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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-10-08 to 2012-11-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008-10-03
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-11-12
Limit test:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
Boron
EC Number:
231-151-2
EC Name:
Boron
Cas Number:
7440-42-8
Molecular formula:
B
IUPAC Name:
Boron
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
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: 32 days; females: 34 days
- Weight at study initiation: males: 126.8 - 159.8 g; females: 120.3 - 143.9 g
- Housing: the animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages.
- Diet (ad libitum): commercial ssniff®-R/M-H V1530 (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 12 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.8% aqueous hydroxyl propyl methylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was dispersed in the vehicle to the appropriate concentration.
The test item-vehicle mixture was freshly prepared every day.
The amount of the test item was adjusted to the animal's current body weight daily.
The test item was used as supplied, i.e. no correction factor was used.

ADMINISTRATION VOLUME: 10 mL/kg b.w./day

VEHICLE
- Batch no.: 11A27-N27 (FAGRON GmbH & Co. KG, 22885 Barsbüttel, Germany)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that was mixed with the vehicle, tests by appropriate analytical methods were conducted to determine the concentration, stability and homogeneity of the test item in the solutions.
For the analysis of the test-vehicle mixtures, samples of approx. 10 mL were taken at the following times and stored at -20°C or colder until analysis.
1) At study initiation (Test day 1):
- Analysis of stability and concentration
Immediately after preparation of the solution as well as after 8 and 24 hours storage of the test item preparations at room temperature (3 samples/dose level group). Total number of samples: 3
- 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).
Total number of samples: 3
2) At study termination (Test day 28):
- Analysis of concentration
During treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group).
Total number of samples: 1
Sum of all samples: 7

Results:
The results of the analysis showed that the test item-vehicle mixtures were correctly prepared and stable for at least 24 hours after storage at room temperature and the concentrations found were in good agreement to those expected. The measured concentrations ranged from 89% to 96% of the theoretical value and were well within the admissible limits.
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males / 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected by the Sponsor based on available toxicological data.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were observed individually at least once daily for any signs of behavioural changes, reaction to treatment or illness. Any signs of illness or reaction to treatment were recorded for each individual animal. Cageside observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns.
In addition, animals were checked regularly throughout the working day from 7.30 a.m. to 4.30 p.m. On Saturdays and Sundays animals were checked regularly from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
Further checks were made early in the morning and again in the afternoon of each working day to look for dead or moribund animals. On Saturdays
and Sundays a similar procedure was followed except that the final check was carried out at approximately 4:00 p.m.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (1, 2, 4, 8 and 24 hours after administration), detailed clinical observations were made in all animals; in test week 4 these observations were performed prior to any laboratory investigations. These observations were made outside the home cage in a standard arena and at the same time, each time. Signs noted included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. selfmutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: the weight of each rat was recorded 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 throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: 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 upon completion of a treatment week. From these data the food consumption (in g/kg b.w./day) was determined using the following formula:
Relative food consumption (g/kg b.w./day) = (Total food given (g) - Total food left (g))/ (Number of animal 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 (visual appraisal throughout the study)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of administration and at the end of test week 4.
Prior to examination, mydriasis was produced after instillation of MYDRUM® (ankerpharm GmbH, 07407 Rudolstadt, Germany) eye drops into the conjunctival sacs.
- Dose groups that were examined: all animals
- Parameter examined: adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body, fundus

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection (test day 29)); blood samples were taken from the retrobulbar venous plexus
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, fasted overnight
- How many animals: all animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, differential blood count (relative), differential blood count (absolute), reticulocytes, platelets, haematocrit value, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration,
Neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes. large unstained cells were simultaneously quantified during measurement of the differential blood count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection (test day 29)); blood samples were taken from the retrobulbar venous plexus
- Animals fasted: Yes, fasted overnight
- How many animals: all animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acid, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (in blood), calcium, chloride, potassium, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase and lactate dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Dose groups that were examined: all animals
- Time schedule for examinations: in test week 4 (TD 25) approx. 1 - 2 hours after dosing and before any blood sampling for laboratory examinations, screening of sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli) (based on GAD, S.C., A Neuromuscular Screen for Use in Industrial Toxicology. Journal of Toxicology and Environmental Health, 9, 691-704 (1982)), as well as the assessment of grip strength (MEYER, O. A., H. A. TILSON, W. C. BYRD AND M. T. RILEY. A method for the routine assessment of fore- and hind limb grip strength of rats and mice. Neurobehavioral Toxicology, Vol. 1, pp. 233-236 (1979)) and motor activity assessment were conducted.
- Parameters examined:
1) Observational screening: rightning reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire maneuver, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, and auditory function
2) Grip strength
3) Locomotor activity
- high sensitivity (slight movements): stereotype, static movement (e.g. grooming, a stationary movement of the animal without leaving its own position)
- low sensitivity (active moving): active locomotion

DETERMINATION OF BORON LEVELS (URINE AND BLOOD):
reported under IUCLID section 7.1: s_Leuschner_2013
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
On test day 29 (one day after the last administration) the animals were dissected following a randomisation scheme.
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. 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.

ORGAN WEIGHTS:
The weights of the following organs of all animals were determined before fixation: adrenal gland (2), brain, epididymis (2), heart, kidney (2), liver, ovary (2), spleen, testicle (2), thymus and as a whole prostate, seminal vesicles with coagulating glands
Paired organs were weighed individually and identified as left or right.

HISTOPATHOLOGY: Yes
The following organs or parts of organs of all animals were fixed in 7% buffered formalin. The eyes were preserved in Davidson’s solution and the testes in Bouin’s solution for optimum fixation.
- adrenal gland (2), bone (os femoris with joint), bone marrow (os femoris), brain (3 levels: cerebrum, cerebellum, medulla / pons), epididymis (2), eye with optic nerve (2), gross lesions observed, heart (3 levels: left and right ventricle, septum), intestine, large (colon, rectum), intestine, small (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 (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal, leg), nerve (sciatic), ovary (2), pituitary, prostate and seminal vesicles with coagulating glands, spinal cord (3 levels: cervical, midthoracic, lumbar), spleen, stomach, testicle (2), thymus, thyroid (2) (incl. parathyroids), tissue masses or tumours (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts) and vagina
The afore-listed organs of all animals were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made and stained with Oil Red O.
Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
Statistics:
The test item group was compared with the control group.
The following statistical methods were used:
- STUDENT's t-test: all numerical functional tests / body weight / food consumption / haematology / clinical biochemistry / relative and absolute organ weights. (p ≤ 0.05 and p ≤ 0.01). The following limits were used: p = 0.05 / 0.01 ^ t = 2.3060 / 3.3554 for 8 degrees of freedom
- Exact test of R. A. FISHER: histopathology (p ≤ 0.05)
These statistical procedures were 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:
not examined
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:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
All male and female animals treated with 1000 mg boron, amorphous/kg bw/day revealed pilo-erection from test days 15 (females) or 16 (males) onwards. The
symptom started 1 hour p.a. lasting for 24 hours.
No premature deaths occurred during the course of the study.
The faeces of all animals were of normal consistency throughout the experimental period.

BODY WEIGHT AND WEIGHT GAIN
Body weight, body weight gain and body weight at autopsy were not influenced in the male and female animals treated with 1000 mg boron, amorphous/kg bw/day compared to the control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No test item-related influence was noted on the food consumption of the male and female animals treated with 1000 mg boron, amorphous/kg bw/day compared to the control group.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
The visual appraisal of the drinking water consumption did not reveal any test item-related changes.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmological examination revealed no changes of the eyes and the optic region in the male and female rats treated with 1000 mg boron, amorphous/kg bw/day. No changes were noted for the control animals, either.

HAEMATOLOGY
No test item-related influence on the haematological parameters was observed for the animals treated with 1000 mg boron, amorphous/kg bw/day compared to the control group.

CLINICAL CHEMISTRY
No test item-related influence was noted for the biochemical parameters of the animals treated with 1000 mg boron, amorphous/kg bw/day compared to the control group.

NEUROBEHAVIOUR
The observational screening performed in test week 4 approximately 1 - 2 hours after dosing did not reveal any test item-related influence on the animals treated with 1000 mg boron, amorphous/kg bw/day.

ORGAN WEIGHTS
The absolute and relative organ weights were not influenced in the male and female animals treated with 1000 mg boron, amorphous/kg bw/day compared to the control group.

GROSS PATHOLOGY
At necropsy, no test item-related changes were noted in the male and female animals treated with 1000 mg boron, amorphous/kg bw/day for 28 days.

HISTOPATHOLOGY: NON-NEOPLASTIC
The histopathological examination of rats treated with boron, amorphous for 28 days by oral administration of 1000 mg boron, amorphous/kg bw/day did not reveal any morphological lesions which are considered to be related to the administration of the test item.
There was no morphological difference between the dose group (1000 mg boron, amorphous/kg bw/day) and the control group.

DETERMINATION OF BORON LEVELS (URINE AND BLOOD):
reported under IUCLID section 7.1: s_Leuschner_2013

Effect levels

Dose descriptor:
NOAEL
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
No adverse effects observed in a limit dose study (1000 mg/lg/day). No NOAEL derived.