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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:
2015-07-22 to 2015-08-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

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

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:
GLP certificate signed 2014-05-14
Limit test:
yes

Test material

Constituent 1
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material: Silicic acid, zirconium salt, cadmium pigment encapsulated
- new EC name: Zirconium zircon with encapsulated cadmium selenium sulphide
-Physical state: solid, bright red powder, odourless
- Storage condition: store separate from food and drinks, in closed containers and protected places. Keep the containers air-tight.
- Generic formular: ZrSiO4 x Cd2S1.23Se0.77

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Rats were 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 Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 55 days; females: 62 days
- Weight at study initiation: males: 291.6 g - 323.2 g; females: 222.6 g - 257.9 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 23 cm and a height of approx. 18 cm; bedding material: Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): Commercial ssniff® R/M-H V1530 diet (ssniff Spezialdiäten GmbG, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 7 days


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

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The route of administration was selected according to the expected route of exposure
Vehicle:
other: 0.8 % aqueous hydroxyl propyl methylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was suspended in the vehicle to the appropriate concentration. The administration formulation was continuously agitated by stirring throughout the entire administration procedure. The administration formulation was freshly prepared every day. Administration volume: 10 mL/kg b.w./day. The amount of the test item was adjusted to each animal's current body weight daily.

VEHICLE
- Source: Fagron GmbH & Co. KG, 22885 Barsbüttel, Germany
- Batch no.: 13D03-N03
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that was mixed with a vehicle, tests by ICP-OES were conducted to determine the concentration, stability and homogeneity of the test item in the formulations (Fraunhofer IME, report no. EBR-214/6-27). 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 until dispatch:
1) At study initiation:
- analysis of stability and concentration: Immediately after preparation of the administration formulation as well as after 8 and 24 hours storage at room temperature (number of samples: 3).
- homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of group 2 (number of samples: 3).
2) at study termination:
- analysis of concentration: During treatment always before administration to the last animal of group 2 (number of samples: 1- sum of all samples: 7).

The samples were digested as follows:

Before the lyophilisation residues were melted, a pre-test with the original pigment was performed to check whether the melting method was applicable. The recoveries of cadmium and zirconium were well within the tolerance limit of 100 % ± 25 %. To confirm that the pigment remained unchanged in the application solutions solely the cadmium and zirconium content of the dissolved melting product was determined because it was assumed that elemental selenium could evaporate due to its relatively low boiling point (685 °C).

Then the digested samples were measured by ICP-OES.

The ICP-OES measurement was performed with an Agilent 720 ICP-OES. The following solutions were used to calibrate the instrument (not all calibration solutions were used in each measurement series): blank, 0.1 μg/L, 0.25 μg/L, 0.5 μg/L, 0.75 μg/L, 1.0 μg/L, 2.5 μg/L, 5.0 μg/L, 7.5 μg/L, 10 μg/L, 25 μg/L, 50μg/L, 75 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L. Calibrations were performed before each measurement and in the respective acid matrix (matrix adaption of calibration solution). The linearity of the calibration was adequate for the lower and higher concentration range. In some measurement series, individual concentrations were excluded from the calibration by the instrument software since the nominal concentration was ± 25% of the measured concentration, i.e. mainly low concentrations for which the difference between background of blank and concentration was not high enough. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument. The correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.999960 (required 0.995). Specific wavelengths for the data evaluation were selected based on the best recovery of cadmium and zirconium (no selenium analysed as only samples of application solutions were measured with ICP-OES) in quality control samples (certified waters, recovery/fortification samples, etc.). Furthermore, the wavelengths were checked for possible interferences and wavelengths with a possible interference were not taken into account for a possible evaluation.

Instrumental and analytical set-up for the ICP-OES instruments:

Instrument: Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer, from Glass Expansion
Spray chamber: Iso Mist with Twister Helix from Glass Expansion
Plasma stabilization time: at least 30 min before start of the measurements
Plasma gas flow: 15.0 L/min
Additional gas flow: 1.50 L/min
Carrier gas flow: 0.75 L/min
RF power: 1200W
Stabilization time of sample: 15 sec
Repetition time: 30 sec (three internal measurements per sample)
Wavelengths: Cd: 214.439 nm, 226.502 nm, and 228.802 nm
Zr: 267.865 nm, 327.307 nm, 327.927 nm, 343.823 nm and 349.619 nm

At least three internal measurements for each sample were performed and the mean was calculated and printed by the instrument software.
The applied LOD/LOQ calculations are (according to DIN 32645) (Chemische Analytik - Nachweis-, Erfassungs- und Bestimmungsgrenze unter Wiederholbedingungen – Begriffe, Verfahren, Auswertung; German version DIN 32645:2008-11. Beuth Verlag.):
LOD: 3 x standard deviation of calibration blank/slope of the calibration
LOQ: 3 x LOD

The resulting LODs/LOQs are as follows:
- LOD: 0.111 - 0.120 µg/L (Cd); 0.235 - 1.01 µg/L (Zr)
- LOQ: 0.334 - 0.361 µg/L (Cd); 0.704 - 3.03 µg/L (Zr)
- correlation coefficient: 0.999964 - 0.999994 (Cd); 0.999960 - 0.999966 (Zr);

Certified reference materials as well as quality control standards, recalibration standards and fortification samples (recovery samples) were analysed as quality assurance samples along with the test samples during the measurement. To meet quality assurance requirements recovery needs to be in the range of ± 15 % of the respective certified value or the nominal/calculated values. Since possible interferences of the quality control samples could not be excluded during a measurement series at least 60% of the quality assurance samples have to be valid. Under specific circumstances, this 60% can also be lowered if specific interferences can be proved for example influence of elements in one specific reference material due to concentration interference.

Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Cd:. 110 - 112
Zr: 85.3 - 88.1

Anaylsis of homogenity (3 samples):
Recovery [%]:
Cd:. 108 - 113
Zr: 86.9 - 88.6

Anaylsis of concentration (1 sample):
Recovery [%]:
Cd:. 111
Zr: 87.7
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Doses / concentrations
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: In agreement with the Sponsor and based on available toxicity data a limit test was performed.
Positive control:
none

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs): before and after dosing at each time of dosing as well as regularly throughout the working day from 7:30 a.m. to 4.30 p.m. and on Saturdays and Sundays from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
- Time schedule (mortality): early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays with a final check at approximately 4.00 p.m.
- Cage side observations checked: clinical signs and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure and once a week thereafter (1, 2, 4, 8 and 24 hours after administration) as well as in the test week 4 prior to any laboratory investigations.

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 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 on completion of a treatment week. The relative food consumption (in g/kg b.w./day) was determined
- 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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the start of administration and at the end of test week 4.
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination (on the day of dissection)
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes , overnight
- How many animals: all animals
- Parameters examined: haemoglobin content, erythrocytes, leucocytes, retriculocytes, platelets, haematocrit value, differential blood count (relative and absolute- neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes), mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, thromboplastin time, activated partical thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- 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 (in blood), calcium, chloride, potassium, sodium, alanine amino-transferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 4 approx. 1 to 2 hours after dosing and before any blood sampling
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
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, tail pinch, wire manoeuvre, hind-leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function
2) Functional tests: grip strength and locomotor activity

IMMUNOLOGY: No

OTHER:
TOXICOKINETICS: Yes (please refer to Fraunhofer IME, report no. EBR-214/6-27)
Urine and plasma samples were obtained at study termination for the determination of the zirconium, silicon, selenium and cadmium levels by ICP-MS.
- urine sample: individual urine samples were collected from all animals for a 24-hour period following the last administration on test day 28 (one 24-hour fraction/animal). For this purpose, the animals were placed in metabolic cages during the 24-hour collection period, directly after the last oral administration.
- plasma sample: 24 hours after the last administration, a terminal blood sample was collected from all animals by puncture from the retrobulbar venous plexus under isoflurane anaesthesia in order to obtain 0.5 mL LiHeparin plasma/animal. Afterwards, the animals were sacrificed and dissected
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes

On test day 29 (approximately one day after the last administration), the animals were dissected following a randomisation scheme. The animals were sacrificed by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically
under the direction of a pathologist. All superficial tissues were examined visually and by palpation and the cranial roof was 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 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, As a whole: Prostate and seminal vesicles with coagulating glands. Paired organs were weighed individually and identified as left or right.

The following organs or parts of organs of all animals were fixed in 7% buffered formalin. The eyes were fixed 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: right and left 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 sections), Spleen, Stomach, Testicle (2), Thymus, Thyroid (2) (incl. parathyroids), Tissue masses or tumours (incl. regional lymph nodes), Trachea (incl. larynx), Urinary bladder, Uterus (incl. cervix and oviducts), Vagina

The above-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, stained with Oil Red O and examined microscopically. 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-treated Group 2 was compared with the control Group 1.
The following statistical methods were used:
1) STUDENT's t-test All numerical functional tests / Body weight / Food consumption / Haematology and coagulation / 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)

2) Exact test of R. A. FISHER Histology (p ≤ 0.05 and p ≤ 0.01)

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
Endocrine findings:
not examined
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:
- No adverse changes in behaviour, external appearance or faeces were noted for the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day or for the animals treated with the vehicle control once daily for 28 days.
- all male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day revealed reddish discoloured faeces as of test day 2. This finding is considered to be due to the test item per se being a neonred powder and, hence, is not an adverse effect.

MORTALITY:
- None of the animals died prematurely during the study

BODY WEIGHT AND WEIGHT CHANGES
- No test item-related influence was observed for the body weight, the body weight gain and body weight at autopsy in the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28 days. All data are regarded to be within the normal range.

FOOD CONSUMPTION AND COMPOUND INTAKE
- No test item-related changes in relative food consumption were noted for the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28 days compared to the control group.

WATER CONSUMPTION AND COMPOUND INTAKE
- The visual appraisal of the drinking water consumption did not reveal any test itemrelated influence.

OPTHALMOLOGICAL FINDINGS
- Ophthalmological examination revealed no changes of the eyes and the optic region in the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day or for the animals treated with the vehicle control once daily for 28 days.

HAEMATOLOGICAL FINDINGS
- No test item-related influence in haematological and coagulation parameters was noted for the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28 days compared to the control group.

CLINICAL BIOCHEMISRY FINDINGS
- No test item-related influence in biochemical parameters was noted for the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28 days compared to the control group.
- Statistically significant differences in biochemical parameters compared to the control which are not considered to be test item-related: males (test day 29): decreased urea; females (test day 29): increased alkaline phosphatase

BEHAVIOUR (FUNCTIONAL FINDINGS)
- The neurological screening performed at the end of the treatment period in test week 4 approximately 1 to 2 hours after dosing did not reveal any test item-related influence in the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28 days, 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. The examination results of the animals treated with the vehicle control were also in the normal range.

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIOS
- No test item-related changes in relative and absolute organ weights were noted for the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28-days compared to the control group.

GROSS PATHOLOGICAL FINDINGS
- None of the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28-days revealed any macroscopic changes at necropsy on test day 29.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
- The histomorphological examination did not reveal any morphological changes which are considered to be related to the administration of the test item. There was no difference between the groups.
- The inflammatory lesions in different organs are considered to be coincidental findings or spontaneous organ changes and are thus not test item-related. No differences were noted between the groups.
- The fatty infiltration in the hepatocytes and in the tubular epithelial cells of the kidney in male and female rats of the control and/or the test item-treated group were within the physiological limits.
- The involution of the thymus in the rats of both groups corresponded in type, incidence and severity to the age of the animals.
- The coincidental findings from different organs in a small number of control and test item-treated animals are considered to be spontaneous organ changes and are thus not test item-related.
- No morphological differences were noted for any of the organs examined between the controls and the high dose group 2. Type, incidence and severity of the lesions were comparable to the control animals.

OTHER
TOXICOKINETICS
The uptake of cadmium, zirconium, and selenium during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.05% of the dose was excreted via urine for all three metals, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that all three elements are not biologically available upon ingestion of the pigment silicic acid, zirconium salt, cadmium pigment encapsulated.

Please also refer to the field "Attached background material" below.

Effect levels

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL (oral; rats) > 1000 mg silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day

No test item-related changes were observed for clinical signs, mortality, neurologically screening, body weight/body weight gain, food consumption, water consumption, haematology, clinical chemistry, organ weights, ophthalmology, gross pathology and histopathology.

The uptake of cadmium, zirconium, and selenium during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.05% of the dose was excreted via urine for all three metals, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that all three elements are not biologically available upon ingestion of the pigment silicic acid, zirconium salt, cadmium pigment encapsulated.