the study report.
Water quality control analysis and microbiological assessment are performed once in every six months by National Public Health and Medical Officer Service (József A. u. 36. 8200 Veszprém, Hungary). The quality control results are available at Toxi-Coop Zrt.’s archives and are presented in the study report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12 - artificial light, from 6 a.m. to 6 p.m. (except days of ophthalmology examinations)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The route of application was selected in compliance with international guidelines

Vehicle:

other: Sunflower oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was administered at appropriate concentrations, prepared with the vehicle. Sunflower oil was dehydrated by adding Na2SO4 (1 g Na2SO4/10 mL sunflower oil). The supernatant was used for formulation after fully sedimentation to avoid as far as possible hydrolysis of the substance before administering to the animals. Preparation of the test item formulations were made every day, using a magnetic stirrer in the Formulation Laboratory of the Test Facility and was administered to the animals within 4 hours.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item is neither soluble nor stable in water. Thus, sunflower oil was used for preparing formulations appropriate for oral administration.
- Concentration in vehicle: 2.5, 6.25, 22.5 mg/mL
- Amount of vehicle (if gavage): 10 mL, 4 mL/kg bw dosing volume
- Lot/batch no. (if required): 8006332001

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability (recovery and stability) of the vehicle Sunflower oil for the test item at the intended concentrations was analytically verified up front.
LiBOB Advanced Battery Grade concentrations in the dose solutions used for administration of animals varied within the range of 90 % to 110 % in comparison to the nominal values, thereby confirming proper dosing. Deviation of the concentration of the samples taken from different places of the vessels was ≤ 14%, therefore the formulations were considered homogeneous.

Duration of treatment / exposure:

90 day exposure

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle only

Doses / concentrations 2

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery group

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 5

Dose / conc.:

90 mg/kg bw/day (nominal)

Doses / concentrations 6

Dose / conc.:

90 mg/kg bw/day (nominal)

Remarks:

Recovery group

No. of animals per sex per dose:

10 males and 10 females.
Recovery group: 5 males and 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose setting with 0, 10, 25 and 90 mg/kg bw/day was based on the results of Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with Lithium-bis(oxalato)borate in the Rat (Study no. 559-439-3243) in agreement with the Sponsor’s representative.
- Rationale for animal assignment (if not random): Selected rats were distributed by randomization according to stratification by body weight
- Fasting period before blood sampling for clinical biochemistry: for approximately 16 hours
- Post-exposure recovery period in satellite groups: 41-42 days post-exposure recovery of recovery groups

Positive control:

none

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected for signs of morbidity and mortality twice daily at the beginning and end of each working day. General clinical observations were made cage-side once a day, after the administration at approximately the same time during the treatment period as well as during the course of the recovery period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly during the treatment and recovery periods.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was determined with the measurement of non-consumed diet with a precision of 1 g once weekly to coincide with body weight measurements. During the recovery period the food consumption was measured once weekly.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined: During the acclimation period, the eyes of all rats being considered for study were examined by focal illumination, by indirect ophthalmoscopy. Prior to test termination, the eyes of the high dose group and control group were examined. There were no treatment related changes in animals at the highest dose, therefore eyes of animals in the low and mid dose groups were not examined. The animals of recovery groups were examined on the last week of recovery period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: conducted at termination of the treatment and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes, Isofluran CP®
- Animals fasted: Yes, all animals were fasted overnight prior to blood collection.
- How many animals: all animals
- Parameters examined: white blood cell count, red blood cell count, hemoglobin concentration, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, mean platelet volume, reticulocytes, differential white blood cell count, APTT, PT

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: conducted at termination of the treatment and at the end of the recovery period.
- Animals fasted: Yes, all animals were fasted overnight prior to blood collection.
- How many animals: all animals
- Parameters examined: ALT, AST, GGT, ALP,TBIL, CREA, UREA, GLUC, CHOL, HDL, LDL, BUN, ALB, TPROT, A/G and inorganic phosphate, calcium, sodium, potassium, chloride concentrations

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: day of necropsy
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: TSH and Thyroid Hormones (FT3 and FT4)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Gross pathology was performed on every experimental animal at termination of the treatment i.e. one day after the last treatment, on Days 90 and 91 (male and female, respectively) or at the end of the recovery period. Animals were anesthetized with Release and were exsanguinated from the abdominal aorta after verification of narcosis. The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size. One dead female animal at 25 mg/kg bw/day was necropsied on day of her death (Day 82).

HISTOPATHOLOGY: Yes
Histopathological examination was performed on the preserved organs and tissues of the animals from both the control and high dose groups (Groups 1 and 4, respectively), in group 3 and in male animals of group 2 the kidney as possible target organ including recovery groups and in one female dead animal from 25 mg/kg bw/day dose group. As there were no test item-related lesions in the organs or tissues of high dose animals, no general histopathological examination was performed for low and mid dose animals. Examination of parathyroids was performed by section plane of thyroids crosses parathyroids. The fixed tissues were trimmed, processed, embedded in paraffin, sectioned with a microtome, placed on glass microscope slides, stained with hematoxylin and eosin and examined by light microscopy.

Other examinations:

Estrous cycle and sperm examinations

At necropsy (at the end of treatment and recovery periods), the estrous cycle was monitored by examining vaginal smears from each female animal. These observations provided information regarding the stage of oestrus cycle at the time of sacrifice and assist in histological evaluation of estrogen sensitive tissues.
The vaginal smear was prepared and stained with 1 % aqueous methylene blue solution. The smear was examined with a light microscope.
At termination, testis and epididymis weights were recorded for all males. One epididymis from each male was reserved for histopathological examination. The remaining epididymis was used for enumeration of cauda epididymis sperm reserves sperm examinations.
Sperm from the ductus deferens was collected for evaluation of sperm motility and morphology at the necropsy in line with the recommendation of OECD 408. Both numbers of motile and immotile sperms were recorded. Two samples were prepared from each animal. For the determination of the sperm motility, the mean percentage of motile sperms were determined.
A morphological evaluation of ductus deferens sperms sample was performed from the same animals. Sperm was examined as fixed, wet preparations and classified as either normal or abnormal (isolated heads, misshapen heads and/or tails).
The epididymis was used for enumeration of cauda epididymis sperm reserves. The total number of sperm in homogenization was enumerated. The testis and epididymidis were frozen and enumeration were performed later.
Treatment related effects were not found in the high dose group, the lower dose groups were not evaluated.

Statistics:

Statistical analysis was done with SPSS PC+ software

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No clinical signs related to the test item were found at general daily or detailed weekly clinical observations at 10, 25 and 90 mg/kg bw/day. The functional observations did not reveal any test item influence on the animal behavior or neurological functions.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female animal of middle dose died during the study. The acute pneumonia noted could be considered as the cause of death and is evaluated as an individual disease. Histological examination revealed acute serous pneumonia and acute focal hemorrhage in the leptomeninx of cerebellum. No degenerative or other toxic lesions were observed in the investigated organs of this animal. The death is likely to be caused by an application error.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no test item related changes in the mean body weight and body weight gain in the male and female animals at 10, 25 and 90 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No adverse effects on the mean food consumption development were observed at 10, 25 or 90 mg/kg bw/day in male or female animals (main and recovery groups).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No alterations were detected in the eyes of the animals of the high dose group at the end of treatment or recovery periods.

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test item related adverse pathological changes in the investigated hematological or blood coagulation parameters in any group (main and recovery groups).

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Pathological test item effects were not detected upon the evaluation of the clinical chemistry parameters in male animals at 10, 25 mg/kg bw/day and in female animals at 10, 25 and 90 mg/kg bw/day (main and recovery groups). However, in males at a dose of 90 mg/kg bw/day, high mean TBIL, CREA, UREA, BUN and high mean calcium (Ca ++) concentrations (statistically significant compared to the concurrent controls) are indicative for an effect on the kidneys, which is further supported by findings noted at necropsy and the histopathological examination. These effects were however not observed in the recovery group and indicate reversibility of the changes.

Endocrine findings:

no effects observed

Description (incidence and severity):

The structure and the cell morphology of the endocrine glands were the same in the control and treated animals. There were no toxicologically relevant changes in the concentrations of FT3 (free T3), FT4 (free T4) and TSH hormones in male and female animals at 10, 25 and 90 mg/kg bw/day at the end of treatment and recovery period.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The functional observations did not reveal any test item influence on the animal behavior or neurological functions.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

No treatment related alterations were found in the organ weight results (absolute weights, relative to the body weight and brain weight) in male and female animals compared to the control parameters. However, in the male group at 90 mg/kg bw/day the higher mean kidney weight (absolute weight and relative to brain weight) with the corresponding histopathological findings is considered a test item related effect.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item related macroscopic changes were not detected in animals at 10 and 25 mg/kg bw/day and in female animals at 90 mg/kg bw/day at the necropsy. However, in the high dose male animals, the pale, enlarged and granulated surface kidneys with the corresponding histopathological findings, the clinical chemistry and organ weight is considered a test item related effect.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The test item did not cause any clear test item related lesions detectable by histological examination of investigated organs of the experimental female animals at 10, 25 and 90 mg/kg/bw/day dose groups and in males of the 10 mg /kg/bw/day dose group. At dose level of 25 mg/kg in two male animals out of ten minimal (grade 1) segmental tubular basophilia, accompanied with intertubular lymphocytic infiltration and mineral deposites and vacuolation were found in the kidneys. At dose group of 90 mg/kg in three male animals out of ten severe (grade 4), in other two animals moderate (greade 3) segmental tubular basophilia, accompanied with intertubular lymphocytic infiltration and mineral deposites and vacuolation were found in the kidneys. In three animals mild (grade 2) and in one animal minimal (grade 1) segmental tubular basophilia, accompanied with intertubular lymphocytic infiltration and mineral deposites and vacuolation in the kidneys were observed. The observed minimal changes noted in the two male animals at a dose level of 25 mg/kg/day (grade 1 only) are not considered as an adverse effect because the minimal histopathological alterations, occurring spontaneously in male rats were not supported by any other interlinked parameters (necropsy findings, clinical chemistry or kidney weight).

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Estrous cycle and sperm examinations

A test item effect on the estrous cycle was not detected at any dose level. There were no notable differences between the control and at 10, 25 or 90 mg/kg bw/day female animals. In accordance with this finding, histopathological examinations did not reveal changes in the morphology of uterus or ovaries in this study.
The morphology and motility of sperm cells (motile sperms, immotile sperms, sperms with normal morphology, sperms with seperated head and tail) were not affected after three- month administration of 90 mg/kg bw/day of the test item.

Details on results:

On basis of international literature, the observed renal changes at 90 mg/kg bw dose levels and potentially at 25 mg/kg bw are likely to represent CPN (chronic progressive nephropathy) a symptom of describing the coexistence of several alterations (segmental tubular basophilia accompanied with intertubular lymphocytic infiltration, mineral deposits in the lumina of descending proximal tubules and segmental vacuolization of tubular epithelial cells). These changes are present in all CPNs, but the grade score depends on their severity. CPN is an important spontaneous renal disease of the commonly used strains of laboratory rat because it is a serious confounding factor in experimental pathology and toxicology studies. Compare to female rats, there is a distinct male predisposition to CPN with respect to onset, incidence and severity progression. In the present study CPN in the male animals of control and at 10 mg/kg bw/day was not found. CPN tend to occur more frequently in long-term studies (in at least 1 year old animals).
Based on the frequency and severity of CPN effects noted at 90 mg/kg bw/day, the test item seems to accelerated the pathogenesis of CPN (chronic progressive nephropathy) in male animals with effects noted in 90% of the animals in contrast to the incidence and severity in other male and female treated and control groups.
Taken together with other effects noted in clinical chemistry, macroscopic examination (enlarged kidneys and granulated surface of kidneys) and in kidney weight of males, this indicates that the test item causes kidney toxicity at this dose level. The kidney was also identified as the most sensitive target in OECD 422 study.
The incidence of alterations in high dose group males remained higher than in controls in the recovery animals (incidence of 40% against 0 in the controls) but only grade 1 effects were noted indicating a tendency to recovery.

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

25 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical biochemistry

gross pathology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

2

Dose descriptor:

NOAEL

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects up to the highest dose tested

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

The severity scores (internal scoring system) of the histopathological kidney alterations (segmental tubular basophilia, accompanied with intertubular lymphocytic infiltration and mineral deposites and vacuolation) were given as follows:

1                  minimal: one circumscribed area

2                  mild: 2 -3 circumscribed areas

3                  moderate: more than 3 circumscribed areas

4                  severe: diffuse lesion, affects the whole examined area

 

Applicant's summary and conclusion

Conclusions:

The test item did not cause adverse effects in male Han:WIST rats after 90/91-day consecutive oral (by gavage) administration of 25 mg/kg bw/day and in female animals at 10, 25 and 90 mg/kg bw/day doses.
In the male animals at 25 mg/kg bw/day and in female animals at 10, 25 and 90 mg/kg bw/day doses, there were no toxicologically relevant changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry, serum levels of thyroid hormones, estrous cycle and sperm examinations, necropsy findings, organ weights or histopathological findings).
However, in the male group of 90 mg/kg bw/day the higher mean kidney weight (absolute weight relative to brain weight), clinical chemistry parameters (high mean TBIL, CREA, UREA, BUN and high mean calcium (Ca ++) concentrations), necropsy (the kidneys pale, enlarged, granulated surface) and histopathological findings (test item accelerated the pathogenesis of CPN in 90% of the animal with up to score 4) are indicative for a test item related renal toxicity. In the male animals at dose level of 25 mg/kg/day the observed low frequency and minimal (score 1) histopathological alterations, known to spontaneous occurring in male rats were not considered as a toxicologically relevant effect of the test item, as the alteration was not supported by other related parameters (necropsy findings, clinical chemistry or organ wieght) yet.
Based on the observations made in this toxicity study the No Observed Adverse Effect Level (NOAEL) was determined as follows.
NOAEL: 25 mg/kg bw/day for male animals
90 mg/kg bw/day for female animals

Executive summary:

A 90 -day repeated dose toxicity study was performed in compliance with GLP and according to OECD Test Guideline 408. Doses of 10, 25 and 90 mg/kg bw/day were applied by gavage to male and female animals over a 90-day period of time covering post-weaning maturation and growth well into adulthoodfollowed by a 42-day recovery period in the high dose and control animals in order to assess reversibility, persistence or delayed occurrence of potential toxicological effects.

Under the conditions of this study, the test item did not cause adverse effects in male Han:WIST rats after 90/91-day consecutive oral (by gavage) administration of 25 mg/kg bw/day and in female animals at 10, 25 and 90 mg/kg bw/day doses. In the male animals at 25 mg/kg bw/day and in female animals at 10, 25 and 90 mg/kg bw/day doses, there were no toxicologically relevant changes in the examined parameters (clinical signs, body weight and body weight gain, food intake, ophthalmology, hematology, blood coagulation and clinical chemistry,serum levels of thyroid hormones,estrous cycle andsperm examinations, necropsy findings, organ weights or histopathological findings). However, in the male group of 90 mg/kg bw/day the higher mean kidney weight (absolute weight relative to brain weight), clinical chemistry parameters(high mean TBIL, CREA, UREA, BUN and high mean calcium (Ca⁺⁺) concentrations), necropsy (the kidneys pale, enlarged, granulated surface) and histopathological findings (test item accelerated the pathogenesis of CPN in 90% of the animals with scores up to 4) are indicative for a test item related renal toxicity. In the male animals at dose level of 25 mg/kg/day the observed low frequency and minimal (score 1) histopathological alterations, known to spontaneous occurring in male rats were not considered as a toxicologically relevant effect of the test item, as the alteration was not supported by other related parameters (necropsy findings, clinical chemistry or organ weight) yet. Based on the observations made in this toxicity study the No Observed Adverse Effect Level (NOAEL) was determined as 25 mg/kg bw/day for male and 90 mg/kg bw/day for female animals.