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Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-23 to 2018-02-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Objective of study:
mass balance
Test guideline
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 2017-05-08

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
Specific details on test material used for the study:
not applicable
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The rat is a commonly used rodent species for such 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 administration: males and females: 63 days
- Weight at administration: males: 284.6 g - 315.2 g; females: 226.0 g - 262.6 g
- Housing (exception: sampling period): 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; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt/Arkeburg, Germany)
- Diet (ad libitum): commercial diet, ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity: 55% ± 10% (maximum range)
- Air changes: 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: test item: 0.5 % aqueous hydroxyl propylmethylcellulose gel; reference item (cadmium chloride): tap water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Silicic acid, zirconium salt, cadmium pigment-encapsulated
On the day of administration, the test item was suspended or dissolved in the designated vehicle to the appropriate concentration.
The application formulations were continuously agitated by stirring throughout the entire administration procedure. The dose of the test item was adjusted to the animal's current body weight on the administration day.

Administration volume: 10 mL/kg bw

2) Reference item (cadmium chloride; purity: 99.99 % (61.0 % cadmium); dose level: 38 mg/kg bw)
On the day of administration, the reference item was suspended or dissolved in the designated vehicle to the appropriate concentration.
The application formulations were continuously agitated by stirring throughout the entire administration procedure. The dose of the reference item was adjusted to the animal's current body weight on the administration day.

Administration volume: 10 mL/kg bw
Duration and frequency of treatment / exposure:
single administration
Doses / concentrations
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 of this study were selected after consultation with the Sponsor based on available toxicity data.
The oral LD50 value for the reference item was as follows:
cadmium chloride: 336 mg/kg bw (as Cd2+: 225 mg/kg bw)
Furthermore, oral biovailabilities of soluble Cd substances are given in the public domain with 0.2 - 3% (Cd), depending on the dose and of the duration of the exposure.

Due to the high acute toxicity of soluble selenium compounds (LD50 1.5-6 mg/kg b.w.) adequate dosing within the framework of this single dose study is not feasible. Hence, only Cd was selected for determination. Suitable soluble zirconium and silicon compounds as reference substances are not available.

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 silicic acid, zirconium salt, cadmium pigment encapsulated equates to a dose of 47 mg Cd/kg bw, corresponding to a dose of 77 mg/kg bw of cadmium chloride. Nevertheless, an additional safety factor of 2 (multiplying by 0.5) was used which leads to a dose of 23 mg Cd/kg bw, corresponding to a dose of 38 mg /kg bw of cadmium chloride.

The dosage for the reference item was set to 5% of the dose of the test item on a stoichiometric basis for each metal, thereby lowering the dose for reasons of tolerability of the test animals. This equates to a dose of 23 mg Cd/kg bw, corresponding to a dose of 38 mg/kg bw of cadmium chloride.
The dose level for the reference item was performed in a preliminary experiment (non-GLP) employing 2 animals. No changes were noted in behaviour or the external appearance after dosing with 38 mg CdCl2/kg bw.

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, 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.

The different biological matrices were handled differently for the analytical measurements. Additionally, in all samples of rat treated with soluble salt cadmium chloride (reference item group) only cadmium was measured.

Urine samples were measured directly by ICP-MS without any pre-treatment except for a dilution (for adjustment of matrix), acidification with HNO3 and filtration (0.2 μm PES filter, polyethersulfone membrane syringe filter, DIA Nielsen) of samples.

For faeces samples and application solutions, no digestion or melting procedure was available resulting in a complete dissolution of the pigment in one digestion or melting step. Therefore, a modified procedure was applied. Faeces samples first were lyophilised to weight constancy to determine the dry weight and afterwards the samples were homogenised either manual milling (mortar) or a planetary mill. Subsequently the samples were microwave-assisted digested with nitric acid to decompose the organic matrix. Cadmium, selenium and zirconium concentrations in the digestion solutions were measured by ICP-MS and ICP-OES. The remaining residues after digestion of pigment-treated rats then were melted with sodium carbonate and sodium tetraborate. Cadmium and zirconium concentrations in the dissolved melting masses were analysed by ICP-MS and ICP-OES.

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: 1550 W
Isotopes: 108Cd, 110Cd, 111Cd, 113Cd, 114Cd, 118Cd, 76Se, 77Se, 78Se, 82Se, 90Zr, 91Zr 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; µg/L): Cd: 0.001 - 0.012; Zr: 0.002 - 0.011; Se: 0.039 - 0.291
LOQ (urine samples; µg/L): Cd: 0.003 - 0.036; Zr: 0.006 - 0.032; Se: 0.117 - 0.874
LOD (faeces samples; µg/L): Cd: 0.002 - 0.006; Se: 0.053 - 0.103
LOQ (faeces samples; µg/L): Cd: 0.006 - 0.018; Se: 0.160 - 0.308

The ICP-OES measurements were performed with an Agilent 720 ICP-OES (Agilent
Technologies, Waldbronn, Germany).

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: 1200 W
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.

LOD (faeces samples; µg/L): Cd: 0.034; Zr: 0.116 - 0.416
LOQ (faeces samples; µg/L): Cd: 0.102; Zr: 0.349 - 1.25

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 cadmium and zirconium excreted via urine in each 24 h time period (in mg/24 h) 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 silicic acid, zirconium salt, cadmium pigment encapsulated or CdCl2, served as the control group. The mean mass of cadmium and zirconium excreted via urine by the control animals over 24 h was <0.01 μg/<0.01 μg /24 h (Cd, male and female).

The measured mean background masses in urine were subtracted from the mass of the respective elements Cd, and Zr 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 Cd and Zr excreted via faeces by the control animals over 24 h, calculated by multiplying the concentrations measured for a specific sample with the total weight of the faeces (wet weight) was 1.39 μg/24 h and 1.04 μg/24 h (Cd, male and female). These background excretions were utilised to correct the masses of Cd 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 cadmium and zirconium was calculated, by multiplying the target dose with the actual body weight of each animal. The respective doses for each dosing group (Group 2: Silicic acid, zirconium salt, cadmium pigment encapsulated; Group 3: CdCl2), please refer to section "Overall remarks, attachments" below.

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

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
- Necrospy and macroscopic inspection: 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 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 were examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes, and accessory reproductive organs were recorded.

The weight of the following organs was determined (where possbile): 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) (including parathyroids).
Paired organs were weighed individually and identified as left or right.
The residual carcasses were weighed.

TEST ITEM FORMULATION ANALYSIS
Remaining application formulation (approx. 5 mL) of the test and reference item that were mixed with a vehicle were stored at ≤- 20°C until analysis (Number of samples: 2). In all samples of rat treated with soluble salt cadmium chloride (reference item group) only cadmium was measured.

The dry weight of undissolved test item was determined gravimetrically for the application solution of pigment-treated rats after lyophilization until weight constancy. In addition, an aliquot of the lyophilisation residue was melted with sodium carbonate and sodium tetraborate in order to measure the cadmium and zirconium content by ICP-OES. Furthermore, the dissolved fractions of zirconium, cadmium and selenium in the 0.5% aqueous hydroxylpropyl methylcellulose matrix were determined after microwave-assisted digestion by ICP-OES for application solutions of control, pigment-treated and CdCl2-treated rats.

The ICP-OES measurements were performed with an Agilent 720 ICP-OES (Agilent
Technologies, Waldbronn, Germany).

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: 1200 W
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.

LOD (µg/L): Cd: 0.098 - 0.111; Zr: 0.020 - 1.01
LOQ (µg/L): Cd: 0.293 - 0.344; Zr: 0.060 - 3.03

Results:
Recovery (test item):
- cadmium: 112 %
- zirconium: 88.1 %
Statistics:
Body weight at autopsy and organ weights were calculated using a departmental computerized system (Provantis® integrated preclinical software, version 9.4.0.1).

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).

Results and discussion

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

Toxicokinetic / pharmacokinetic studies

Details on absorption:
Test item (silicic acid, zirconium salt, cadmium pigment encapsulated):
The calculation of the mass balances show that the mass balances for cadmiun (Cd) is essentially complete and indicates that Cd contained in the pigment " silicic acid, zirconium salt, cadmium pigment encapsulated ", present as Cd2+ is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.
Urinary excretion for the element was negligible and below 0.000001 % for Cd.

Please also refer to the section "Overall remarks, attachments".
Details on distribution in tissues:
not examined
Details on excretion:
1) Test item (silicic acid, zirconium salt, cadmium pigment encapsulated)
Animals that received 1000 mg pigment/kg bw excreted 86.3 % Cd 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 85.2 % of Cd were excreted via faeces as largest fraction. Urinary excretion for the element was negligible and below 0.000001 % for Cd.

2) Reference item (cadmium chloride)
Animals that received 23.3 mg Cd/kg bw (administered as CdCl2) excreted 93.3 % (Cd) of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure. The largest fraction (78.4% Cd) was excreted via faeces and urine (0.008 % Cd) within the first 48 h.


Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
not measured

Enzymatic activity

Enzymatic activity measured:
not measured

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
not measured

Any other information on results incl. tables

CLINICAL SIGNS, MORTALITY, BODY WEIGHT, GROSS PATHOLOGY


1) Vehicle control group:


- no premature death was noted in the control group.


- no changes in behaviour, the external appearance or the faeces were noted.


- no differences in the body weight at autopsy were noted between the animals of the control group and the animals treated with the test item or the reference item.


- a dilated uterus that was noted for 1 female of the control group, which was considered to be spontaneous.


 


2) Silicic acid, zirconium salt, cadmium pigment-encapsulated:


- no premature death was noted in the group treated with the test item.


- no changes in behaviour, the external appearance or the faeces were noted.


- on both test days (day of group allocation and day of administration) no differences in body weight were noted between the animals of the control group and the animals treated with the test item.


- no differences in the body weight at autopsy were noted between the animals of the control group and the animals treated with the test item.


- four test days after the administration of the test item increased absolute and relative organ weights of the left and right testis (statistically significant absolute weight (left and right testis) and relative weight (left testis only)) were noted for the animals treated with the test item in comparison to the control group. Please also refer to section "Overall remarks, attachments" below


- no increased liver weights were noted for the female animals of the test item group. Please also refer to section "Overall remarks, attachments" below


- no changes were noted during the macroscopic examination of the internal organs and tissues of the male and female animals treated with the test item.


 


3) Reference item (cadmium chloride):


- no premature death was noted in the group treated with the reference item.


- no changes in behaviour, the external appearance or the faeces were noted.


- on both test days (day of group allocation and day of administration) no differences in body weight were noted between the animals of the control group and the animals treated with the reference item.


- no differences in the body weight at autopsy were noted between the animals of the control group and the animals treated with the reference item.


- four test days after the administration of the reference item increased absolute and relative organ weights of the left and right testis (statistically significant absolute left testis weight only) were noted for the animals treated with the reference item in comparison to the control group.


- increased absolute and relative organ weights of the liver (statistically significant for the relative liver weight) were noted for the female animals treated with the reference item in comparison to the control group. Since in the test item group, which contained a comparable amount of cadmium, no increase liver weights were observed, the result was considered to be spontaneous. Please also refer to section "Overall remarks, attachments" below.


- no changes were noted during the macroscopic examination of the internal organs and tissues of the male and female animals treated with the reference item.

Applicant's summary and conclusion

Conclusions:
Animals that received 1000 mg pigment/kg bw excreted 86.3 % Cd 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 85.2 % of Cd were excreted via faeces as largest fraction. Urinary excretion for the element was negligible and below 0.000001 % for Cd.

The calculation of the mass balances show that the mass balances for cadmiun (Cd) is essentially complete and indicates that Cd contained in the pigment " silicic acid, zirconium salt, cadmium pigment encapsulated ", present as Cd2+ is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.