Lithium bis(oxalato)borate

Endpoint summary

Description of key information

oral

oral gavage, rat, OECD 408, GLP, NOAEL = 25 mg/kg bw/day (males) and 90 mg/kg bw/day (females)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Reason / purpose for cross-reference:

reference to other study

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

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

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

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)

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

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle only

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Recovery group

Dose / conc.:

10 mg/kg bw/day (nominal)

Dose / conc.:

25 mg/kg bw/day (nominal)

Dose / conc.:

90 mg/kg bw/day (nominal)

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

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

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.

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

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

Critical effects observed:

no

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

 

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.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

GLP and Test Guideline compliant studies are available. Thus, the quality of the database is high.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

repeated oral toxicity

key study

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 (TOXICOOP 2021).

supporting study

LiBOB was tested in a for repeated dose toxicity in a 28 days oral gavage test according to OECD guideline 407. LiBOB was administered to male and female Sprague-Dawley rats (n= 10/sex/dose) at dose levels of 0 (control), 10, 40 and 160 mg/kg bw/day for consecutive 28 days. To observe the reversibility and delayed effects, an additional high dosage group (160 mg/kg bw/day) and an additional control group (0 mg/kg bw/day) were applied and observed for another 14 days. During the period of experiment body weights and food consumption were recorded weekly, and overt signs of toxicity including quality of hair, general condition of eyes, mouth, teeth, nose and ears, posture, gait, behavior or activity, character of excreta, etc. were observed daily. At the end of experiment, urine samples were collected for routine assays. All rats were sacrificed by CO2. Blood samples were collected for routine assays and specified organs were taken for pathological examination. The data was statistically analyzed by ANOVA between groups and t-test for the additional groups, respectively.

No clinical signs or death were found in any groups throughout the experimental period. A significant decrease in body weight of males of high dose group was observed from the first to the fourth weeks, compared with the control group (P<0.05). For females of high dose group, absolute organ weights of kidney and relative organ weights of heart, liver, spleen, kidney, adrenal gland and lung were significantly increased (P<0.05). For females of mid dose group, relative organ weights of lung were significantly increased (P<0.05). For males of high dose group, absolute organ weights of spleen and kidney were significantly increased, while relative organ weights of brain, heart, spleen, kidney and adrenal gland were significantly increased, compared with the control group (P<0.05). The results of blood analysis showed a significantly lower value of hemoglobin in females of high dose group and significantly lower values of RBC and hemoglobin in males of high dose group, compared with the control group (P<0.05). In the urinalysis, significant lower pH values were observed in both genders of high dose groups, compared with their respective control groups (P<0.05). On serum biochemical examination, values of TP, ALB were significantly decreased in females of high dose group, compared with the control group (P<0.05). Significant decreases in values of TP, ALB and Na and significant increases in BUN in male rats of high dose group, and a significant decrease in values of Na in males of mid dose group were noted, compared with their respective control groups (P<0.05). All rats were subjected to a full gross necropsy and no obvious abnormalities were found during macroscopical observation. The kidneys in all rats of both genders from high dose group exhibited different extent of degeneration and necrosis of partial renal tubule, renal tubule crystal retention and infiltration of inflammatory cells of interstitial tissue, while such observations did not occur for rats of both genders from mid or low dose groups. The findings in the mid dose group, in particular the higher relative lung weights in female and the relative lower concentration of the Na concentration in male animals, are considered to be not test substance-related. The lung is not a target organ of the test substance as there were no concentration depending effects in the lung to brain ratios, no histopathological findings even in the high dose group and no correlation between the sexes. The decrease in the Na concentration was considered as incidental as the differences were minimal and the values exhibited a high standard deviation and showed no clear concentration dependency.

Additional high dose group and additional control group were kept for two more weeks, examinations and records were performed as dosage groups. A significant decrease in body weight of males of additional high dose group was observed from the second to the fourth weeks, compared with the additional control group (P<0.05). For females of additional high dose group, absolute organ weights of spleen and kidney and the relative organ weights of liver, spleen, kidney, adrenal gland were significantly increased, compared with the control group (P<0.05). For males of additional high dose group, absolute organ weights of kidney, adrenal gland and relative organ weights of heart, liver, spleen, kidney, adrenal gland were significantly increased (P<0.05). A significant decrease of hemoglobin in females and significant decreases in RBC and hemoglobin in male rats of treated group were noted (P<0.05). In males of additional high dose group, values of Na were significantly decreased, values of BUN, CREA, Cl were significantly increased, compared with their respective control groups (P<0.05). No obvious abnormalities were found during macroscopical observation. During pathological examination, the kidneys in all rats of both genders from high dose group exhibited different extent of degeneration and necrosis of partial renal tubule, renal tubule crystal retention and infiltration of inflammatory cells of interstitial tissue.

Based on the results above, a NOAEL of LIBOB PREMIUM BATTERY GRADE in repeated dose 28-day oral toxicity test in SD rats is 40 mg/kg bw/d in this study. There was no significant trend to recovery toward the end of the prolonged 14-day observation. (IOAT, 2010)

This finding is supported by a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental toxicity screening test 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)

Justification for classification or non-classification

Based on the results obtained from repeated dose testing, lithium bis(oxalato)borate was not classified and labelled for long-term toxicity according to Regulation (EC) No 1272/2008 (CLP).