Lithium tetrafluoroborate, anhydrous

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Lithium tetrafluoroborate, anhydrous

EC Number:

238-178-9

EC Name:

Lithium tetrafluoroborate, anhydrous

Cas Number:

14283-07-9

Molecular formula:

BF4.Li

IUPAC Name:

lithium tetrafluoroborate, anhydrous

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The study was designed to evaluate the potential toxic effect of the test itemLithium Tetrafluoroborate when administered to rats for a minimum of 28 days and to evaluate the potential of the test item to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition, and early postnatal development.

Sex:

male/female

Details on test animals or test system and environmental conditions:

All animals were observed twice daily for mortality and morbidity. Detailed clinical observations, body weights, and food consumption were recorded weekly. Functional Observational Battery (Open field test, Tail flick test and Grip-strength test) were recorded for 5males at the end of dosing, for 5females during the last week of lactation shortly before scheduled kill and in satellite animals at the end of in-life phase.
Females were allowed to deliver and rear their pups until lactation Day 13. Live pups were counted and sexed and litters weighed. The anogenital distance (AGD) of each pup was measured on Day 4 post-partum. Any abnormal behavior of the offspring was recorded. The number of nipples/areolae in male and female pups were counted on post-natal Day 13 (PND 13).
The urinalysis was performed in five randomly selected males of each group during the last week of the treatment and in satellite animals using urine volume collected from animals in metabolic cages during 6 hours.
Clinical pathology evaluations (haematology, coagulation, and clinical chemistry) were performed in half males and all females and in satellite animals at the end of in-life phase. Thyroxine level was assayed in parent rats and pups PND 13 and PND 4.
The pups were euthanized on PND 13 and examinated for gross abnormalities with particular attention to the the reproduction organs and the thyroid glands were preserved.From all adult males and females and one male and female pup Day 13 from each litter thyroid glands were preserved.The weight of thyroid glands in adults was determined.
Animals were subjected to gross necropsy. Complete necropsies were conducted on all animals and satellite animals, and selected organs were weighed.Selected tissues were examined microscopically from 5 males and 5 females in the vehicle control and high-dose groups from main subgroup; organs of the reproductive system in all males and females in the vehicle control and high-dose groups.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test item, Lithium Tetrafluoroboratein the vehicle aqua pro injection, was administered by gavage once daily to three groups ofWistar rats at the doses of 50, 150 and 250 mg/kg. A concurrent vehicle control group received the vehicle (aqua pro injection) on a comparable regimen and in the same volume of 2mL/kg. The females (13per group) were screened for normal oestrous cycles in a 2-week pre-treatment period.
Each group consisted of 10 males; all females were used for dosing and mating. After mating, 10 females, showing evidence copulation were used in each group.
Males were administrated 14 daily doses prior to mating and continuing throughout the mating period, for a total of 28 doses. Females received 14 daily doses prior to mating and were dosed through mating, gestation and lactation (13 days).
Satellite animals (five males and five females from control group and high dose group) were not mated, administrated for a total 28 doses, males and 54 doses, females, and euthanized after two-week recovery period.

Vehicle:

other: aqua pro injection

Details on oral exposure:

The test item, Lithium Tetrafluoroboratein the vehicle aqua pro injection, was administered by gavage once daily to three groups ofWistar rats at the doses of 50, 150 and 250 mg/kg. A concurrent vehicle control group received the vehicle (aqua pro injection) on a comparable regimen and in the same volume of 2mL/kg. The females (13per group) were screened for normal oestrous cycles in a 2-week pre-treatment period.
Each group consisted of 10 males; all females were used for dosing and mating. After mating, 10 females, showing evidence copulation were used in each group.
Males were administrated 14 daily doses prior to mating and continuing throughout the mating period, for a total of 28 doses. Females received 14 daily doses prior to mating and were dosed through mating, gestation and lactation (13 days).
Satellite animals (five males and five females from control group and high dose group) were not mated, administrated for a total 28 doses, males and 54 doses, females, and euthanized after two-week recovery period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each animal was marked with an ID number. Each cage was affixed with a cage card containing pertinent animal and study information. The animals in cages were marked by a line on the tail with an ink marker.
Number of animals in cage was according period of study:
• Pre-treatment period (14 days) – 5 animals per cage
• Pre-mating (14 days) –5 animals per cage
• Mating (maximum 14 days)- 1 male/1 female per cage
• Gestation (approximately 22 days) - 1 female per cage
• Post-partum (13 days) - 1 female with offspring per cage
Satellite animals – 5 animals per cage during all the study
The test item was administered in a single dose by gavage using a metal stomach tube.Administration volume was 2 mL/kg. For each animal the individual dosing volume was calculated on the basis of the actual body weight.

Duration of treatment / exposure:

The animals were dosed daily for 7 days per week until the end of treatment

Frequency of treatment:

daily

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

One day before the treatment, all animals were randomized. Within the frame of treatment groups, each rat was marked by code on the tail root to identify animal individually. Each cage was provided with a cage card.
Rats were inspected twice daily for evidence of reaction to treatment or ill-health. The observation focused on changes in skin, fur, eyes and mucous membranes, occurrence of secretions and excretions and autonomic activity as lacrimation, piloerection, pupil size, and unusual respiratory pattern. Moreover, possible changes in gait, posture and response to handling as well as the presence of clonic or tonic movements or bizarre behaviour (self-mutilation, walking backwards) were observed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

During the study mortality of animals was recorded. Two males of High dose, male ID43 died on Day 14 and male ID 39 on Day 15 of the treatment. Males of High dose satellite (ID 46, 47, 48 and 50) died between Day 16 and 23 of the treatment. Lethargy, piloerection and nose-bleed in males was registered before death.All other males at lower dosage levels survived without significant visible clinical signs.

Mortality:

no mortality observed

Description (incidence):

No mortality in females was registered. All females at all dosage levels survived to the scheduled necropsy without significant visible clinical signs.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

At the start of the study average body weights were 284.9 g males and 217.15 g females. The animals were divided into the groups according to the body weight. The variation of animal body weights no exceeded ± 5% of the mean weight for each sex.
No differences between body weight of control and dose groups were registered in males.
At the commencement of the study, the weight variation of animals in the dose groups was about 5%; after statistical evaluation significant differences were observed.Statistically significant differences between Control and Low andMid dose on Day 1 of treatment in females were observed.
Decrease of body weight of all dose group against Control was registered from Day 7 of the treatment. Decrease of the body weight of all dose groups continued up to the end of treatment (lactation Day 13).The body weight of High dose-satellite females was similar in comparison with recovery Control females during the whole study.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of males and females of all dose groups was similar to thecontrol males during the whole study.Food consumption of satellite females was similar in comparison with Control.

Haematological findings:

no effects observed

Description (incidence and severity):

Only decreased value of prothrombin time (PT) of males of High dose against Control was recorded.
Statistically significant differences between Control and Mid dose of erythrocytes (RBC), haematocrit (HCT) and monocytes (Mon) in females of Low dose; in case of Mon also in females of High dose were registered. The changes were of small magnitude and was not considered as a toxicologically important.
No differences in satellite females were noticed.
During the study, haematology parameters in both sexes were within or close to the historical control data for this species. No test item related effects on the haematology parameters were observed in this study.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Statistically significant higher concentration of total cholesterol (CHOL) and triacylglycerol (TAG) and lower concentration of urea, calcium (Cal) and inorganic phosphorus (PHOS) were observed in males of High dose when compared to the Control group. Males of Mid dose had significant increase of concentration of urea and decrease concentration of Cal and PHOS against Control. Statistically significant decrease of CHOL and PHOS and increase of urea in males of Low dose against Control were registered.
No significant differences were observed in monitored parameters of female rats.
These sporadic changes had no dose relationship; they were considered to be a result of intra-individual and inter-individual variability for this species or they have only statistical character.
The average values of clinical chemistry parameters in all animals were within or slightly outside the serum historical control data.There were no findings in clinical chemistry parameters which could be definitively attributed to the treatment.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

In the urine of some animals, small amounts of protein, ketones and presence of leukocytes were observed. There are no differences between Control and the dose groups and these findings can be consideredclose to normal (7).In males of Mid and High dose groups the increased diuresis was observed. The volume of urine, collected during 6 hours, was about 3 times higher than in males of Control group.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Relative weights of the liver and prostate in the Low dose males were statistically significant increased compared to Control. Statistically significant increase of relative weight adrenal, prostate and thyroid in males of Mid dose, and significant increase of relative weight of kidney, adrenal, prostate and thyroid in males of High dose were registered.In case of prostate and thyroid with a dose-related response. Reversibility was not possible to compare, due to the death of four from 5 males of the High satellite group.
Increase relative weight of adrenal right in females of Low dose and decrease of adrenal left of females of High dose and significant increase of relative weight of thyroid and brain in females of High dose were registered.
No changes between Control and High dose were seen in satellite females.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

During the study, two males of High dose (ID 43, 39) and four males of High dose-satellite (ID 46, 47, 48 and 50) died. Male ID 39 and males of satellite group was not necropsied (autolytic changes); male ID 43 was dehydrated, cachectic, with hyperaemic intestinal tract, coagulated blood in thoracal and abdominal cavity.
In two males of Low dose, multifocal red coloration of thymus (ID 20, 22), enormously increased content of stomach (ID 20, 23) and adrenomegaly in male of High dose-satellite (ID 49) were observed.
In females only brown-red content of the stomach and duodenum (ID 93-Mid dose) and pale liver (ID 102-High dose) were observed.Other animals were without visible pathological findings.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Incidental finding of lymphoepithelioid granuloma (37 M/HD) in CE (caecum) is supposed to be induced by stress factors. Psychological stress may increase gastrointestinal permeability allowing luminal constituents access to the mucosal immune system (9). We have found lymphoepitheliod granulomas also in earlier examined toxicological studies in control rats (non-published). Lesions are not related with the test item administration.

Effect levels

Dose descriptor:

NOAEL

Effect level:

ca. 50 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

body weight and weight gain

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

After consideration of the study results following conclusions were maderegarding the test itemLithium Tetrafluoroborate.

The test item administration

-      had no visible toxic effect on females and male of Low and Mid dose

-      had no impact on body weight and food intake in males and females

-      had no influence on monitored haematological and clinical chemistry parameters males and females

-      had no influence on the reaction time males and females and grip strength in males

-      did not cause histopathological changes on the examined organs

The test item administration

-      caused mortalityand had toxic effecton males of High dose (250 mg/kg/bw)

-      caused increase of diuresis in males of Mid (150 mg/kg/bw) and High dose (250 mg/kg/bw)

-      caused decreaseof muscle strength infemales of Mid (150 mg/kg/bw) and High dose (250 mg/kg/bw)

-      caused depress oflocomotor activity males of High dose (250 mg/kg/bw), females of Mid (150 mg/kg/bw) and High dose (250 mg/kg/bw)

-      caused increase relative weight adrenal, prostate and thyroid in males of Mid dose (150mg/kg/bw) and increase relative weight of kidney, adrenal, prostate and thyroid in males of  High dose (250 mg/kg/bw)

-      caused increase relative weight of thyroid in females of High dose (250 mg/kg/bw)

The test item administration

-      caused decrease number of implants in High dose (250 mg/kg/bw)

-      caused decrease oflive pups at Day 1 and Day 4 post-partum in Mid (150 mg/kg/bw) and High  dose (250 mg/kg/bw)

-      caused decrease oflitter weight at Day 1 post- and at Day 4 PP in High dose (250 mg/kg/bw)

-      impacted the length of pregnancy in all doses

-      caused decrease bodyweight of mothers at birth in all doses

-      caused increase post-implantation losses in High dose (250 mg/kg/bw)

-      caused increase concentration T4 in F1 offspring at Day 13 post-partum; male pups of Mid (150mg/kg/bw) and High dose (250 mg/kg/bw) and female pups of all doses

Applicant's summary and conclusion

Conclusions:

After consideration of the study results following conclusions were made regarding the test item.
• The High dose of Ethyl lactate 800 mg/kg b.w. induced systemic parental toxicity which
overreached 10% mortality, therefore test doses were reduced since 10th day of application.
• Test item did not induce significant clinical findings or behavioural deviations in the
experimental animals exposed to doses of Low dose - 75 mg/kg b.w., Mid dose -300 mg/kg
b.w. and High dose - 600 mg/kg b.w. No test item-related mortality in significant extent
was recorded during the study. No moribund animals were isolated due to exposure to three
abovementioned doses of test item.
• No statistically significant differences in body weight amongst the treated groups of adult
male and female rats were revealed. The animals grew over time in all experimental groups,
no significant reduction or stagnation of body weight was recorded.
• The test item caused statistically significant increase of body weight in offspring males at
the high dose (600 mg/kg) at Day 13 post-partum.
• Test item did not affect the food consumption. Food consumption was not significantly
different amongst the experimental groups.
• The clinical chemistry parameters measured in serum of male and female rats showed some
statistically significant differences. These differences were minimal in nature and had no
biological or toxicological significance.
• No significant differences in haematological parameters among groups exposed to the test
item and controls were found in all evaluated parameters with exception of PT and APTT
values. The differences were minor, had no biological or toxicological significance.
• In the urine no significant changes against normal physiological conditions were detected.
• The observed significant reduction in T4 levels in the male and female offspring not
accompanied with significant alterations in the mean relative weight of thyroids (after
fixation) as well as with no serious morphological alterations were considered to be findings
without biological/toxicological significance.
• Functional battery observation tests (tail flick test and grip strength test) conducted at the
end of the dosing period and at the end of recovery period revealed no abnormalities.
• Macroscopic findings described during the gross necropsy, were observed in the all treated
groups but incidence was sporadic without clear dose dependency. From microscopic point
of view no significant item related alterations in the sense of the prevalence, severity and
character were described.
• Test item did not cause histopathological changes on examined reproduction organs
Significant changes of mean relative organ weights in male rats, being sensitive indicator of
the adverse effect of the test substance, were observed in Cowper`s glands, musculus
bulbocavernosus, kidneys and spleen. The histological examinations revealed no serious
morphological alterations and statistically significant differences (when compared to the
control animals) were considered to be findings without biological significance.
• Dose-dependent statistically significant reduction of AGI with adverse potential in male
pups (feminization in Low and Mid dose group male pups) and probably realized via non-
traditional mechanisms resulting in inverted-U-shape dose-response curve (because of
mechanism deletion in High dose group) might be regarded as specific developmental effect
of prenatal exposure to test item and should be considered for setting of NOAEL.
• Records of pre-implantation and early post-implantation loss might suggest potential effect
of Low- and Medium-dose of the test item during a very short critical developmental
window.
• Changes of number live pups per dam on postnatal day 0 and 4 and changes in litter weight
at birth described in Mid dose offspring and analysed as statistically nonsignificant should
not be omitted due to high differences in litter characteristics
• Nonsignificant post-natal loss (higher incidence of records of clinical observations, cases of
morbidity and mortality) described in the Mid dose group could point out on potential to
induce non-monotonous dose response effects by the action of the test item.
Based on these results,
- the no-observed-adverse-effect-level (NOAEL) was 600 mg/kg/day for parental systemic
toxicity.
- the NOAEL for reproductive toxicity was considered to be less than 75 mg/kg/day.