Potassium hexahydroxoantimonate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-01-10 to 2013-05-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 1998-09-21)

Deviations:

no

GLP compliance:

yes

Remarks:

signed 2009-11-12

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Sodium hexahydroxoantimonate

EC Number:

251-735-0

EC Name:

Sodium hexahydroxoantimonate

Cas Number:

33908-66-6

Molecular formula:

NaSb(OH)6

IUPAC Name:

sodium hexahydroxoantimonate

Test material form:

not specified

Test animals

Species:

rat

Strain:

other: Crl:CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Research Models and Services Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 60 days; females: 60 days
- Weight at study initiation: males: 190.2 - 214.8 g; females: 150.7 - 174.9 g
- Housing: animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approximately 39 cm x 23 cm and a height of approximately 18 cm. Granulated textured wood (Granulate A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages. The cages were changed and cleaned once a week.
- Diet (ad libitum): Commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany); Food residue was removed and weighed.
- Water (ad libitum): drinking water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Humidity: 55% ± 15% (maximum range)
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 0.8% aqueous hydroxypropylmethylcellulose gel

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to the appropriate concentrations and was administered orally at a constant volume of 2 mL/kg bw. The application formulations were freshly prepared every day. The amount of the test item was adjusted to the animal's current body weight daily up to and including test week 6 and weekly thereafter.

The control animals received the vehicle at the same administration volume daily for 90 days.

VEHICLE - Methocel
- Supplier: FAGRON GmbH & Co., 22885 Barsbüttel, Germany
- Batch no.: 12A30-N02

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Tests by appropriate analytical methods were conducted to determine the concentration and homogeneity of the test item in the formulations.
For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at -20°C or colder:
1) At study initiation:
- Analysis of concentration: immediately after preparation of the suspension as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose level group; 1 sample of the control group).
- Homogeneity: at the start of administration, during (middle) administration and before administration to the last animal of the dose level group (3 samples/dose level group).
2) At study termination:
- Analysis of concentration: during treatment with the test item always before administration to the last animal of the dose level group (1 sample/group).

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Main study groups: 10 males / 10 females
Recovery group (only vehicle control group and 1000 mg/kg bw group): 5 males / 5 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on the results of a 28-day dose-range-finding study in rats (please refer to the endpoint study record k_Hansen_2012_28 days (Dose range finder)). In this dose-range-finding study male and female CD® rats treated once orally (by gavage) with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day for 28 days did not reveal any test item-related influences on systemic tolerance, body weight, food and drinking water consumption. At necropsy, no test item-related changes were noted during macroscopic examination.

- Post-exposure recovery period in satellite groups: 28 recovery days for the scheduled animals

Positive control:

not applicable

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations checked: cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.
The animals were checked early in the morning and again in the afternoon of each working day for premortal symptoms or exitus.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once a week thereafter
Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking back-wards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter always on the same day of the week

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean weekly diet consumption calculated as g food/kg body weight/week: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. The relative food consumption (in g/kg b.w.) was determined using the following formula:
Relative food consumption (g/kg bw): (Total food given [g] - Total food left [g])/ (Number of animals days# x Body weight [kg])
#: The term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

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: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual appraisal

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once predose and at study termination (main study and recovery period)
- Dose groups that were examined: all animals
The eyes were examined with a HEINE ophthalmoscope. Prior to examination, mydriasis was produced after instillation of MYDRUM® eye drops into the conjunctival sacs. The following ocular structures were examined: Adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples were taken from the retrobulbar venous plexus at the end of the dosing period (main study animals) or at the end of the recovery period (all recovery animals)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute; neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes, large unstained cells were simultaneously quantified during measurement of the differential blood count), reticulocytes, platelets, haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular hemoglobin and mean corpuscular haemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood samples were taken from the retrobulbar venous plexus at the end of the dosing period (main study animals) or at the end of the recovery period (all recovery animals)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase

URINALYSIS: Yes
- Time schedule for collection of urine: urine samples were collected from animals at the end of the dosing period (all main study animals) or at the end of the recovery period (all recovery animals). The urine was collected for 24 hours in an URIMAX funnel cage and the total volume/animal was determined. The collection of urine was terminated immediately prior starting the blood withdrawals for the haematological and clinical biochemical examinations.
- Parameters examined: total antimony (Sb), creatinine

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: main study animals and recovery animals were screened in test week 13 and test week 17, respectively. The screening was performed approximately 1 to 2 hours after dosing.
- Dose groups that were examined: all animals
- Battery of functions tested:
1) Observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions,pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation and auditory function
2) Functional tests: grip strength and locomotor activity

Sacrifice and pathology:

Starting on test day 91 (one day after the last administration) the main study animals were dissected. Main study animals not dissected on test day 91 were dosed again until one day before sacrifice.
Necropsy of all animals allocated to the recovery period was performed on test day 119.

GROSS PATHOLOGY: Yes
The animals were sacrificed under ether anaesthesia by cutting the aorta abdominalis, exsanguinated, weighed, dissected and inspected macroscopically.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

The following organs or parts of organs (with the exception of the eyes, epididymides and testicles) of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson's solution for optimum fixation. One epididymis and one testicle were preserved in Bouin's fixative. The second epididymis and testicle were not preserved but used for the spermiogram.
Organs: adrenal gland (2), aorta abdominalis, bone marrow (os femoris), brain (cerebrum, cerebellum, medulla/pons), coagulating gland with seminal vesicle, epididymis (2), eye with optic nerve (2), gross lesions observed, heart (left and right ventricle, septum), large intestine (colon, rectum), small intestine (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles; preserved by inflation with fixative and then immersion), lymph node (cervical) (1), lymph node (mesenteric) (1), mammary gland, nerve (sciatic), oesophagus, ovary (2), pancreas, pituitary, prostate, salivary glands (mandibular, parotid, sublingual), skin (left flank), spinal cord (cervical, mid-thoracic, lumbar), spleen, stomach, testicle (1), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (incl. regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts) and vagina.

HISTOPATHOLOGY: Yes
The organs stated above of all main study and recovery animals of groups 1 to 4 were examined histologically after preparation of Haematoxylin-eosin stained paraffin sections. Parathyroids were examined microscopically if in the plane of section and in all cases where they are noted as grossly enlarged.
In addition, frozen sections of the liver and one kidney were prepared, stained with Oil Red O, and examined microscopically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology of interstitial testicular structure) of all males of groups 1 and 4 following H-E and PAS staining.

Other examinations:

ORGAN WEIGHTS:
The weights of the following organs of all animals were recorded before fixation: adrenal gland (2), brain, epididymis, heart, kidney (2), liver, ovary (2), spleen, testicle (2), thymus and uterus.
Paired organs were weighed individually and identified as left or right.

SPERM VIABILITY AND MORPHOLOGY
One epididymis and one testicle were used for the sperm count. The sperm viability was de-termined and the sperm morphology was examined for all male animals (n=50) according to the method described by I. Chahoud and R. Franz (1993)* as well as by S. Plassmann and H. Urwyler (2001)*.

Statistics:

The test item groups were compared to the control group.
The following statistical methods are used:
1) STUDENT's t-test (all numerical functional tests / sperm count (p ≤ 0.05 and p ≤ 0.01))
2) Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control, Biometrics, 482-491 (Sept 1964): body weight / food consumption / haematology / clinical chemistry / urinalysis / relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01))
3) chi2-test: sperm motility and morphology

For all numerical values (e.g. body weight, food consumption and organ weight data) homogeneity of variances was tested by using the BARTLETT chi-square test. If the variances were homogeneous, the DUNNETT test (p ≤ 0.01) was used to compare the experimental groups with the control group. In case of heterogeneity of variances, the STUDENT's t-test was carried out; limit of significance is p ≤ 0.01.

Exact test of R. A. FISHER: histology (p ≤ 0.01)

This statistical procedure was used for all data.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY (treatment period / recovery period)
The behaviour or external appearance of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day did not reveal any changes during the 13-week treatment period or during the 4-week recovery period.

No premature deaths occurred during the treatment or recovery period. The faeces of all animals were of normal consistency during the experimental period.

The left eye of one female rat treated with a dose of 300 mg/kg bw/day was haemorrhagic in the last treatment week (test days 89 to 91) which is considered to be an incidental and thus not test item-related finding.

BODY WEIGHT AND WEIGHT GAIN (treatment period / recovery period)
The body weight, body weight gain and body weight at autopsy of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day were within the normal range of variation during the 13-week treatment period or during the 4-week recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE (treatment period / recovery period)
The relative food consumption of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day were within the normal range of variation during the 13-week treatment period or during the 4-week recovery period.

WATER CONSUMPTION AND COMPOUND INTAKE (treatment period / recovery period)
The visual appraisal of the drinking water consumption did not reveal any test item-related influence in any of the dose groups.

OPHTHALMOSCOPIC EXAMINATION (treatment period / recovery period)
No changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus were noted in the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day during the 13-week treatment period or during the 4-week recovery period.

HAEMATOLOGY (treatment period / recovery period)
No test item-related influence was noted on the haematological parameters of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day compared to the control group during the 13-week treatment period or during the 4-week recovery period.

CLINICAL CHEMISTRY (treatment period / recovery period)
No test item-related influence was noted on the biochemical parameters of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day compared to the control group during the 13-week treatment period or during the 4-week recovery period.

URINALYSIS (treatment period / recovery period)
No test item-related influence was noted on the urinary parameters of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day compared to the control group during the 13-week treatment period or during the 4-week recovery period.

The antimony content in the 24-h urine was measured in all animals following the last gavage on test day 90. The total urinary animony excretion compared with the administered total antimony is 0.52% (males 0.51%, females 0.53%) in the 100mg/kg bw/day dose group, 0.35% (males 0.31%, females 0.39%) and 0.17% (males 0.14%, females 0.20%).

NEUROBEHAVIOUR (treatment period / recovery period)
The neurological screening performed at the end of the treatment period in test week 13 did not reveal any test item-related influence in the male and female rats treated with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility.

ORGAN WEIGHTS (treatment period / recovery period)
No test item-related influence was noted on the relative and absolute organ weights of the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day at the end of the 13-week treatment period or at the end of the 4-week recovery period.

GROSS PATHOLOGY (treatment period / recovery period)
At necropsy, no test item-related influence was observed for the male and female rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day at the end of the 13-week treatment period or at the end of the 4-week recovery period.

HISTOPATHOLOGY: NON-NEOPLASTIC (treatment period / recovery period)
The histopathological examination of rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day did not reveal any morphological microscopic lesions which are considered to be related to the administration of the test item.

SPREM VIABILITY AND MORPHOLOGY (treatment period / recovery period)
No treatment-related influence was noted on the number of ultrasound-resistant spermatids, the number of motile spermatozoa and the sperm morphology of the male rats treated orally with 100, 300 or 1000 mg sodium hexahydroxoantimonate/kg bw/day or previously treated with 1000 mg sodium hexahydroxoantimonate/kg bw/day.

Effect levels

Dose descriptor:

NOEL

Effect level:

> 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

dermal irritation

food consumption and compound intake

gross pathology

histopathology: neoplastic

mortality

ophthalmological examination

organ weights and organ / body weight ratios

water consumption and compound intake

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

NOEL (male and female rats) > 1000 mg sodium hexahydroxoantimonate/kg bw/day
None of the rats died prematurely and no test item-related influence was noted on the behaviour or external appearance of the male and female rats. The observational screening, the examination of the fore- and hindlimb grip strength as well as the spontaneous motility did not reveal any test item-related changes. The body weight, body weight gain, food and drinking water consumption, the haematological, biochemical and urinary parameters examined were in the normal range of variation. No test item-related changes were observed at ophthalmological examination. No treatment-related influence was noted on the number of ultrasound resistant spermatids, the number of motile spermatozoa and the sperm morphology of the male rats.
The necropsy as well as the histopathological examination of the organs examined did not reveal any macroscopic or microscopic lesions. The relative and absolute organ weights were in the normal range of variation.
The antimony content in the 24-h urine was measured in all animals following the last gavage on test day 90. The total urinary antimony excretion compared with the administered total antimony is 0.52% (males 0.51%, females 0.53%) in the 100mg/kg bw/day dose group, 0.35% (males 0.31%, females 0.39%) and 0.17% (males 0.14%, females 0.20%), indicating minimal systemic bioavailability.