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REACH

Lithium bis(oxalato)borate

EC number: 456-990-3 | CAS number: 244761-29-3 LITHIUM-BIS(OXALATO)BORATE

Life Cycle description
Uses advised against

Toxicological information

Developmental toxicity / teratogenicity

Administrative data

Endpoint:: developmental toxicity
Remarks:: Prenatal Developmental Toxicity Study
Type of information:: experimental study
Adequacy of study:: key study
Study period:: 13 NOV 2020 to 14 JUN 2021
Reliability:: 1 (reliable without restriction)
Rationale for reliability incl. deficiencies:: guideline study

Cross-referenceopen all close all

Cross-reference 1

Reason / purpose for cross-reference:: reference to other study

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-03-19 to 2012-10-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant Guideline study.

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:

Qualifier:

according to guideline

Guideline:

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

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

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 exposure:

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

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

Details on mating procedure:

- M/F ratio per cage: 1 / 1
- Length of cohabitation: Each female was placed with the same male until mating was assumed or 14 days had elapsed
- Proof of pregnancy: detection of vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After 14 days of unsuccessful pairing, replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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

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

Duration of treatment / exposure:

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

Frequency of treatment:

Once daily (7 days / week)

Details on study schedule:

- Animal age at mating in the study: 14 weeks

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

Parental animals: Observations and examinations:

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

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

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

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

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

WATER CONSUMPTION: No

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

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

URINALYSIS: No

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

Sperm parameters (parental animals):

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

Litter observations:

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

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

Postmortem examinations (parental animals):

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

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

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

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

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

Postmortem examinations (offspring):

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

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

Statistics:

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

Reproductive indices:

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

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

Offspring viability indices:

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

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

salivation, piloerection, activity decrease, decreased body tone

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

high dose females

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

high dose females

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

kidneys

Other effects:

not examined

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

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

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

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

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

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

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

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

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

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

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

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

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

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

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

30 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

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

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

> 100 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: No effects at all dosages applied

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

at dosage with maternal toxicity

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

at dosage with maternal toxicity

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

at dosage with maternal toxicity

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

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

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

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

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

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

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Generation:

Effect level:

30 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

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

Reproductive effects observed:

not specified

Conclusions:

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

Executive summary:

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

Hematology

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

Clinical chemistry

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

Necropsy

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

Organ weight

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

Histopathology

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

Reproduction

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

Offspring

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

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

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

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

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

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

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

Cross-reference 2

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:

Qualifier:

according to guideline

Guideline:

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

Deviations:

GLP compliance:

yes (incl. QA statement)

Limit test:

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Route of administration:

oral: gavage

Vehicle:

other: dehydrated sunflower oil

Details on oral exposure:

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Duration of treatment / exposure:

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:

Positive control:

No positive control

Observations and examinations performed and frequency:

Sacrifice and pathology:

Statistics:

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:

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

Clinical chemistry

Necropsy

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

Reproduction

Offspring

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)

Cross-reference 3

Reason / purpose for cross-reference:: reference to other study

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-08-17 to 2010-02-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline compliant study

Qualifier:

according to guideline

Guideline:

OECD Guideline 425 (Acute Oral Toxicity: Up-and-Down Procedure)

Version / remarks:

October 2008

Deviations:

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

April 29, 2004

Deviations:

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Version / remarks:

December, 2002

Deviations:

GLP compliance:

yes (incl. QA statement)

Test type:

up-and-down procedure

Limit test:

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (Europe) Laboratories Inc. TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 203-229 g
- Fasting period before study: food but not water was withheld for 24h before treatment
- Housing: 1 animal/cage (Type II. polypropylene/polycarbonate)
- Diet (e.g. ad libitum): ssniff SM R/M-Z+H, ad libitum
- Water (e.g. ad libitum): tap water from municipal supply, ad libitum
- Acclimation period: at least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-24.7
- Humidity (%): 38-67%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: corn oil

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 17.5, 55, 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): 058K0070

Doses:

175, 550, 2000 mg/kg bw

No. of animals per sex per dose:

treatment group 1 (175 mg/kg bw): 2 females
treatment group 2 (550 mg/kg bw): 4 females
treatment group 3 (2000 mg/kg bw): 1 female

Control animals:

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations were done daily
- Necropsy of survivors performed: yes
- clinical signs: were observed 30 min, 1, 2, 3, 4 and 6 hours after treatment and once each day for 14 days thereafter
- body weight: were recorded on days -1, 0 (day of experiment), and on days 7 and 14

Sex:

female

Dose descriptor:

LD50

Effect level:

550 mg/kg bw

Based on:

test mat.

95% CL:

88.94 - 2 430

Sex:

female

Dose descriptor:

LD0

Effect level:

175 mg/kg bw

Based on:

test mat.

Mortality:

Treatment group 1: 175 mg/kg bw
No mortality occurred in this group.

Treatment group 2: 550 mg/kg bw
Treatment with lithium bis(oxalato)borate (LiBOB) at a dose level of 550 mg/kg bw caused mortality in case of 2 animals (2/3).
One female died died shortly after treatment due to a dosing accident (misgavage) and/or gastro-esophageal reflux (not test item related).

Treatment group 3: 2000 mg/kg bw
Treatment with lithium bis(oxalato)borate (LiBOB) at a dose level of 2000 mg/kg bw caused mortality in this dose group.

Clinical signs:

other: In Treatment group 1 LiBOB caused decreased activity in one out of two rats. In Treatment group 2 a decreased activity in one out of 4 rats was observed.

Gross pathology:

Treatment group 1: 175 mg/kg bw
Treatment with lithium bis(oxalato)borate (LiBOB) at a dose level of 175 mg/kg bw caused dilatation, (pelvis, right) and white cloudy fluid in one rat’s kidneys in this dose group.

Treatment group 2: 550 mg/kg bw
Treatment with lithium bis(oxalato)borate (LiBOB) at a dose level of 550 mg/kg bw caused diffuse dark red discoloration of the lungs and liver, dark, punctuate red discoloration of the thymus (bilateral) and dark, red, diffuse discoloration and mucosa in ileum in this dose group.

Treatment group 3: 2000 mg/kg bw
Treatment with lithium bis(oxalato)borate (LiBOB) at a dose level of 2000 mg/kg bw caused diffuse dark red discoloration of the liver and dark, red, diffuse discoloration and mucosa in ileum, duodenum and jejunum in this dose group.

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

Lithium bis(oxalato)borate (LiBOB) was ranked into Category 4 of Globally Harmonized Classification System as the estimated LD50 is between 300 and 2000 mg/kg bw.

Executive summary:

An acute oral toxicity study (up-and-down procedure) was performed with lithium bis(oxalato)borate (LiBOB) according to OECD Guideline 425. 7 young female CRL:(WI) BR Wistar rats were treated with the test item by oral gavage at dose levels of 175, 550 and 2000 mg/kg bw in corn oil. The concentrations of the formulations in corn oil were 200, 55 and 17.5 mg/mL with a constant treatment volume of 10 mL/kg bw.

Lithium bis(oxalato)borate (LiBOB) caused 100% mortality at 2000 mg/kg bw (1/1 animal) and 67% (2/3 animals) mortality at 550 mg/kg bw. There was no mortality at 175 mg/kg bw.

Treatment with lithium bis(oxalato)borate (LiBOB) caused mortality at a dose level of 2000 mg/kg bw, decreased activity (1/3) and mortality (2/3) at 550 mg/kg bw and decreased activity (1/2) at 175 mg/kg bw.

The body weight gain of the animals was considered to be normal with no indication of test item related effect.

The macroscopic examination revealed some minor alterations including diffuse dark red discoloration of the lungs (1/6) and liver (3/6), dark, punctuate red discoloration of the thymus (bilateral) (1/6), dark, red, discoloration, diffuse, mucosa in ileum (2/6), duodenum, jejunum (1/6). Dilatation, (pelvis, right) and white cloudy fluid was found in one rat’s kidneys.

The study was marked reliable without restrictions and LD50 and LD0 values of 550 and 175 mg/kg bw respectively were derived. (LAB, 2010)

Cross-reference 4

Reason / purpose for cross-reference:: reference to other study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2010-09-14 to 2010-11-02

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No GLP but OECD compliant study with sufficient scientific documentation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

October 3rd 2008

Deviations:

GLP compliance:

yes

Remarks:

Procedures similar to GLP were assessed.

Limit test:

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Shanghai SLAC Laboratory Animal Co., Ltd., China
- Weight at study initiation: 120 - 143 g
- Fasting period before study: no
- Housing: two rats per polycarbonate box
- Diet (e.g. ad libitum): powdered diet from Suzhou Shuangshi Laboratory Animal Fedd Co., Ltd., ad libitum
- Water (e.g. ad libitum): sterilized drinking water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 40 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed with automatic balance, and then diluted with corn oil to designed concentrations. The volume of liquid administered daily was 5 mL/kg bw. The gavage dose was adjusted at the beginning of each week based on the individual body weights.

VEHICLE
- Concentration in vehicle: 160, 40, 10, 0 mg/kg bw per day and additional groups of 0 and 160 mg/kg bw per day (observed for another 14 days after exposure period)
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

Duration of treatment / exposure:

Doses were administered on 28 consecutive days.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
160, 40, 10, 0 mg/kg bw per day
Basis:
actual ingested

No. of animals per sex per dose:

10 animals per sex and dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Doses were selected in accordance with the results of the acute oral toxicity study.

Positive control:

No.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the experiment as part of the termination
- How many animals: all animals
- Parameters checked: red blood cells (RBC), hemoglobin (Hb) content, white blood cells (WBC), platelet (PLT) counts, plasma prothrombin time (PT) and activated partial thromboplatin (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the experiment as part of the termination
- How many animals: all animals
- Parameters checked: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total protein (TP), albumin (ALB), bilirubin (TBIL), blood urea nitrogen (BUN), creatinine (CREA), cholinesterase (CHE), cholesterol total (CHOL), glucose (GLU), Na, K, Cl, Ca, P

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the experiment as part of the termination
- Parameters checked: specific gravity, pH, protein content, blood/blood cells, glucose

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all rats were subjected to a full necropsy and macroscopically observed in detail at the end of the experiment.
HISTOPATHOLOGY: Yes, the following tissues were used for histological examination: brain, heart, lung, liver, thymus, spleen, kidney, adrenal gland, testis or ovaries, nervus ischiadicus, cervical, spinal cord, thyroid, pancreas, stomach, duodenum, jejunum, ileum, colon, rectum, mesenteric lymph node, prostate, epididymides, urinary bladder, uterus

Statistics:

Data was analyzed by ANOVA, and the additional high dosage group and control group were analyzed by a t-test using SPSS 10.0.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

see details below

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

see details below

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

see details below

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

see details below

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see details below

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see details below

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY

No abnormalities or deaths were observed during the whole experimental period.

BODY WEIGHT AND WEIGHT GAIN

A significant decrease in body weights of males of the highest dose group were observed from the first up to the fourth week of exposure in comparance to the control group (P<0.05). A significant decrease in body weights of males of the additional high dose group was observed from the second to the forth week, too, compared to the additional control group (P<0.05).

HAEMATOLOGY

The results of the blood analysis showed a significantly lower value of hemoglobin and significantly higher values of PLT in females of the high dose group. The values of RBC and hemoglobin in males of the high dose group were significantly decreased. In the mid dose group the PLT values of the males were decreased, too. The additional high dose group showed a significant decrease of hemoglobin in both males and females and a decrease of RBC in the male rats. All values had been put into relation with the corresponding control group (P<0.05).

CLINICAL CHEMISTRY

On serum examination , values of ALT, TP, ALB were significantly decreased in females of the high dose group. Significant decreases were also detected for the values of ALT, AST, TP, ALB and Na in male rats of the high dose group. ALP and Na were also decreased in the mid dose group. A significant increase was noted for BUN and CHE in the male rats of the high dose group. A significant decrease of CHE was detected in the females of the additional high dose group. The additional male high dose group showed a decrease of ALT and Na, while the values of BUN, CREA, CHE and Cl were significantly increased. All values and findings had been put into relation with the corresponding control groups (P<0.05).

URINALYSIS

Both males and females of the high dose group showed a lower pH value compared with their respective control groups (P<0.05).

ORGAN WEIGHTS

For females of the high dose group, absolute organ weights of kidney and relative organ weights of heart, liver, spleen, kidney, adrenal gland and lung were significantly increased, while absolute organ weights and relative organ weights of the thymus were significantly decreased. For females of the mid dose group, relative organ weights of the lung were significantly increased. For males of the high dose group , absolute organ weights of spleen and kidneys were significantly increased, while relative organ weights of brain, heart, spleen, kidney and adrenal glands were significantly increased. Significant increases of absolute organ weights of spleen and kidney and significant increases of relative organ weights of liver, spleen, kidneys and adrenal glands in females of the additional high dose group were found. Significant increases of absolute organ weights of kidneys, adrenal glands and relative organ weights of heart, liver, spleen, kidneys, adrenal glands in males of the additional high dose group were found, while absolute organ weights of testis were significantly decreased.
As brain weight is known to be mainly a function of actual age and to alter little with changing body weights, organ/brain weight ratios were calculated based on raw data. Organ/brain weight ratios of kidneys were significantly decreased in females of the high dose group. For the males of this dose group , the organ/brain weight ratios of spleen and kidneys were significantly increased. Organ/brain weight ratios of spleen, kidneys and ovaries were significantly increased in females of the additional high dose group. For the males of this group, the ratios of adrenal glands were significantly increased, while the ratio for the testes was significantly decreased. All values and findings had been put into relation with the corresponding control groups (P<0.05).

GROSS PATHOLOGY

Results from the gross necropsy showed no overt abnormalities in all animals.

HISTOPATHOLOGY: NON-NEOPLASTIC

Control group (10 females): No overt abnormalities.
High dosage group (10 females): Mid-grade degeneration and necrosis of the partial renal tubule, mid-grade renal tubule crystal retention with light-grade infiltration of inflammatory cells of interstitial tissue in 3 cases; mid-grade degeneration and necrosis of the partial renal tubule, mid-grade renal tubule crystal retention in 7 cases.
Control group (10 males): No overt abnormalities.
High dosage group (10 males): Mid-grade degeneration and necrosis of the partial renal tubule with mid-grade renal tubule crystal retention in 6 cases; mid-grade degeneration and necrosis of the partial renal tubule, mid-grade renal tubule crystal retention with light-grade infiltration of inflammatory cells of interstitial tissue in 4 cases
Mid dosage group (10 females): No overt abnormalities in the kidney.
Mid dosage group (10 males): No overt abnormalities in the kidney.
Low dosage group (10 females): No overt abnormalities in the kidney.
Low dosage group (10 males): No overt abnormalities in the kidney.
Additional control group (10 females): No overt abnormalities.
Additional high dosage group (10 females): partial light-grade renal tubule degeneration and necrosis with mid-grade renal tubule crystal retention in 1 case; partial light-grade renal tubule degeneration and necrosis with light-grade renal tubule crystal retention in 6 cases; partial mid-grade renal tubule degeneration and necrosis with light-grade renal tubule crystal retention in 1 case; partial light-grade renal tubule degeneration and necrosis, light-grade renal tubule crystal retention with light-grade infiltration of inflammatory cells of interstitial tissue in 1 case; partial light-grade renal tubule degeneration and necrosis, light-grade renal tubule crystal retention with mid-grade infiltration of inflammatory cells of interstitial tissue in 1 case.
Additional control group (10 males): light-grade infiltration of inflammatory cells of interstitial tissue of prostate in 1 case; mid-grade infiltration of inflammatory cells of interstitial tissue of prostate in 1 case.
Additional high dosage group (10 males): partial light-grade renal tubule degeneration and necrosis with light-grade renal tubule crystal retention in 6 cases; partial light-grade renal tubule degeneration and necrosis with mid-grade renal tubule crystal retention in 1 case; partial light-grade renal tubule degeneration and necrosis, light-grade renal tubule crystal retention with mid-grade infiltration of inflammatory cells of interstitial tissue in 1 case; partial mid-grade renal tubule degeneration and necrosis, light-grade renal tubule crystal retention with light-grade infiltration of inflammatory cells of interstitial tissue in 1 case; partial light-grade renal tubule degeneration and necrosis, light-grade renal tubule crystal retention with light-grade infiltration of inflammatory cells of interstitial tissue in 1 case; light-grade infiltration of inflammatory cells of interstitial tissue of prostate in 2 cases.

Dose descriptor:

NOAEL

Effect level:

ca. 40 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects clinical signs; mortality; body weight; food efficiency; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; organ weights; histopathology;

Critical effects observed:

not specified

Conclusions:

Lithium bis(oxalato)borate (LiBOB) was tested for toxic effects in male and female Sprague-Dawley rats after consecutive 28-day oral administration with a prolonged observation period of 14 days. The derived NOAEL is 40 mg/kg bw/day.

Executive summary:

LiBOB was tested 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)

Data source

Reference

Reference Type:: study report
Title:: Unnamed
Year:: 2021
Report date:: 2021

Materials and methods

Test guidelineopen all close all

Test guideline 1

Qualifier:: according to guideline
Guideline:: OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:: 25 JUN 2018
Deviations:: no

Test guideline 2

Qualifier:: according to guideline
Guideline:: other: OECD Guidance No. 43 on Mammalian Reproductive Toxicity Testing and Assessment
Version / remarks:: 24 JUL 2008
Deviations:: no

Test guideline 3

Qualifier:: according to guideline
Guideline:: EU Method B.31 (Prenatal Developmental Toxicity Study)
Version / remarks:: 30 MAY 2008
Deviations:: no

Test guideline 4

Qualifier:: according to guideline
Guideline:: EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)
Version / remarks:: AUG 1998
Deviations:: no

GLP compliance:: yes (incl. QA statement)
Remarks:: 22 MAY 2019; Ref. no: OGYÉI/8623-5/2019
Limit test:: no

Test material

Test material information

Test material form:: solid: particulate/powder

Test animals

Species:: rat
Strain:: Wistar
Remarks:: Han: WIST
Details on test animals or test system and environmental conditions:: TEST ANIMALS
- Source: TOXI-COOP ZRT. Cserkesz u. 90.1103 Budapest, Hungary
- Age of females at arrival: Females: ca. 8 weeks (55-58 days)
- Age of animals at mating: Females: Young adult and nulliparous females, ca. 10 weeks of age at start of the mating period; males: ca. 57-58 weeks of age at start of the mating period
- Weight of females at mating: The group averages of the body weight of the females were as similar as possible on the first day of gestation
- Housing: before mating: 2 females per cage; 1-2 males per cage; during mating: 1 male and 1-2 females / cage; during gestation: 2 sperm positive females per cage, if not possible 1 sperm positive female per cage; Cage type: Type II polypropylene/polycarbonate; Bedding: Certified laboratory wood bedding.
- Diet: ad libitum (ssniff® SM R/M-Z+H "Autoclavable complete food for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany)
- Water: ad libitum (tap water as for human consumptionfrom municipal supply from 500 ml bottles)
- Acclimation period: yes, 15 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 °C to 23.8 °C
- relative Humidity (%): 40 % to 67 %
- Air changes (per hr): above 10 air exchanges/hour by central air-condition system
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 a..m to 6.00 p.m.

At the end of each working day floors were swept and then mopped with an acceptable disinfectant. Water bottles were cleaned on a rota basis as required during the course of the study.
The temperature and relative humidity were checked and recorded once daily during the study.

IN-LIFE DATES:
Start of experimental phase: November 17, 2020
Date of end of in-life phase: January 06, 2021

Administration / exposure

Route of administration:: oral: gavage
Vehicle:: other: Sunflower oil
Remarks:: dehydrated by adding Na2SO4 (1 g Na2SO4/10 mL sunflower oil); supernatant used
Details on exposure:: PREPARATION OF DOSING SOLUTIONS:
Sunflower oil was dehydrated by adding Na2SO4 (1 g Na2SO4/10 mL sunflower oil). The supernatant was used for formulation after full sedimentation to avoid as far as possible hydrolysis of the substance before administering to the animals.
Frequency of Preparation: Preparation of the test item formulations was 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 therefore dehydrated sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil has been shown to be a suitable vehicle to facilitate formulation analysis for the test item.
- Concentration in vehicle: 5 mg/mL, 15 mg/mL and 25 mg/mL.
- Amount of vehicle (if gavage): 4 mL/kg bw; The actual volume of administration was adjusted for each animal based on the most recent individual body weight measured.
- Lot/batch no. (if required): 8006332001
Analytical verification of doses or concentrations:: yes
Details on analytical verification of doses or concentrations:: Analytical control of dosing solutions (control of test item concentration and homogeneity) was performed in the Analytical Laboratory of the Test Facility two times during the study.
Five aliquots of 10 mL of each formulation to be administered to the animals and five aliquots of 10 mL control item (vehicle) were taken from different places (top, middle and bottom).
The test item content of the formulations was determined based on the lithium content (determined by ICP-OES analysis) and on the boric acid content (analysed by a GC-MS method).
The determined test item concentrations were within 10% of the nominal concentration and, therefore, the dosing formulations were considered acceptable.
A homogenous distribution of the test item in the vehicle was demonstrated for all analysed dosing formulations. The test item was neither soluble nor stable in water therefore dehydrated sunflower oil was used for preparing formulations appropriate for oral administration. Sunflower oil has been shown to be a suitable vehicle of the test item and an analytical method for this vehicle was established and validated.
Details on mating procedure:: - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: one male: one to two females
- Length of cohabitation: the females were paired to males in the mornings for two to four hours until the number of sperm positive females and females with implantations per group achieved at least twenty- two and sixteen respectively
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy. Vaginal smears were prepared from each female, stained with 1 % aqueous methylene blue solution and examined for presence of sperm and for estrus cycle
Sperm positive females were separated and caged maximum in groups of 2 animals. Caging of the females individually was avoided if possible.
Duration of treatment / exposure:: The sperm positive females were treated from gestational day 5 to 19.
Frequency of treatment:: single daily oral gavage (stomach tube), 7 days/week at similar time.
Duration of test:: till gestation day 20

Doses / concentrationsopen all close all

Doses / concentrations 1

Dose / conc.:: 0 mg/kg bw/day
Remarks:: Control

Doses / concentrations 2

Dose / conc.:: 20 mg/kg bw/day
Remarks:: low dose

Doses / concentrations 3

Dose / conc.:: 60 mg/kg bw/day
Remarks:: mid dose

Doses / concentrations 4

Dose / conc.:: 100 mg/kg bw/day
Remarks:: high dose

No. of animals per sex per dose:: control: 32; low: 29; mid: 28; high: 28
Control animals:: yes, concurrent vehicle
Details on study design:: - Dose selection rationale:
The selection of the dose levels was based on the results on the dose range finding study with the test idem (Study No.: 976-410-5645) where body weight effects starting at 125 mg/kg bw/day and clear kidney effects (macroscopic and histopathological) at 75 mg/kg bw/day and above were noted as well as at the dose level of 40 mg/kg bw/day (1 of 7 animals was affected) according to the histopathological evaluation.

The highest dose was chosen accordingly with the aim to induce some maternal and/or developmental toxicity but not death or severe suffering. The low dose level was chosen with the aim not to produce any evidence of either maternal or developmental toxicity. The mid dose was interpolated geometrically.Furthermore, the results of the studies below were also taken into consideration: “Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test with Lithium-bis(oxalato)borate in the Rat “(Study number: 559.439.3243, Year: 2012) where the dose levels of 100, 30 and 10 mg/kg bw/day were investigated and the NO(A)EL (No Observed (Adverse) Effect Level) for male and female rats as well as F1 offspring was 30 mg/kg bw/day, the NO(A)EL for reproductive performance was 100 mg/kg bw/day for male and female rats. In this study also pronounced kidney effects were noted at 100 mg/kg bw/day. Based on the information at the ECHA homepage (https://echa.europa.eu/hu/registrationdossier/-/registered-dossier/3137/7/6/1), according to the oral toxicity study, the LD50 and LD0 were determined as 550 and 175 mg/kg bw/day respectively (LAB 2010). In the Repeated Dose 28-Day Oral Toxicity in Rats, (IOAT, year 2010), where the dose levels of 160, 40 and 10 mg/kg bw/day were investigated, the NOAEL was derived as 40 mg/kg bw/day for male and female animals also based primarily on kidney effects.

- Time of day for (rat) dam blood sampling: Blood samples were collected from all sperm positive females from the sublingual vein in the morning on the day of necropsy within a short timeframe (not exceeding two hours).

Examinations

Maternal examinations:: CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made from gestation day 0, once a day, on treatment days after treatment at approximately the same time, considering the peak period of anticipated effects after dosing. Individual observation included the check of behavior and general condition. Observations for signs of morbidity and for mortality were made twice daily, at the beginning and before the end of the working period.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of the female rats was measured once in the pre-mating period, but was not statistically evaluated. Body weight of sperm positive females was measured on gestation days 0, 3, 5, 8, 11, 14, 17 and 20 (accuracy of 1 g).
The corrected body weight was calculated for the 20th day of pregnancy (body weight on day 20 minus the weight of the gravid uterus)

FOOD CONSUMPTION: Yes
- Food consumption was measured between gestation days 0 to 3, 3 to 5, 5 to 8, 8 to 11, 11 to 14, 14 to 17 and 17 to 20 by re-weighing the non-consumed diet (accuracy: 1 g).

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined:
Weight of thyroid glands and kidneys (mean ± S.D.) of all sperm positive females
Histological examination (sum, %) was performed on the thyroid glands of all females as well as on the kidneys and forestomach of the control and high dose females. In addition, the kidneys were investigated for the mid and low dose too, as the kidney the primary target organ for systemic toxicity of the test item.
- Gross pathology observation (sum, %)

OTHER:
- Examination for Sign of Implantation: On gestation days 13 the sperm positive females were checked for the presence of vaginal bleeding which indicates the implantation of conceptuses. If negative on day 13, the examination was repeated on day 14 of gestation.
Ovaries and uterine content:: The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterine weight: Yes (mean ± S.D.)
- Number of corpora lutea: Yes(mean ± S.D.)
- Number of implantations: Yes (mean ± S.D.)
- Number of early resorptions: Yes (mean ± S.D.)
- Number of late resorptions: Yes (mean ± S.D.)
- Number and percent of live fetuses (mean ± S.D.)
Blood sampling:: Blood samples were collected for TSH and Thyroid Hormones (FT3 and FT4) measurements from all sperm positive females from the sublingual vein in the morning on the day of necropsy within a short timeframe (not exceeding two hours). Blood samples were kept in tubes Vacuette 2.5 mL Z Serum Sep C/A (no anticoagulant; manufactured by Greiner Bio-One International AG, Kremsmünster, Austria; or equivalent). At least 2.5 mL blood was collected into clinical chemistry tubes. Samples were stored in a dark place at room temperature for 30-40 minutes and then centrifuged at 4000 rpm (2860 g) for 15 minutes. The samples were portioned into three aliquots (one for FT3 and FT4 and two for TSH level determination). Serum samples were stored at -15--30°oC until measurement. The quantitative determination of thyroid hormones (FT3 and FT4) was performed from one aliquot per animal by electrochemiluminescence immunoassay with COBAS e411 immunochemistry analyzer.
One other aliquot per animal was transported frosted protected from light to the Test Site where the quantitative determination of TSH hormone levels in serum samples was performed by Berthold Multi-Crystal Gamma Counter LBIS 12 detectors, including LBIS software, 230W. Another aliquot per animal was retained frozen at the Test Facility until the successful measurement of the samples at the Test Site. These samples were destroyed since not needed for a second measurement (shipment).
Fetal examinations:: - External examinations: Yes
- Soft tissue examinations: Yes, half of each litter
- Skeletal examinations: Yes, half of each litter
- Head examinations: Yes, by Wilson's freehand razor blade method
- Anogenital distance of each fetus: yes

Other:
- Sex distribution %, group mean
- Fetal body weight (by sex) litter mean ± S.D and placenta weight
- Fetal body weight (with sexes combined) litter mean ± S.D
Statistics:: The statistical evaluation of data was performed with the program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity is detected a one-way analysis of variance (ANOVA) was carried out. If the obtained result is significant Duncan’s Multiple Range test was used to assess the significance of inter-group differences. If the result of the Bartlett’s test was significant, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test.
Historical control data:: yes

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:: no effects observed
Description (incidence and severity):: None of the animals showed clinical signs during the in-life phase.
Dermal irritation (if dermal study):: not examined
Mortality:: no mortality observed
Description (incidence):: None of the pregnant females died before scheduled necropsy.
Body weight and weight changes:: effects observed, treatment-related
Description (incidence and severity):: Before the treatment period, between G.D. 0 and 3, a statistically significantly (p<0.05) higher body weight gain was seen in the 100 mg/kg bw/day dose group.
Between G.D. 14 to 17 and 5 to 20 a statistically significantly lower body weight gain as well as corrected body weight gain was observed in the 100 mg/kg bw/day group, which was attributed to the test item.
Food consumption and compound intake (if feeding study):: effects observed, treatment-related
Description (incidence and severity):: Reduced, (-10, -9 and -18 % (p<0.01, 0.05, 0.01) between G.D. 8 to 11, 14 to 17 and 17 to 20 respectively) mean food consumption was indicated in the 100 mg/kg bw/day group which was reflected in the body weight parameters and it was judged to be test item related.
Higher mean food consumption (p<0.05, +6%) before the treatment period was judged as not relevant.
Food efficiency:: not examined
Water consumption and compound intake (if drinking water study):: not examined
Ophthalmological findings:: not examined
Haematological findings:: not examined
Clinical biochemistry findings:: not examined
Endocrine findings:: no effects observed
Description (incidence and severity):: There were no significant differences in the FT3, FT4 levels and TSH levels between treated and control animals.
There were no significant or dose related differences revealed at statistical evaluation in the mean values of thyroid weight (absolute and relative).
Urinalysis findings:: not examined
Behaviour (functional findings):: not examined
Immunological findings:: not examined
Organ weight findings including organ / body weight ratios:: effects observed, treatment-related
Description (incidence and severity):: The mean relative kidney weight was slightly and statistically higher in the 60 and 100 mg/kg bw/day group (p<0.05 and p<0.01, respectively). This finding is in line with the appearance of larger of slightly larger kidneys at necropsy in these groups, and was considered test item related.
There were no significant or dose related differences revealed at statistical evaluation in the mean values of thyroid weight (absolute and relative).
Gross pathological findings:: effects observed, treatment-related
Description (incidence and severity):: At 60 and 100 mg/kg bw/day test-item related findings in the kidneys such as increased incidence of dilated renal pelvis, pale, light coloured foci on the surface were observed (accompanied by slightly larger or larger kidneys and histopathological changes) (for details see below).

The incidence of dilated renal pelvis was 3, 1, 4 and 7 in the control, 20, 60 and 100 mg/kg bw/day group.
Observations revealed pyelectasia for the majority of these animals (except No.: 232, No.: 207 and 163 in the control, 60 and 100 mg/kg bw/day group respectively). The pyelectasia – without other lesions, for example degeneration, inflammation, and fibrosis (histopathology) – can be considered in laboratory rats as a slight disorder without toxicological significance according to the (histo)pathologist.
Pale kidneys were found only in the 100 mg/kg bw/day dose group (accompanied by histopathological findings)
Larger or slightly larger kidneys accompanied with light coloured foci were recorded for one pregnant female in the 60 mg/kg bw/day group (accompanied by histopathological findings).
Light coloured foci were observed on the surface of the kidneys in case of two other animals in the 60 as well as in three pregnant femals in the 100 mg/kg bw/day group (accompanied by histopathological findings).
Smaller kidney (one sided) was observed in one female in the 20 mg/kg bw/day (without histopathological changes).
Full blooded thymus was found in one animal in the 60 mg/kg bw/day group. Considering the low incidence and the lack of dose-dependence it was not considered as test item related
Full-blooded stomach mucosa was found in one pregnant female in the 20, two in the 60 and three females in the 100 mg/kg bw/day group. Histopathology revealed congestions in case of two of the three 100 mg/kg bw/day animals, which was not considered as pathological as no histological lesions were noted.
Neuropathological findings:: not examined
Histopathological findings: non-neoplastic:: effects observed, treatment-related
Description (incidence and severity):: At 60 and 100 mg/kg bw/day the following test-item related effects were observed: renal tubular dilatation, accompanied with basophilic tubules and occasionally focal interstitial mononuclear cell infiltration (for details see below).

For animals with pyelectasia (except No.: 189 in the 60 mg/kg bw/day group and No.: 131 and 104 in the 100 mg/kg bw/day dose group) tubular dilatation was recorded in the 60 and 100 mg/kg bw/day group, and, in addition, two of these animals had lymphocytic and histiocytic infiltration in the kidneys.
In the 100 mg/kg bw/day dose group for animals with pale kindeys were found, histopathology revealed tubular dilatation.
In one pregnent female in the 60 mg/kg bw/day group with larger or slightly larger kidneys accompanied with light coloured foci, histopathology revealed tubular dilatation and lymphocytic and histiocytic infiltration. Similar findings were noted in two females in the 100 mg/kg bw/day group (both accompanied with pale kidneys and one with dilated renal pelvis). Histopathology revealed pyelectasia and tubular dilatation in both of these females.
In two other animals with light coloured foci were observed on the surface of the kidneys in the 60 as well as in three pregnant femals in the 100 mg/kg bw/day group, histopathology revealed tubular dilatation for all of these animals and lymphocytic and histiocytic infiltration for one of the females in the 60 mg/kg bw/day group and both in the 100 mg/kg bw/day group.
According to the histopathological examination tubular dilatation and lymphocytic and histiocytic infiltration was found only in the 60 and 100 mg/kg bw/day group. The renal tubular dilatation (focal, grade 1; multifocal, grade 2, grade 3; or diffuse, grade 4) accompanied with flattened tubular epithelial cells, basophilic tubule and occasionally interstitial mononuclear (lymphocytic and histiocytic) cell infiltration are a common type of rat nephropathy as toxic tubule injury in rat studies. These findings could be considered as a nonspecific form of degeneration and are commonly seen in early cases of chronic progressive nephropathy (CPN) of rats as well. These findings supported by the increased kidney weights are likely to be in connection with the effect of higher (60 and 100 mg/kg bw/day) doses of test item as the kidney is the primary and most sensitive target of the test item’s toxicity.

No histological lesions were noted in thyroid glands of treated and control animals.
Histopathological findings: neoplastic:: not examined
Other effects:: no effects observed

Maternal developmental toxicity

Number of abortions:: no effects observed
Description (incidence and severity):: Chi square test revealed statistically significant (p<0.05) increase in the incidence early embryonic death in the 100 mg/kg bw/day dose group compared to the concurrent control, however it was not significant according to the ANOVA test. Moreover, according to the historical control data the numbers obtained (mean percentage 8.4%) even at the clearly systemic toxic dose of 100 mg/kg bw/day were well within the historical control range (mean 6.3%, range: 1.7-11.3%) and the mean number of viable fetuses was similar in all groups.
Pre- and post-implantation loss:: no effects observed
Description (incidence and severity):: There was no statistically significant increase indicated in the pre-, and post-implantation loss.
Total litter losses by resorption:: no effects observed
Early or late resorptions:: no effects observed
Dead fetuses:: no effects observed
Description (incidence and severity):: There was no statistically significant increase indicated in the late embryonic and fetal death.
Changes in pregnancy duration:: not examined
Changes in number of pregnant:: not specified
Other effects:: no effects observed

Effect levels (maternal animals)

Dose descriptor:: NOAEL
Effect level:: 20 mg/kg bw/day
Based on:: test mat.
Basis for effect level:: body weight and weight gain; gross pathology; histopathology: non-neoplastic; organ weights and organ / body weight ratios

Results (fetuses)

Fetal body weight changes:: no effects observed
Description (incidence and severity):: Mean fetal weight was slightly lower in the 100 mg/kg bw/day group, which could be a secondary effect due to maternal toxicity (body weight, food consumption, increased kidney weight and kidney lesions). However, the values were without a statistical significance.
There were no significant differences in the placental weight.
Reduction in number of live offspring:: no effects observed
Description (incidence and severity):: Chi square test revealed statistically significant (p<0.05) increase in the incidence early embryonic death in the 100 mg/kg bw/day dose group compared to the concurrent control, however it was not significant according to the ANOVA test. Moreover, according to the historical control data the numbers obtained (mean percentage 8.4%) even at the clearly systemic toxic dose of 100 mg/kg bw/day were well within the historical control range (mean 6.3%, range: 1.7-11.3% (see Appendix 19.1)) and the mean number of viable fetuses was similar in all groups.
Changes in sex ratio:: no effects observed
Description (incidence and severity):: The sex distribution of fetuses was similar in the groups and not affected by the test item.
Changes in litter size and weights:: no effects observed
Description (incidence and severity):: Mean fetal weight was slightly lower in the 100 mg/kg bw/day group, which could be a secondary effect due to maternal toxicity (body weight, food consumption, increased kidney weight and kidney lesions). However, the values were without a statistical significance. There was no increase indicated in the incidence of smaller fetuses (retarded in body weight).
Anogenital distance of all rodent fetuses:: no effects observed
Description (incidence and severity):: There were no significant differences in the placental weight and ano-genital distance including the absolute and relative/normalized values.
Changes in postnatal survival:: not examined
External malformations:: no effects observed
Description (incidence and severity):: There were no malformations found at external examinations.
The number of evaluated fetuses was 214, 222, 232 and 216 in the control, 20, 60 and 100 mg/kg bw/day groups respectively.
Skeletal malformations:: effects observed, non-treatment-related
Description (incidence and severity):: There was no statistically significant increase of skeletal variations observed.

Malformations were found with low incidences (one in the control and two in the 60 mg/kg bw/day group) and judged as unrelated to the test item. The second rib was not connected to the sternum in the control fetus. Misshapen supraoccipital bone was recorded for one of the 60 mg/kg bw/day fetuses and bent scapula, as well as thicker, slightly shorter and misshapen humerus for the other. According to the background data of the lab, malformations of the pectoral girdle and forelimbs may occur with low incidences in control fetuses.
The number of examined fetuses was 105, 111, 117 and 108 in the control, 20, 60 and 100 mg/kg bw/ day groups respectively.
Visceral malformations:: no effects observed
Description (incidence and severity):: There were no malformations found in the course of visceral examinations.
The number of examined fetuses was 109, 111, 115 and 108 in the control, 20, 60 and 100 mg/kg bw/day groups respectively.
Other effects:: effects observed, treatment-related
Description (incidence and severity):: - Skeletal Examination: Statistically significantly increased incidence of variations such as delayed ossification of the skull bones (i.e. markedly incomplete ossification or absence of ossification of one or more skull bones) (p<0.05 in the 60 and p<0.01 in the 100 mg/kg bw/day group respectively) was indicated according to the Chi square evaluation. Based on this evaluation, the incidence of affected litters increased also in the 100 mg/kg bw/day group (p<0.01) but not in the 60 mg/kg bw/day group. According to the mean litter percentages, only the increase in the 100 mg/kg bw/day was statistically significant (p<0.01) and the value was not statistically significant in the 60 mg/kg bw/day group.

According to the Chi square test the incidence with unossified hyoid bone or less than 3 ossified sternal bodies increased statistically significantly (p<0.05) in the 100 mg/kg bw/day dose group. In case of both variations, the result was not significant if the mean litter percentages were evaluated. Hence, these findings were not considered as adverse. The incidence of incompletely or unossified sacral vertebra from SII increased statistically significantly, either by Chi square test (p<0.01 for fetal incidence and p<0.05 for litter incidence) or if the mean litter percentages were evaluated (p<0.05) in the 100 mg/kg bw/day group. This variation was also significantly higher according to the Chi square test but not by the mean litter incidence evaluation in the 20 mg/kg bw/day group and without a dose response. This finding together with the markedly incomplete ossification or absence of ossification of one or more skull bones was judged as test item related and could be in relationship with the maternal toxicity associated lower fetal weight. All of the dams of the five affected litters had tubular dilatation, two of them with lymphocytic and histiocytic infiltration in addition. One of these five dams (No.: 2085181) had weight loss and reduced food consumption. Twelve of the thirteen affected fetuses were below 3 g, and 6 of 13 was considered as body weight retarded (see table 4). As can be seen in table 4, the effects are mainly seen in 2 litters, representing 75% of the total variations noted.
Haemorrhage in the eye was observed in one fetus in the 20 mg/kg bw/day group. Considering the low incidence and the fact that this was only noted in the low dose group, this was considered as an accidental finding.
One placenta was found as fused of two in the 60 mg/kg bw/day group which was considered as biologically relevant.

Visceral Examination: At Wilson’s sections, slightly dilated brain ventricles (III and/or lateral) were found in 5 fetuses in the control and one in the 60 mg/kg bw/day dose group which was considered as unrelated to the test item based on lack of dose response. Also, occurrence of hydro- or convoluted ureter not attributed to the treatment considering occurrence in the control group and the lack of dose response.

There was no statistical significance indicated in case of other variations.
Details on embryotoxic / teratogenic effects:: There were no test item effects revealed by external and visceral examination of fetuses. A higher incidence of delayed ossification of the skull bones and sacral vertebrae were noted at the 100 mg/kg bw/day group. This increase was noted almost exclusively in fetuses with lower fetal body weight and in litters of dams with clear signs of maternal toxicity (kidney toxicity or/and reduced food consumption and reduced body weight development). Thus the indications for a slightly delayed development noted at this dose is likely to be linked to the systemic toxicity and a malnutrition during gestation.

Effect levels (fetuses)

Dose descriptor:: NOAEL
Effect level:: 60 mg/kg bw/day
Based on:: test mat.
Sex:: male/female
Basis for effect level:: fetal/pup body weight changes; other: delayed ossification of the skull bones and sacral vertebrae in the 100 mg/kg bw/day group

Overall developmental toxicity

Developmental effects observed:: no

Applicant's summary and conclusion

Conclusions:

At dose levels of 60 and 100 mg/kg bw/day maternal toxicity was observed (kidney toxicity or/and reduced food consumption and reduced body weight development). Pre- and post-implantation loss, sex distribution, pacental weight, ano-genital distance were not affected. There were no test item effects revealed by external and visceral examination of fetuses. A higher incidence of delayed ossification of the skull bones and sacral vertebrae were noted at the 100 mg/kg bw/day group. This increase was noted almost exclusively in fetuses with lower fetal body weight and in litters of dams with clear signs of maternal toxicity. Thus the indications for a slightly delayed development noted at this dose is likely to be linked to the systemic toxicity and a malnutrition during gestation.
Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:
NOAEL (maternal toxicity): 20 mg/kg bw/day
NOAEL (developmental toxicity including teratogenicity): 60 mg/kg bw/day

Executive summary:

This Prenatal Developmental Toxicity Study was conduced in accordance with the OECD 414 and in compliance with GLP. In the study groups of 28 and 29 sperm-positive female Han: WIST rats were treated with Lithium-bis(oxalato)borate in sunflower oil by oral gavage administration at dose levels of 0 (vehicle) ,20, 60 and 100 mg/kg bw/day, respectively, from gestation day 5 to 19. The selection of the dose levels was based on the results of preliminary studies.

Females were cohoused with males (1 male :1-2 females) and the day of detection of sperm in the vaginal smear of females was regarded as gestation day 0.

During the study animals were checked for mortality, clinical signs, body weight, food consumption. Blood collection for determination of thyroid hormone FT3 (free T3) and FT4 (free T4), as well as TSH level was performed on gestation day 20. A Caesarean section and gross pathology were performed on gestation day 20. Organs of the dams were examined macroscopically. Thyroid glands and kidneys of all sperm positive females were weighed and histological examination was performed on the thyroid glands and kidneys of all females as well as on the stomach of the control and high dose animals. The number of implantations, early and late resorptions, live and dead fetuses in each uterine horn and the number of corpora lutea were recorded. Each fetus was weighed and examined for sex and external abnormalities. The ano-genital distance was measured. The placentas were weighed and examined externally. The body of about half of each litter was subjected to visceral examination by means of a dissecting microscope after fixation in Sanomiya mixture. The heads were examined by Wilson's free-hand razor blade method. After double staining, the skeletons of the remaining fetuses (not assigned to visceral examination) were examined by means of a dissecting microscope. All abnormalities found during the fetal examinations were recorded.

On gestation day 20 female animals were sacrificed. There were 20, 21, 23 and 20 females/litters with live fetuses in the control, 20, 60 and 100 mg/kg bw/day groups respectively. No death or clinical signs were noted and FT3, FT4 and TSH values were not influenced by the treatment and there were no differences noted for the absolute and relative thyroid weight. Slightly lower food consumption and lower body weight gain (G.D. 14 to 17 and 5 to 20) including corrected body weight gain was considered as treatment related in the 100 mg/kg bw/day dose group. A slight and dose related increase of kidney weight (including statistically significantly higher relative kidney weight) was test evaluated as item related in the 60 and 100 mg/kg bw/day groups as histopathological changes were also noted. The test item at 60 and 100 mg/kg bw/day caused necropsy findings in the kidneys such as increased incidence of dilated renal pelvis, pale, slightly larger or larger kidneys as well as light coloured foci on the surface accompanied with histopathological changes such as increased incidence of pyelectasia, tubular dilatation, lymphotic and histiocytic infiltration (observed also in animals without necropsy findings). The test item caused no lesions in the thyroid glands and stomach of the animals which could have been detected at histopathology.

The test item did not affect the pre- and post-implantation loss or the sex distribution. A slightly and statistically not significantly lower mean fetal weight in the 100 mg/kg bw/day group was noted. Placental weight as well as ano-genital distance were not adversely affected by the test item. There were no test item effects revealed by external and visceral examination of fetuses. A higher incidence of delayed ossification of the skull bones and sacral vertebrae were noted at the 100 mg/kg bw/day group. This increase was noted almost exclusively in fetuses with lower fetal body weight and in litters of dams with clear signs of maternal toxicity (kidney toxicity or/and reduced food consumption and reduced body weight development). Thus the indications for a slightly delayed development noted at this dose is likely to be linked to the systemic toxicity and a malnutrition during gestation. Based on the observations the No Observed Adverse Effect Level (NOAEL) was determined as follows:

NOAEL (maternal toxicity): 20 mg/kg bw/day

NOAEL (developmental toxicity including teratogenicity): 60 mg/kg bw/day

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