Chromium tin calcium silicon sphene

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and there is no or no significant human exposure

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-07-24 to 2015-08-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 2014-05-14

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry and stored in a tightly closed container

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

Rats were selected because of their 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: 57 days; females: 64 days
- Weight at study initiation: males: 294.5 g- 331.2 g; females: 230.0 g- 264.8 g
- Housing: animals were 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 GmbH, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 9 days

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

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: 0.5 % aqueous hydroxyl prpyl 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 Barybüttel, Germany
- Batch no.:13DO3-NO3

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 appropriate analytical methods were conducted to determine the concentration, stability 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 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 prepared for digestion and then the digested samples were measured by ICP-OES and ICP-MS.

Before total digestion and measurement of total chromium and tin in the application solutions the samples were retrieved from the cryogenic storage container and thawed at least overnight. Afterwards, the total amount of each application solution was transferred into a 50 mL centrifugation vial and concentrated nitric acid was added. After shaking for 72 h the samples were centrifuged to speed-up the sedimentation of the pigment (15 minutes at 4500
rpm, Allegra X-15R). Therefore, centrifugation time and speed were determined as required. Subsequently, as much supernatant as possible (without taking up pigment) was transferred into a 50 mL vial and filled up with ultra-pure water to 50 mL, and measured by ICP-MS and ICP-OES. Furthermore, concentrated HNO3 was added to each empty original application solution vial, which were also shaken for 72 hours. The nitric acid was also measured by ICP-OES and ICP-MS. This procedure was performed for each application solution.

After the remove of the supernatant the remaining pigment was shared into three digestion vials. 5 mL concentrated HNO3 were filled in each digestion vial and the samples were digested by a microwave procedure (UltraClave II and UltraClave V). After digestion in all three subsamples as much supernatant as possible without transfer of pigment was removed by a pipette and combined in a 15 mL vial and was filled up exactly to 15 mL. After the first digestion ten further digestions were performed. Therefore, the remaining pigment was filled up with the respective digestion medium. Besides 69 % HNO3, mixtures of HNO3/H2O2 or HNO3/HF were used as digestion medium; also different microwave programmes were applied. The supernatants of each digestion were measured by ICP-OES. After the 11th digestion, the procedure was aborted as it turned out to be too time-consuming and not promising. Subsequently, the remaining pigment was lyophilised and the residual amount of pigment determined gravimetrically.

The ICP-OES measurements were performed with an Agilent 720 ICP-OES and an Agilent 5110 VDS ICP-OES. The following solutions were used to calibrate the instrument (not all calibration solutions were used in each measurement series): blank, 1.0 μg/L, 2.0 μg/L, 2.5 μg/L, 4.0 μg/L, 5.0 μg/L, 6.0 μg/L, 7.5 μg/L, 8.0 μg/L, 10.0 μg/L, 20 μg/L, 25 μg/L, 40 μg/L, 50μg/L, 60 μg/L, 75 μg/L, 80 μg/L, 100 μg/L, 200 μg/L, 250 μg/L, 400 μg/L, 500 μg/L, 600 μg/L, 750 μg/L, 800 μg/L, 1000 μg/L, 2000 μg/L, 4000 μg/L and 6000 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 0.9998 (required 0.995). Specific wavelengths for the data evaluation were selected based on the best recovery of chromium and tin 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: Cr: 205.560 nm, 267.716 nm, 283.563 nm, 284.984 nm,
285.567 nm and 357.868 nm
Sn: 183.113 nm, 189.925 nm, 215.152 nm, 226.893 nm, 235.485 nm, 242.950 nm, 283.998 nm and 326.233 nm

Instrument: Agilent 5110 VDS, 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: 12.0 L/min
Additional gas flow: 1.0 L/min
Carrier gas flow: 0.7 L/min
RF power: 1200 W
Stabilization time of sample: 20 sec
Repetition time: 30 sec (three internal measurements per sample)
Wavelengths: Cr: 205.560 nm, 206.158 nm, 206.550 nm, 267.716 nm,
283.563 nm
Sn: 181.059 nm, 189.925 nm, 235.485 nm, 242.950 nm and 283.998 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 × standard deviation of calibration blank/slope of the calibration
LOQ: 3 × LOD
The resulting LODs/LOQs are as follows:
- LOD: 0.106 - 0.646 µg/L (Cr); 0.729 - 19.2 µg/L (Sn)
- LOQ: 0.318 - 1.94 µg/L (Cr); 2.19 - 57.5 µg/L (Sn)
- correlation coefficient: 0.9998 - 1.00000 (Cr); 0.9998 - 1.00000 (Sn)

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 (according to internal laboratory SOPs). 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:
Results:
Analysis of stability and concentration (3 samples):
Recovery [%]:
Cr:. 20.6 - 24.8
Sn: 13.4 - 15.8

Anaylsis of homogenity (3 samples):
Recovery [%]:
Cr:. 21.9 - 26.1
Sn: 11.8 - 21.8

Anaylsis of concentration (1 sample):
Recovery [%]:
Cr:. 24.9
Sn: 16.7

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

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

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, prothrombin 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 (total), 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-213/6-27)
Urine and plasma samples were obtained at study termination for the determination of the chrome and tin levels by ICP-OES and 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)

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:
- No adverse changes in behaviour, external appearance or faeces were noted for the male and female rats treated with 1000 mg Chromium tin calcium silicon sphene (Pigment red 233)/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 Chromium tin calcium silicon sphene (Pigment red 233)/kg b.w./day revealed purple discoloured faeces as of test day 3. This finding is considered to be due to the test item per se being a pink 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 Chromium tin calcium silicon sphene (Pigment red 233)/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 Chromium tin calcium silicon sphene (Pigment red 233)/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 item related 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 Chromium tin calcium silicon sphene (Pigment red 233)/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 Chromium tin calcium silicon sphene (Pigment red 233)/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 Chromium tin calcium silicon sphene (Pigment red 233)/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 cholesterol; males (test day 29): decreased calcium

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 Chromium tin calcium silicon sphene (Pigment red 233)/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 Chromium tin calcium silicon sphene (Pigment red 233)/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 Chromium tin calcium silicon sphene (Pigment red 233)/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 tin and chromium 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 both metals, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that both elements are not biologically available upon ingestion of the pigment chromium tin calcium silicon sphene.

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

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Critical effects observed:

no

Conclusions:

NOAEL (oral; rats) > 1000 mg Chromium tin calcium silicon sphene (Pigment red 233)/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 tin and chromium 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 both metals, mirrored by either minimal or no increases in blood plasma concentrations. This supports the assumption that both elements are not biologically available upon ingestion of the pigment chromium tin calcium silicon sphene.

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-05-25 to 2020-05-28

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

toxicokinetics

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

not applicable

Radiolabelling:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for toxicokinetic studies.

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 dosing: 61 days
- Weight at dosing: males: 299.4 to 430.3 g; females: 168.7 to 294.3 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of
approx. 39 cm × 23 cm and a height of approx. 18 cm
- Diet (ad libitum): certified commercial pellet diet (ssniff® R/M-H V1534, ssniff Spezialdiäten GmbH, 59494 Soest, Germany); bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 5 days
- Health status: an initial health check was performed upon delivery of the animals. Only animals free of signs of illness were selected for the study.

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

Route of administration:

other: chrome tin pink sphene: oral (gavage); reference item: oral (gavage) & intravenously injected

Vehicle:

other: chrome tin pink sphene: 0.5% aqueous hydroxylpropyl methylcellulose gel; reference item: tap water (oral adminsitration) or 0.9% NaCl solution (intravenous administration)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1) Chromium tin calcium silicon sphene
The test item was freshly suspended in the vehicle to achieve the appropriate concentration on the administration day. The administration formulation was continuously agitated by stirring throughout the entire administration procedure.

Administration volume: 10 mL/kg bw

The amount of test item was adjusted to each animal's current body weight on the administration day.

2) Reference item (mixture of chromium (III) acetate hydroxide (Purity: Cr: 23.5 %) and sodium stannate trihydrate (Purity: Sn: 43.8 %)
The reference item compounds - chromium(III) acetate hydroxide and sodium stannate trihydrate - were combined according to the following dosage regimen:
- Reference item (oral adminsitration): 15.5 mg/kg bw of chromium(III) acetate hydroxide and 80.5 mg/kg bw of sodium stannate trihydrate
- Reference item (intravenous adminsitration): 0.78 mg/kg bw of chromium(III) acetate hydroxide and 0.81 mg/kg bw of sodium stannate trihydrate

The mixture was dissolved or suspended in the respective vehicle for oral/intravenous administration.The administration formulations were continuously agitated by stirring throughout the entire administration procedure.

Administration volume: 10 mL/kg bw
Injection speed (intravenous administration): approx. 15 seconds
The amount of reference item was adjusted to each animal's current body weight on the administration day.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

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

No. of animals per sex per dose / concentration:

15 males / 15 femlaes

Control animals:

no

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: the dose levels for this preliminary study had been selected based on available toxicity data:

The oral LD50 values for chromium acetate, basic and sodium stannate trihydrate are stated as being > 5000 and approximately 2400 mg/kg bw (recalculated from SnCl2 and SnSO4), respectively. Oral bioavailabilities of soluble Sn and Cr(III) substances are given in the public domain with approximately 2% (Sn) with as much as 98% being excreted directly in the faeces at intakes around 10 mg/day or higher, and 0.1% to 2% (Cr), respectively.

The test item oral dose of 1000 mg/kg bw corresponded to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of chrome tin pink sphene equates to a dose of 4 mg Cr/kg bw and 358.4 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 805 mg sodium stannate trihydrate/kg bw, respectively.

The dosage for oral administration (gavage) of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) was set equal to the dose of the test item on a stoichiometric basis from the test item for Cr(III) and was reduced to 10% for Sn. This equates to a dose of 4 mg Cr/kg bw and 35.8 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 80.5 mg sodium stannate trihydrate/kg bw, respectively.

The dosage for intravenous injection of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) was set to 5 % of the dose of the reference item administered via gavage on a stoichiometric basis for Cr(III) and to 1% for Sn, thereby lowering the dose for reasons of tolerability of the test animals. This equates to doses of 0.36 mg Sn/kg bw and 0.2 mg Cr/kg bw, respectively, corresponding to doses of 0.81 mg sodium stannate trihydrate/kg bw, and 0.78 mg chromium acetate, basic/kg bw, respectively.

The dose levels for the reference item (gavage and intravenous administration) were confirmed in a preliminary experiment (non-GLP) employing two animals per group.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: in order to obtain approx. 100 μL LiHeparin plasma per animals and sampling time, a sufficient volume of blood was collected from the retrobulbar vvenous plexus of all animals under isoflurane anaesthesia. The blood was withdrawn at the following sampling times: 0 (predose), 1, 2, 4, 8, 12, 24, 48 and 72 hours after administration. At each sampling time point 5 animals/sex/group were used and three sampling points used the same animals.

The blood samples were cooled using an IsoTherm-Rack system until centrifugation. Immediately after centrifugation the isolated plasma was frozen at -20 °C ± 2 °C and stored at this temperature until analysis. The total time between blood withdrawal and freezing the plasma did not exceed 30 minutes.

For plasma samples, a pre-treatment by a microwave digestion with HNO3 was necessary to digest the proteins in plasma. Afterwards chromium and tin concentrations in digested samples were determined by ICP-MS.

The ICP-MS measurement were performed with an Agilent 7700 ICP-MS (Agilent
Technologies, Waldbronn, Germany).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Conical nebulizer, from Glass Expansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Dilution Gas flow: 0.11 L/min
RF power: 1500 W
Isotopes: 52Cr; 53Cr; 116Sn; 118Sn; 120Sn and 103Rh (internal standard)

LOD: Cr: 0.003 - 0.011; Sn: 0.005 - 0.077
LOQ: Cr: 0.009 - 0.033; Sn: 0.014 - 0.232

Toxicokinetic evaluation of the data from the plasma analysis was performed and a non-compartment model was employed. The following parameters were determined:
AUC0-inf = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from zero to the last quantifiable plasma concentration > LLOQ
Kel = elimination rate constant
t1/2 = elimination half-life
Cmax values are the highest measured plasma concentrations and tmax values are the time points of highest plasma concentrations.

Cmax values are the highest measured plasma concentrations and tmax values are the time points of highest plasma concentrations.

Elimination rate constants (Kel) and plasma elimination half-lives (t½) were calculated by linear regression analysis of the log/linear portion of the individual plasma concentration-time curves (c = concentration, t = time).

Half-life:
t0.5 = ln2/Kel [h]
(dc/dt) = Kel * c [h]

Area under the curve (AUC) values were calculated using the linear trapezoidal method and extrapolated to infinite time by dividing the last measurable plasma concentration by the elimination rate constant. Plasma concentrations at time zero were taken to be those at the first blood sampling time.

A dose correction to the analyte content for AUC values was performed for chromium and tin.

As far as possible based on the available data, the bioavailability was calculated for the oral route in comparison to the intravenous route based on AUC0-t last/dose and AUC0-inf/dose values as follows:

- reference item (oral adminsitration) vs. reference item (intravenous administration)
Bioavailability (abs.) = (AUC/dose (reference item; oral administration) / AUC/dose (reference item; intravenous administration)) x 100
- test item (oral adminsitration) vs. reference item (intravenous administration)
Bioavailability (rel..) = (AUC/dose (test item; oral administration) / AUC/dose (reference item; intravenous administration)) x 100

OBSERVATIONS:
- clinical signs: before and after dosing as well as regularly throughout the working day (7:00 a.m. to 3:45 p.m.) and on Saturdays and Sundays (7:00 a.m. to 11:00 a.m.; final check at approx. 3:30 p.m).
- mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 3:00 p.m).
- body weight: at the time of group allocation and on the administration day.

Statistics:

Reference item (oral administration) and reference item (intravenous administration were compared to the test item.

The following statistical method was used for body weight (p ≤ 0.05 and p ≤ 0.01): Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964)

The statistical evaluation of the parametrical values captured by Provantis was done using the following settings:

Homogeneity of variances and normality of distribution were tested using the
BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Preliminary studies:

Please refer to the field "Details on study design" above.

Details on absorption:

BIOANALYSIS
1) Chromium
Following oral administration of 1000 mg Chromium tin calcium silicon sphene/kg bw (test item, oral administration), peak mean chromium concentrations were noted at 72 hours p.a. for the male animals and at 1 hour p.a. for the female animals. The mean peak values were 0.0060 μg chromium/g sample for the males and 0.0195 μg chromium/g sample for the females.

The peak mean chromium plasma concentrations observed for the animals orally treated with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate; oral administration) were slightly higher compared to the test item-treated group with Cmax values of 0.0132 and 0.0258 μg chromium/g sample for males and females, respectively.

The male and female animals treated intravenously with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) revealed peak mean plasma concentrations of 0.0884 and 0.0752 μg chromium/g sample for males and females, respectively, corresponding approximately to the 15-fold (males) and 4-fold (females) of the peak mean concentrations observed for the test item-treated animals.

Please also refer to the section "Overall remarks, attachments".

2) Tin
Following oral administration of 1000 mg Chromium tin caclium silicon sphene/kg bw (test item, oral administration), peak mean tin concentrations of 0.0635 μg tin/g sample for the males and 0.0290 μg tin/g sample for the females were observed at 1 hour p.a.

The peak mean tin plasma concentrations observed for the animals orally treated with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) were slightly lower compared to the test item-treated group for the male animals (0.0458 vs. 0.0635 μg tin/g sample) but slightly higher when the values of the females are compared (0.0645 vs. 0.0290 μg tin/g sample).

The male and female animals treated intravenously with the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate) revealed peak plasma concentrations of 0.0180 and 0.0354 μg tin/g sample for males and females, respectively, being lower with regard to the males and slightly higher with regard to the females compared to the peak concentrations observed for the test item-treated animals.

Please also refer to the section "Overall remarks, attachments".

TOXICOKINETIC EVALUATION
1) Chromium
The highest Cmax value for chromium in the plasma samples was noted following the intravenous administration of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate), followed by the oral administration of the reference item and the oral administration of the test item (chrome tin pink sphene).

The plasma concentrations of chromium declined post dosing with an elimination half-life (t½) ranging approximately from 20.1 to 43.5 hours across sexes and treatments.

The AUC0-t last values ranged from 0.1469 to 1.6272 h μg/g across sexes and treatments. The animals intravenously treated with the reference item revealed the highest exposure towards chromium, whereas the lowest exposure was observed following treatment with the test item chromium tin caclium silicon sphene.

Although the female animals orally treated with the reference item appeared to show a slightly higher exposure to chromium than the male animals, no general differences with regard to exposure were noted between the sexes at all treatments.

The calculation of t½, Kel, and AUC0-∞ was not possible for the male animals of test item group (oral administration) and the female animals of the reference item group (oral administration) because the chromium concentrations measured increased towards the end of the kinetics period. Further, the AUC%extrapolated for the male animals of the reference item group (oral administration) was larger than 20%, rendering the AUC0-inf value unreliable.

Please also refer to the section "Overall remarks, attachments".

2) Tin
The highest Cmax value for tin in the plasma samples was noted following the oral administration of the test item chromium tin calcium silicon sphene, followed by the oral administration of the reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate), and the intravenous administration of the reference item.

The elimination half-life (t½) could be calculated only for the test item-treatment of the male animals and the intravenous treatment with the reference item of both sexes, and ranged from 4.3 to 23.5 hours.

The AUC0-t last values ranged from 0.0872 to 0.3522 h μg/g across sexes and treatments. The male animals revealed the highest exposure towards tin following intravenous administration of the reference item, the lowest exposure was noted following oral treatment with the reference item. For the female animals, the highest exposure was observed following oral administration of the test item chromium tin calcium silicon sphene, whereas the oral treatment with the reference item resulted in the lowest exposure towards tin.

The calculation of t½, Kel, and AUC0-∞ was not possible for the female animals of test item group (oral adminsitration) and the male and female animals of reference item group (oral administration) because there were insufficient measurement data and/or because the tin concentrations measured increased towards the end of the kinetics period. Further, for both the males and the females of the reference item group (intravenous administration), the AUC%extrapolated was larger than 20%, rendering the AUC0-inf values unreliable.

Please also refer to the section "Overall remarks, attachments".

Details on distribution in tissues:

not applicable

Details on excretion:

not applicable

Toxicokinetic parameters:

other: "see Remarks"

Remarks:

Abs. bioavailability of 0.63/2.27% (m/f) was calculated for Cr(III) acetate hydroxide following the oral administration of the reference item compared to the i.v. administration, & a rel. bioavailability of approx. 0.43/1.17% (m/f) for the test item.

Toxicokinetic parameters:

other: see "Remarks"

Remarks:

Rel. bioavailability of tin was calculated to be approx. 0.05%/0.25% (m/f) following treatment with the test item. For the oral treatment with the reference item (Na stannate trihydrate), an abs. bioavailability of 0.56%/0.63% (m/f) was calculated.

Metabolites identified:

not measured

Details on metabolites:

not measured

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability) testing results:

1) Chromium
For chromium, an absolute bioavailability (based on AUC0-t last/dose) of 0.63/2.27% (males/females) was calculated from soluble chromium(III) acetate hydroxide following the oral administration of the reference item compared to the intravenous administration, and a relative bioavailability of approximately 0.43/1.17% (males/females) for the test item (chromium tin calcium silicon sphene).

2) Tin
The relative bioavailability (based on AUC0-t last/dose) of tin was calculated to be approx. 0.05% and 0.25% for male and female animals, respectively, following treatment with the test item chromium tin calcium silicon sphene. For the oral treatment with the reference item, an absolute bioavailability (based on AUC0-t last/dose) of 0.56% was calculated for the males and of 0.63% for the females.

CLINICAL SIGNS AND MORTALITY (males and females)

1) Chromium tin calcium silicon sphene (oral administration)

- no deaths were noted during the study course.

- no abnormal findings with regard to behaviour, external appearance, or consistency of faeces.

2) Reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate; oral administration)

- no deaths were noted during the study course.

- no abnormal findings with regard to behaviour, external appearance, or consistency of faeces.

3) Reference item (mixture of chromium (III) acetate hydroxide and sodium stannate trihydrate; intravenous administration)

- no deaths were noted during the study course.

- no abnormal findings with regard to behaviour, external appearance, or consistency of faeces.

- none of the animals treated intravenously with the reference item revealed any signs of local intolerance reactions at the injection site.

 

BODY WEIGHT (males and females)

The body weight of all animals employed in the study was in the normal range. There were no noteworthy differences between the three test groups.

Conclusions:

For chromium, a relative bioavailability of approximately 0.43/1.17% (males/females) was calculated for chromium tin calcium silicon sphene. Furthermore, the relative bioavailability of tin was calculated to be approx. 0.05% and 0.25% for male and female animals, respectively, following treatment with chromium tin calcium silicon sphene.

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

basic toxicokinetics, other

Remarks:

mass balance

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-11-19 to 2019-12-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Objective of study:

mass balance

Qualifier:

according to guideline

Guideline:

OECD Guideline 417 (Toxicokinetics)

Version / remarks:

2010-07-22

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed on 2017-05-08

Specific details on test material used for the study:

not applicable

Radiolabelling:

no

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for toxicity studies.

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 dosing: males: 62 - 75 days; females: 55 - 68 days
- Weight at dosing: males: 396.9 – 459.8 g; females: 240.9 – 284.1 g
- Housing (exception: sampling period): 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 V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany
- Water (ad libitum): drinking water
- Acclimation period: males: 20 days; females: 13 days
- Health status: an initial health check was performed upon delivery of the animals. Only animals free of signs of illness were selected for the study.

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

Route of administration:

oral: gavage

Vehicle:

other: test item: 0.5 % aqueous hydroxyl propyl methylcellulose gel; reference item (mixture of chromium(III) acetate hydroxide and sodium stannate trihydrate): tap water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
1) Chromium tin calcium silicon sphene
The formulations were freshly prepared on the administration day by dissolving the test item in the respective vehicle to the appropriate concentrations. During and after preparation the formulation was continuously stirred and the homogeneity of the suspension was checked by visual appraisal.

Administration volume: 10 mL/kg bw

The amount of the test item was adjusted to each animal's current body weight on the administration day.

2) Reference item (mixture of chromium(III) acetate hydroxide (Purity: Cr: 23.5 %; dose level:15.5 mg/kg bw) and sodium stannate trihydrate (Purity: Sn: 43.8 %: dose level: 80.5 mg/kg bw))
The formulations were freshly prepared on the administration day by dissolving the reference item in the respective vehicle to the appropriate concentrations. During and after preparation the formulation was continuously stirred and the homogeneity of the suspension was checked by visual appraisal.

Administration volume: 10 mL/kg bw

The amount of the reference item was adjusted to each animal's current body weight on the administration day.

Duration and frequency of treatment / exposure:

single administration

Dose / conc.:

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

No. of animals per sex per dose / concentration:

5 males / 5 females

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

none

Details on study design:

- Dose selection rationale: the dose levels for this study had been selected based on available toxicity data:

The oral LD50 values for chromium acetate, basic and sodium stannate trihydrate are stated as being > 5000 and approximately 2400 mg/kg bw (recalculated from SnCl2 and SnSO4), respectively; oral bioavailabilities of soluble Sn and Cr(III) substances are given in the public domain with approximately 2 % (Sn) with as much as 98% being excreted directly in the faeces at intakes around 10 mg/day or higher, and 0.1 – 2 % (Cr), respectively.

The test item oral dose of 1000 mg/kg bw corresponds to the limit dose used in a separate 28-day oral toxicity study, which is considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of chrome tin pink sphene equates to a dose of 4 mg Cr/kg bw and 358.4 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 80.5 mg sodium stannate trihydrate/kg bw, respectively.

The dosage for the reference item group was set equal to the dose of test item group on a stoichiometric basis from the test item for Cr(III) and was reduced to 10% for Sn. This equates to a dose of 4 mg Cr/kg bw and 35.8 mg Sn/kg bw, corresponding to a dose of 15.5 mg chromium acetate, basic/kg bw and 80.5 mg sodium stannate trihydrate/kg bw, respectively.

The dose level for the reference item have been confirmed in a preliminary experiment (non-GLP) employing two animals.

Details on dosing and sampling:

TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling: all animals of the test item, vehicle and reference item groups were scheduled for urine and faeces sampling. After the single administration, the animals were kept in metabolism cages. Urine and faeces were collected in 3 fractions/animal (sampling periods: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours).
The urine and faeces weight per collected fraction and animal were determined upon removal of the sample fraction. All samples were frozen at - 20 °C or colder until analysis.

The different biological matrices were handled differently for the analytical measurements. Urine samples were directly analysed for chromium and tin content by ICP-MS without any pre-treatment except for an acidification with HNO3 and dilution of samples (according to concentration of chromium and tin) and filtration. Faeces samples were initially freeze-dried (lyophilisation), followed by homogenization and microwave digestion and were analysed for chromium and tin content using ICP-MS.

The ICP-MS measurements were performed with an Agilent 7700 ICP-MS (Agilent Technologies, Waldbronn, Germany).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Concentric nebulizer, from Agilent
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Make-up/Dilution Gas gas flow: 0.11 L/min
RF power: 1550 W
Isotopes: 52Cr, 53Cr, 54Cr, 116Sn, 118Sn, 120Sn and 103Rh (internal standard)
Gas mode: [NoGas] = no gas mode; [He] = Helium (flow rate 4.3 mL/min) gas mode; [HEHe]
= high Helium mode (flow rate 10 mL/min)

At least three internal measurements for each sample were performed and the mean was calculated and printed by the instrument software.

LOD (urine samples): Cr: 0.005 - 0.122; Sn: 0.013 - 0.028
LOQ (urine samples): Cr: 0.016 - 0.366; Sn: 0.040 - 0.085
LOD (faeces samples): Cr: 0.002 - 0.033; Sn: 0.006 - 0.153
LOQ (faeces samples): Cr: 0.005 - 0.098; Sn: 0.017 - 0.460

The mass balance was calculated based on analytical information on urine and faeces that were measured, as described above, and using the raw data on urine and faeces weight. The calculation procedure was as follows:

For each animal, the mass of chromium and tin excreted via urine in each 24h time period (in mg/24h) was calculated by multiplying the concentration measured in urine with the volume of urine that was sampled for each individual animal. The volume of urine was obtained by correcting the recorded mass of urine with the density of urine (1.036 g/mL; reference: Ferrets, Rabbits and Rodents, 2nd Edition, Quesenberry and Carpenter,ISBN: 978-0-7216-9377-4).

Animals not treated with either chrome tin pink sphene or a mixture of
Cr3(OH)2(CH3COO)7 and Na2SnO3 · 3H2O served as the control group. The mean mass of chromium and tin excreted via urine by the control animals over 24h was <0.07 μg/<0.04 μg /24h (Cr, male and female), and <0.04 μg/<0.02 μg/24h (Sn, male and female).

The measured mean background masses in urine were subtracted from the mass of the respective elements Cr and Sn excreted by the treated animals (for m/f respectively). If this background correction resulted in a negative figure, the corrected excretion was set to zero for that animal for further calculations.

The mean mass of Cr, and Sn excreted via faeces by the control animals over 24h, calculated by multiplying the concentrations measured for a specific sample with the total weight of the faeces (wet weight) was 27.04 μg/24h and 10.22 μg/24h (Cr, male and female), and 0.97 μg/24h and 0.57 μg/24h (Sn, male and female). These background excretions were utilised to correct the masses of Cr, and Sn excreted by the treated animals (for m/f respectively). If this background correction resulted in a negative figure, the corrected excretion was set to zero for that animal.

The received actual dose of chromium, and tin was calculated, by multiplying the target dose with the actual body weight of each animal. For the respective doses for each dosing group (Group 2: Chrome tin pink sphene; Group 3: mixture Cr3(OH)2(CH3COO)7 and Na2SnO3 · 3H2O) please refer to section "Overall remarks, attachments" below.

For each treated animal the fractions of received chromium and tin that was excreted via urine or faeces was calculated for each 24h time period (0-24h, 24-48h, 48-72h).

OBSERVATIONS
- clinical signs: before and after dosing as well as regularly throughout the working day (7.30 a.m. to 4.30 p.m.) and on Saturdays and Sundays (8.00 a.m. to 12.00 noon; final check at approx. 4.00 p.m).
- mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays (final check at approx. 4.00 p.m).
- body weight: at the time of group allocation and on the day of administration

GROSS PATHLOLOGY / HISTOPATHOLOGY
On test day 4 (approx. 72 hours after the administration) the animals were dissected. The animals were sacrificed, weighed, dissected, and inspected macroscopically.

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. 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: adrenal gland (2), brain, heart, kidney (2), liver, lungs, lymph nodes (cervical (1), mesenteric (1)), ovary (2), pituitary, prostate, spleen, testicle (2), thymus, and thyroid (1).
Paired organs were weighed individually and identified as left or right.
The residual carcasses were weighed.

Statistics:

The test and reference item treated groups were compared statistically to the control group.

The following statistical method was used for body weight/relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01): Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (September 1964)

The statistical evaluation of the parametrical values captured by Provantis was done using the following settings:

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non-normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

Preliminary studies:

Please refer to the field "Details on study design" above.

Details on absorption:

The calculation of the mass balance show that the elements Cr3+ and Sn4+ of chromium tin calcium silicon sphene are not absorbed in the gastrointestinal tract to any significant extent but pass the animal effectively unchanged. Urinary excretion for all two elements was negligible and below 0.001 % for Cr and 0.002 % for Sn.

Please also refer to the section "Overall remarks, attachments".

Details on distribution in tissues:

not examined

Details on excretion:

1) Test item (Chromium tin calcium silicon sphene)
Animals that received 1000 mg test item /kg bw excreted 91.6 % Cr, and 79.4% Sn of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 84 % of Cr, and 71.6 % of Sn were excreted via faeces as largest fraction. Further 7.43 % and 0.13 % (Cr), and 7.63 % and 0.17 % (Sn) were excreted via faeces on the second and third day. Urinary excretion for all two elements was negligible and below 0.001 % for Cr, and 0.002 % for Sn.

2) Reference item (mixture of chromium(III) acetate hydroxide and sodium stannate trihydrate)
Animals that received a mixture of 4 mg Cr /kg bw (administered as Cr3(OH)2(CH3COO)7), and 35.8 mg Sn/Kg bw (administered as Na2SnO3 · 3H2O) excreted 86 % (Cr) and 95.6 % (Sn) of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure. The largest fraction (72.5 % for Cr, and 79.39 % for Sn) was excreted via faeces and urine (0.13 % for Cr and 0.01 % for Sn) already within the first 24h.

Please also refer to the section "Overall remarks, attachments".

Metabolites identified:

not measured

Details on metabolites:

not measured

Enzymatic activity measured:

not measured

Bioaccessibility (or Bioavailability) testing results:

not measured

CLINICAL SIGNS / MORTALITY /BODY WEIGHTS / GROSS PATHOLOGY

1) Control group

- none of the rats died prematurely.

- no signs of systemic intolerance.

- faeces of test item-treated animals were normally formed.

- body weight of the male and female animals were within the normal range on test day 1.

- no pathological findings were recorded.

 

2) Test item group (chromium tin calcium silicon sphene)

- none of the rats died prematurely.

- none of the rats showed any changes in behaviour or external appearance during the study.

- faeces of test item-treated animals were normally formed. However, male and female animals treated with the test item showed a reversible purple discolouring of the faeces on TD 2 caused by excreted amount of the test item.

- body weight of the male and female animals were within the normal range on test day 1.

- no pathological findings were recorded.

- no test item-related changes in relative and absolute organ weights were
noted for the male and female rats. However, statistically significant differences in relative and absolute organ weights compared to the
control which are not considered to be test item-related were noted, as follows:

Relative liver weight of male animals was statistically significant (p ≤ 0.05) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Absolute liver weight of male animals was statistically significant (p ≤ 0.05) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

 

Please also refer to the section "Overall remarks, attachments".

 

3) Reference item group (mixture of chromium(III) acetate hydroxide and sodium stannate trihydrate)

- none of the rats died prematurely.

- none of the rats showed any changes in behaviour or external appearance during the study.

- faeces of test item-treated animals were normally formed.

- body weight of the male and female animals were within the normal range on test day 1.

- no pathological findings were recorded, except for a slight reduced size of the left adrenal gland of one male animal (no reference item-related finding).

- no reference item-related changes in relative and absolute organ weights were
noted for the male and female rats. However, statistically significant differences in relative and absolute organ weights compared to the
control which are not considered to be reference item-related were noted, as follows:

Relative liver weight of male animals was statistically significant (p ≤ 0.01) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Relative liver weight of female animals was statistically significant (p ≤ 0.05) decreased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Relative (right) kidney weight of female animals was statistically significant (p ≤ 0.05) decreased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

Absolute liver weight of male animals was statistically significant (p ≤ 0.01) increased on test day 4. The slight alteration in comparison to the animals of the control group is without biological relevance.

 

Please also refer to the section "Overall remarks, attachments".

 

Conclusions:

Animals that received 1000 mg chromium tin calcium silicon sphene /kg bw excreted 91.6 % Cr, and 79.4% Sn of the administered dose via urine and faeces during the first three days after exposure (mean for 10 animals). Within the first 24 hours approximately 84 % of Cr, and 71.6 % of Sn were excreted via faeces as largest fraction. Further 7.43 % and 0.13 % (Cr), and 7.63 % and 0.17 % (Sn) were excreted via faeces on the second and third day. Urinary excretion for all two elements was negligible and below 0.001 % for Cr, and 0.002 % for Sn. In total, the calculation of the mass balance show that the elements Cr3+ and Sn4+ of chromium tin calcium silicon sphene are not absorbed in the gastrointestinal tract to any significant extent but pass the animal effectively unchanged.

Materials and methods

Test animals

Species:

rat

Results and discussion

Results (fetuses)

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified