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


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 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.

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.
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2018-02-26 to 2018-03-01
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 toxicity 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: 59 days
- Weight at administration: males: 262.5 g - 292.4 g; females: 217.4 g - 253.7 g
- Housing: 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: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Humidity: 55 % ± 10 % (maximum range).
- Air changes (per hr): 15 - 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
other: silicic acid, zirconium salt, cadmium pigment encapsulated: oral (gavage); reference item: oral (gavage) & intravenously injected
Vehicle:
other: silicic acid, zirconium salt, cadmium pigment-encapsulated: 0.5% aqueous hydroxypropylmethylcellulose gel; reference item: tap water (oral administration) or 0.9 % NaCl solution (intravenous administration)
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 vehicle to the appropriate concentration. The application formulation was continuously stirred throughout the entire administration procedure. The application formulation was administered orally, by gavage, at a constant volume. The dose of the test item was adjusted to the animal's current body weight on the administration day.

2) Reference item (cadmium chloride; purity: 99.9 %; cadmium content: 61 %)
On the day of administration, the reference item was dissolved in the designated vehicle for oral/intravenous administration. The application formulations were continuously stirred throughout the entire administration procedure. The application formulation was administered orally, by gavage, at a constant volume. The dose of the reference item was adjusted to the 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:
no
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 cadmium (as Cd2+) is stated as being 225 mg/kg bw (366 mg/kg bw for cadmium chloride). Oral biovailabilities of soluble cadmium substances are given in the public domain with 0.2 - 0.3% (cadmium), 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 bw) adequate dosing within the framework of this single dose study is not feasible. Hence, only cadmium 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 cadmium/kg bw, corresponding to a dose of 77 mg/kg bw of cadmium chloride. Nevertheless, an additional factor of 2 is used which leads to a dose of 23 mg cadmium/kg bw, corresponding to a dose of 38 mg/kg bw of cadmium chloride.

The dosage for intravenous injection of the reference item (cadmium chloride) was set to 1% of the dose of the reference item administered via gavage on a stoichiometric basis, thereby lowering the dose for reasons of tolerability of the test animals. This equates to a dose of 0.23 mg cadmium/kg bw, corresponding to a dose of 0.4 mg/kg bw of cadmium chloride.

The dose level for the reference item was confirmed in a preliminary experiment (non-GLP) employing 2 animals. No changes were noted in behaviour or the external appearance after dosing with 38 mg cadmium chloride/kg bw.
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 preferably from the vena jugularis 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.

The whole blood samples were cooled using an IsoTherm-Rack system until centrifugation. Immediately after centrifugation the isolated plasma was frozen at ≤ -20°C and stored at this temperature until analysis.

Toxicokinetic evaluation of plasma data was performed and a non-compartment model was employed. The following parameters were determined, if possible:
AUC0-∞ = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from time zero to the last quantifiable plasma/serum concentration > LOQ
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.

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
(dc/dt) = Kel * c

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.

Furthermore, the AUC0-t last was calculated according to the linear trapezoidal rule. Values below or at the limit of quantification (LOQ) were excluded from calculation.

In addition, the bioavailability was calculated for the mixture (for cadmium only).

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 administration day.

TEST ITEM FORMULATION ANALYSIS
The remaining administration formulations (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: 3).
Statistics:
The statistical evaluation of the parametrical values was done by Provantis 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 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:
1) Zirconium
Analysis of zirconium in plasma was only suitable for the test item group animals. Low plasma levels of zirconium were detected only for single animals at a few blood withdrawal time-points. The mean values of calculated toxicokinetic parameters for a dose of 455 mg zirconium/kg are as follows:
males: 0.0011 µg/g (Cmax); 5.3 hours (tmax)
females: 0.0010 µg/g (Cmax); 17.2 hours (tmax)

2) Cadmium
For test item group animals treated once with 1000 mg test item/kg bw (i.e. 47 mg cadmium/kg bw), the mean cadmium levels were generally very low. Cmax values of 0.0005 μg/g or 0.0004 μg/g were noted for the male and the female animals, respectively. After single dosing with 38 mg reference item/kg bw via gavage (i.e. 23 mg cadmium/kg bw), Cmax values of 0.1318 μg/g or 0.1564 μg/g were observed for the male and female animals, respectively. For the animals of reference item group treated intravenously with 0.4 mg reference item/kg bw (i.e. 0.23 mg cadmium/kg bw), Cmax values of 0.0224 μg/g or 0.0204 μg/g were noted for the male and female animals, respectively. Mean tmax occurred between 1.0 hour (i.e. first sampling time-point) and 1.2 hours after administration.

The mean terminal elimination half-life ranged from 41.9 hours to 64.3 hours for the test item (p.o. administration), from 14.3 hours to 14.7 hours for the reference item (p.o. administration) and from 13.8 hours to 17.4 hours for the reference item (intravenous administration).

3) Selenium
For test item group animals treated once with 1000 mg test item/kg bw (i.e. 15 mg selenium/kg bw), Cmax values of 0.4294 μg/g or 0.4036 μg/g were observed for the male and female animals, respectively. Mean tmax occurred between 19.2 hours and 24 hours after administration. The mean terminal elimination half-life ranged from 184.9 hours to 234.5 hours for the test item.

Please also refer to the field "Attached background material" below.
Details on distribution in tissues:
not applicable
Details on excretion:
not applicable
Toxicokinetic parameters:
other: Absolute bioavailabilities of 0.032% or 0.033% (males or females) were calculated for cadmium within the test item silicic acid, zirconium salt, cadmium pigment encapsulated compared to intravenous administration of the reference item cadmium chloride.
Toxicokinetic parameters:
other: Absolute bioavailabilities of 4.81% or 4.89% (males or females) were calculated for cadmium within the reference item cadmium chloride administered orally compared to intravenous administration of the reference item cadmium chloride.
Metabolites identified:
not measured
Details on metabolites:
not measured
Enzymatic activity measured:
not measured
Bioaccessibility (or Bioavailability) testing results:
Absolute bioavailabilities of 0.032% or 0.033% (males or females) were calculated for cadmium within the test item silicic acid, zirconium salt, cadmium pigment encapsulated and of 4.81% or 4.89% (males or females) for cadmium within the reference item cadmium chloride administered orally compared to intravenous administration of the reference item cadmium chloride.

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

CLINICAL SIGNS AND MORTALITY (males and females)


Silicic acid, zirconium salt, cadmium pigment-encapsulated


- no premature death was noted for the animals treated once with the test item.


- no changes in behaviour, the external appearance or the faeces were noted for the animals treated once with the test item after administration on test day 1 until study termination on test day 4.


Reference item (oral administration)


- no premature death was noted for the animals treated once with the reference item (38 mg cadmium chloride/kg bw).


- no changes in behaviour, the external appearance or the faeces were noted for the animals treated once with the reference item (38 mg cadmium chloride/kg bw) after administration on test day 1 until study termination on test day 4.


Reference item (intravenous administration)


- no premature death was noted for the animals treated once with the reference item (0.4 mg cadmium chloride/kg bw).


- no changes in behaviour, the external appearance or the faeces were noted for the animals treated once with the reference item (0.4 mg cadmium chloride/kg bw) after administration on test day 1 until study termination on test day 4. The animals treated with the reference item (0.4 mg cadmium chloride/kg bw) did not reveal any signs of local intolerance reactions.


 


BODY WEIGHT (males and females)


No differences in body weight were noted between the animals of the test groups.


 


TEST ITEM FORMULATION ANALYSIS


For the application formulations / solutions, a digestion or melting procedure had to be developed. None of the typical digestions (e.g. digestion with HNO3 or with mixture of HNO3/HF) or melting procedures resulted in a total dissolution of the pigment. After consultation with the study monitor the application formulations / solutions were not digested and not measured.

Conclusions:
Absolute bioavailabilities of 0.032% or 0.033% (males or females) were calculated for cadmium within the test item silicic acid, zirconium salt, cadmium pigment encapsulated and of 4.81% or 4.89% (males or females) for cadmiun within the reference item cadmium chloride administered orally compared to intravenous administration of the reference item cadmium chloride.

Due to the high acute toxicity of soluble selenium compounds adequate dosing within the framework of this single dose study is not feasible. Hence, only cadmium was selected for determination. Suitable soluble zirconium and silicon
compounds as reference substances are not available.
Endpoint:
basic toxicokinetics in vitro / ex vivo
Remarks:
Bioaccessibility - transformation/dissolution in artificial physiological media
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-09-23 to 2012-09-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Objective of study:
bioaccessibility (or bioavailability)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media
Deviations:
yes
Remarks:
Bioaccessibility testing: loading of 100 mg/L; five artificial physiological media agitated at 100 rpm, at 37 °C ± 2 °C; sampling after 2h and 24h; determination of Cd, Se and Zr concentrations after filtration by ICP-MS and ICP-OES.
Principles of method if other than guideline:
An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds, including steels, in previous risk assessments (completed under Regulation (EEC) No 793/93) and in recent preparation for REACH regulation (EC) No 1907/2006.
The test was performed on the basis of the guidance for OECD-Series on testing and assessment Number 29 and according to the bioaccessibility test protocol provided by the study monitor.
GLP compliance:
yes (incl. QA statement)
Remarks:
2011-02-07
Species:
other: in vitro (simulated human body fluids)
Details on exposure:
Test principle in brief:
- five different artificial physiological media,
- single loading of test substance of 100 mg/L,
- samples taken after 2 and 24 hours agitation (100 rpm) at 37 ± 2 °C,
- two method blanks per artificial media were tested; measurement (ICP-MS and ICP-OES) of dissolved cadmium, selenium and zirconium concentrations after filtration (0.2 µm, Supor membrane)
- the study was performed in duplicates

The aim of this test was to assess the dissolution of IPC-2013-001 (Silicic acid, zirconium salt, cadmium pigment-encapsulated) in five artificial physiological media: Artificial lysosomal fluid (ALF, pH = 4.5), Artificial sweat solution (ASW, pH = 6.5), Gamble´s solution (GMB, pH = 7.4), Artificial gastric fluid (GST, pH = 1.5), Phosphate buffered saline (PBS, pH = 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion into the gastrointestinal tract and by inhalation.
Duration and frequency of treatment / exposure:
Samples were taken after 2 h and 24h.
Dose / conc.:
100 other: mg of the test item /L artificial media
Details on study design:
Reagents
The water (resistivity >18 MΩ·cm.) used for this test was purified with a Pure Lab Ultra water purification system from ELGA LabWater, Celle, Germany.
- Nitric acid - “Supra” quality (ROTIPURAN® supplied by Roth, Karlsruhe, Germany).
- Hydrochloric acid – “instra-analyzed plus” quality (J.T. Baker, Griesheim, Germany).

Metal analysis
- Standards: Multielement cadmium and selenium standard containing 100 mg/L Cd and Se (19 elements, CPI, Amsterdam, The Netherlands, lot no. 12A089) in 5% nitric acid was utilised. For zirconium a commercially available single element standard was used (CPI, Amsterdam, The Netherlands, lot no. ZR9301).
- Certified reference materials: TMDA-70 and TM 15.2 obtained from Environment Canada (lot no. 0310; lot no. 1011), single element standards (Merck Certipur, Cd standard, lot no. HC123668; Merck Certipur, Se standard, lot no. HC095730) and zirconium single element standard (Fluka TraceCERT, lot no. BCBD9418)

Instrumental and analytical set-up for the ICP-OES instrument:
Thermo IRIS Intrepid II from Thermo Electron Corporation, Germany
Nebulizer: Concentric glass nebulizer, from Thermo
Spray chamber: Glass cyclonic spray chamber, from Thermo
Nebulizer gas flow: 0.68 L/min
Make-up gas flow: 0.5 L/min
RF power: 1150 W
Wavelengths: Cd 226.502 nm and 228.802 nm; Se 196.090 nm and 203.985 nm;
Zr 339.198 nm, 343.823 nm and 349.621 nm.
Calibration: blank, 1.0 µg/L, 5.0 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, 200 µg/L, 300 µg/L, 400 µg/L and 500 µg/L.
Correlation coefficients (r): at least 0.9999
One measurement series was performed for the determination of cadmium, selenium and zirconium concentrations in the mass balance and filter/syringe samples.
The applied LOD/LOQ calculations are:
LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.
These data were read directly from the Thermo IRIS Intrepid II ICP-OES instrument output (data calculated by internal algorithms of the instrument software).

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn, Germany
Nebulizer: Concentric glass nebulizer, from GlassExpansion
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.91 L/min
Dilution/Make-up gas flow: 0.13 L/min
RF power: 1500 W
Isotopes: 111Cd and 114Cd; 78Se and 82Se; 90Zr and 91Zr and 103Rh (internal standard)
Calibrations: 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 and 10.0 µg/L.
Correlation factors (r): at least 0.9965
In sum, six series of measurements were performed for the determination of total dissolved cadmium, selenium and zirconium concentrations in samples including blanks to determine background levels of elements and fortified samples.
The LOD and LOQ for cadmium, selenium and zirconium were calculated using the internal instrument algorithm. This calculation is according to DIN 32645. For this the standard deviation of calibration blanks is multiplied by 3 and divided by the slope of the calibration line.
Details on dosing and sampling:
Loading:
The nominal loading in this test was 100 mg/L. However, due to weighing uncertainties the actual loadings range from 100.148 mg/L to 101.892 mg/L in the test vessels.
Type:
other: Bioaccessibility
Results:
Highest dissolution (loading: 0.1g/L, after 24h) in ASW: Cd: 0.93 ± 0.20 µg/L, Se: 0.79 ± 0.1 µg/L; in GST: Cd: 6.13 ± 0.42 µg/L; in PBS: Se: 0.82 ± 0.03 µg/L; in ALF Zr: 1.25 ± 0.11 µg/L

Method validation summary (ICP-OES)

validation parameter

results

Comment

Selectivity

similar data with different element wavelengths for ICP-OES method

no interferences observed

Linearity

applied calibration functions were linear

correlation coefficient at least 0.9999

limit of detection

Cd: 3.30 µg/L

Se: 4.66 µg/L

Zr: 3.51 µg/L

limit of quantification

Cd: 11.0 µg/L

Se: 15.5 µg/L

Zr: 11.7 µg/L

Accuracy

mean recovery for CRM TMDA-70: Mass balance
Cd: 103 ± 1.1 (n = 4)

Se: 107 ± 5.2 (n = 4)

higher concentration range for Cd and lower concentration range for Se:

Cd: 145 µg/L

Se: 13.0 µg/L

Trueness Cd and Se

mean recovery for recalibration standard: mass balance

Cd: 97.5 ± 1.2 % (n = 4)

Se: 96.5 ± 1.9 % (n = 4)

high concentration range (100 µg/L)

Trueness Zr

mean recovery for recalibration standard: mass balance

Zr: 98.7 ± 1.9 % (n = 4)

high concentration range (50 µg/L)

Reproducibility

mean recovery for CRM TMDA-70: Mass balance
Cd: 103 ± 1.1 (n = 4)

Se: 107 ± 5.2 (n = 4)

higher concentration range for Cd and lower concentration range for Se:

Cd: 145 µg/L

Se: 13.0 µg/L

Method validation summary (ICP-MS)

validation parameter

results

Comment

Selectivity

similar data for the different gas modes

appropriate Isotope and gas mode were chosen for interference free measurements

Linearity

applied calibration functions were linear

correlation coefficient at least 0.9965

limit of detection

Cd: 0.02 – 0.03 µg/L

Se: 0.02 – 0.23 µg/L

Zr: 0.01 – 0.16 µg/L

limit of quantification

Cd: 0.01 – 0.08 µg/L

Se: 0.05 -0.68 µg/L

Zr: 0.04 – 0.47 µg/L

method blanks

Mainly all method blanks are below LOD/LOQ except for Se in GMB and ALF media and for Zr in ASW media

Elevated concentrations of in method blanks origin from the applied chemicals for preparation of different media

Accuracy

mean recovery for CRM TMDA-70:
Cd: 105 ± 8.3 % (n = 15)

Se: 102 ± 10 % (n = 15)

higher concentration range for Cd and lower concentration range for Se:

Cd: 145 µg/L

Se: 13.0 µg/L

Accuracy

mean recovery for CRM TM 15.2:
Cd: 101 ± 4.6 % (n = 15)

Se: 90.6 ± 6.8 % (n = 15)

lower concentration range for Cd and higher concentration range for Se:

Cd: 25.8 µg/L

Se: 15.1 µg/L

Trueness Cd and Se

mean recovery for recalibration standard:

Cd: 98.3 ± 1.7 % (n = 3)

Se: 102 ± 2.2 % (n = 3)

higher concentration
(5 µg/L)

Trueness Cd and Se

mean recovery for recalibration standard:

Cd: 92.4 ± 4.6 % (n = 12)

Se: 92.9 ± 6.8 % (n = 12)

mid concentration range (2 µg/L)

Trueness Cd and Se

mean recovery for recalibration standard:

Cd: 94.0 ± 4.5 % (n = 15)

Se: 105 ± 10.5 % (n = 15)

low concentration range (0.5 µg/L)

Trueness Zr

mean recovery for recalibration standard:

Zr: 104 ± 1.6 % (n = 3)

higher concentration
(20 µg/L)

Trueness Zr

mean recovery for recalibration standard:

Zr: 88.3 ± 2.4 % (n = 6)

mid high concentration range (5 µg/L)

Trueness Zr

mean recovery for recalibration standard:

Zr: 101 ± 12.5 % (n = 9)

mid concentration range (2 µg/L)

Trueness Zr

mean recovery for recalibration standard:

Zr: 96.9 ± 6.9 % (n = 12)

low concentration range (0.5 µg/L)

Trueness

Fortification of samples:

Cd: 92.0 – 108 %

Se: 85.0 – 98.6 %

Zr: 95.4 – 106 %

Trueness Zr

mean recovery for recalibration standard: mass balance

Zr: 98.7 ± 1.9 % (n = 4)

high concentration range (50 µg/L)

Reproducibility

mean recovery for CRM TMDA-70:
Cd: 105 ± 8.3 % (n = 15)

Se: 101 ± 4.6 % (n = 15)

higher concentration range for Cd and lower concentration range for Se:

Cd: 145 µg/L

Se: 13.0 µg/L

Reproducibility

mean recovery for CRM TM 15.2:
Cd: 102 ± 10 % (n = 15)

Se: 90.6 ± 6.8 % (n = 15)

lower concentration range for Cd and higher concentration range for Se:

Cd: 25.8 µg/L

Se: 15.1 µg/L

Solution pH values

After preparation of the artificial physiological media, solution pHs were adjusted to their respective target pH. The target pH in all media before addition of test substance is in the nominal range. During the study, the pH of ALF, GST, GMB and PBS solutions remained stable. The pH of the ASW solutions in all vessels decreased after 24h. The pHs had decreased to 5.8 in all vessels. Therefore, an effect of the test substance can be excluded.

pHs of the physiological test media prior to the test

media

target pH

measured pH

temp. [°C]

ALF

4.5

4.5

30.2

ASW

6.5

6.5

34.0

GMB

7.4

7.4

31.5

GST

1.5 – 1.6

1.5

36.3

PBS

7.2 - 7.4

7.3

35.8

 

Concentration of cadmium in artificial media, calculated nominal cadmium concentration in 100 mg/L silicic acid, zirconium salt, cadmium pigment-encapsulated and dissolved amount of cadmium.

media and sample

total Cd ± SD in method blanks [µg/L]

total Cd ±SD in sample vessels [µg/L]

Cd ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Cd concentration in [µg/L]#

dissolved amount of Cd in artificial media [%] normalizedfor measured background in method blank

ALF 2h

<LOD

0.38 ± <0.01

0.38 ± <0.01

4699

0.01 ± <0.01

ALF 24h

<LOD

0.89 ± 0.10

0.89 ± 0.10

4699

0.02 ± <0.01

ASW 2h

<LOD

0.29 ± 0.09

0.29 ± 0.09

4722

0.01 ± <0.01

ASW 24h

<LOD

0.93 ± 0.20

0.93 ± 0.20

4722

0.02 ± <0.01

GMB 2h

<LOD

0.07 ± <0.01

0.07 ± <0.01

4709

0.002 ± <0.001

GMB 24h

<LOD

0.07 ± <0.01

0.07 ± <0.01

4709

0.001 ± <0.001

GST 2h

<LOD

2.58 ± 0.20

2.58 ± 0.20

4733

0.05 ± <0.01

GST 24h

<LOD

6.13 ± 0.42

6.13 ± 0.42

4733

0.13 ± 0.01

PBS 2h

<LOD

0.11 ± 0.02

0.11 ± 0.02

4711

0.002

PBS 24h

<LOD

0.43 ± 0.15

0.43 ± 0.15

4711

0.01 ± <0.01

# (initial weight (e.g. 50 mg) * 4.66 § (percentage cadmium in test item) / 100) * 2 (multiplication to calculate cadmium amount in one litre -> 100 mg/L) = nominal cadmium concentration in [mg/L] / 1000 = nominal cadmium concentration in [µg/L]

§ according to CoA 4.66 % Cd in test item

In five different artificial physiological media, between 0.001 and 0.13 % of cadmium was dissolved from the test item IPC-2013-001 Silicic acid, zirconium salt, cadmium pigment-encapsulated depending on solution parameters and test duration.

Concentration of selenium in artificial media, calculated nominal selenium concentration in 100 mg/L silicic acid, zirconium salt, cadmium pigment-encapsulated and dissolved amount of selenium.

media and sample

total Se ± SD in method blanks [µg/L]

total Se ±SD in sample vessels [µg/L]

Se ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Se concentration in [µg/L]#

dissolved amount of Se in artificial media [%] normalized for measured background in method blank

ALF 2h

0.08 ± 0.01

0.71 ± 0.02

0.64 ± 0.02

1543

0.04 ± <0.01

ALF 24h

0.08 ± 0.01

0.77 ± 0.05

0.69 ± 0.05

1543

0.04 ± <0.01

ASW 2h

<LOD/LOQ

0.59 ± 0.01

0.59 ± 0.01

1550

0.04 ± <0.01

ASW 24h

<LOD/LOQ

0.79 ± 0.1

0.79 ± 0.1

1550

0.05 ± <0.01

GMB 2h

<LOD/LOQ

0.68 ± 0.04

0.68 ± 0.04

1546

0.04 ± <0.01

GMB 24h

0.25

0.90 ± <0.01

0.65 ± <0.01

1546

0.04 ± <0.01

GST 2h

<LOD/LOQ

0.12 ± 0.02

0.12 ± 0.02

1554

0.01 ± <0.01

GST 24h

<LOD/LOQ

0.20 ± 0.02

0.20 ± 0.02

1554

0.01 ± <0.01

PBS 2h

<LOD/LOQ

0.74 ± <0.01

0.74 ± <0.01

1547

0.05 ± <0.01

PBS 24h

<LOD/LOQ

0.82 ± 0.03

0.82 ± 0.03

1547

0.05 ± <0.01

# (initial weight (e.g. 50 mg) * 1.53 § (percentage selenium in test item) / 100) * 2 (multiplication to calculate selenium amount in one litre -> 100 mg/L) = nominal selenium concentration in [mg/L] / 1000 = nominal selenium concentration in [µg/L]

§ according to CoA 1.53 % Se in test item

In five different artificial physiological media, between 0.01 and 0.05 % of selenium was dissolved from the test item IPC-2013-001 Silicic acid, zirconium salt, cadmium pigment-encapsulated depending on solution parameters and test duration.

Concentration of zirconium in artificial media, calculated nominal zirconium concentration in 100 mg/L silicic acid, zirconium salt, cadmium pigment-encapsulated and dissolved amount of zirconium.

media and sample

total Zr ± SD in method blanks [µg/L]

total Zr ±SD in sample vessels [µg/L]

Zr ± SD in sample vessels with blank subtraction [µg/L]

calculated nominal Zr concentration in [µg/L]#

dissolved amount of Zr in artificial media [%] normalized for measured background in method blank

ALF 2h

<LOD

0.52 ± 0.01

0.52 ± 0.01

45913

0.001 ± <0.001

ALF 24h

<LOD

1.25 ± 0.11

1.25 ± 0.11

45913

0.003 ± <0.001

ASW 2h

0.18 ± 0.13

0.07

-

46134

-

ASW 24h

0.47

0.13 ± 0.12

-

46134

-

GMB 2h

<LOD

<LOD

<LOD

46007

-

GMB 24h

<LOD

<LOD

<LOD

46007

-

GST 2h

<LOD

<LOD

<LOD

46244

-

GST 24h

<LOD

<LOD/LOQ

<LOD/LOQ

46244

-

PBS 2h

<LOD

<LOD

<LOD

46033

-

PBS 24h

<LOD

<LOD

<LOD

46033

-

# (initial weight (e.g. 50 mg) * 45.53 § (percentage zirconium in test item) / 100) * 2 (multiplication to calculate zirconium amount in one litre -> 100 mg/L) = nominal zirconium concentration in [mg/L] / 1000 = nominal zirconium concentration in [µg/L]

§ according to CoA 61.50 % Zr as ZrO2 => 74.03 % zirconium in ZrO2 => (74.03 % * 61.50 %) / 100 % = 45.53 % Zr in test item

In five different artificial physiological media, between 0.001 and 0.003 % of zirconium was dissolved from the test item IPC-2013-001 Silicic acid, zirconium salt, cadmium pigment-encapsulated depending on solution parameters and test duration.

Mass balance calculation

Total dissolved cadmium, selenium and zirconium concentrations in test samples measured by ICP-OES indicated no complete dissolution of Silicic acid, zirconium salt, cadmium pigment-encapsulated in all physiological media after addition of aqua the sample vessels. The concentrations for selenium and zirconium in mass balance samples and filter and syringe samples were below the LOD/LOQ.

Calculation of cadmium mass balance

media

value for dissolved Cd after addition of aqua regia
[mg]

nominal
concentration
[mg] #

recovery
[%]

ALF 24h A

0.01

2.33

0.33

ALF 24h B

0.01

2.37

0.32

ASW 24h A

0.01

2.35

0.42

ASW 24h B

0.01

2.37

0.41

GMB 24h A

0.01

2.36

0.43

GMB 24h B

0.01

2.35

0.39

GST 24 A

0.01

2.36

0.56

GST 24h B

0.01

2.37

0.55

PBS 24h A

0.01

2.34

0.39

PBS 24h B

0.01

2.37

0.39

# nominal concentration Cr = 4.66 % as Cd in test item => 2.33 mg Cd in 50 mg test item => 2.33 * initial weight / 50 mg

Conclusions:
The bioaccessibility of Silicic acid, zirconium salt, cadmium pigment-encapsulated has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation). Dissolved Cd, Zr and Se concentrations were below 6.13 µg/L, 1.25 µg/L and 0.82 µg/L, respectively, even at the highest loading of 0.1 g/L, representing a solubility of 0.006 %, 0.001 % and 0.0008 %, respectively. Therefore, silicic acid, zirconium salt, cadmium pigment-encapsulated may reasonably be considered not bioaccessible.
Executive summary:

The bioaccessibility of silicic acid, zirconium salt, cadmium pigment-encapsulated has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows:

- Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,

- Phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,

- Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,

- Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and

- Artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.

 

Under the conditions of this test (flasks with different artificial physiological media; loadings of 50 mg silicic acid, zirconium salt, cadmium pigment-encapsulated/ 500 mL, 37 °C, sampling after 2h and 24h), the concentrations of dissolved cadmium, selenium and zirconium were as follows: In five different artificial physiological media dissolved cadmium concentrations were between 0.001 and 0.13 %, dissolved selenium concentrations were between 0.01 and 0.05 % and dissolved zirconium concentrations were between 0.001 and 0.003 %, based on contained elements.

Therefore, Silicic acid, zirconium salt, cadmium pigment-encapsulated may reasonably be considered not bioaccessible.

Description of key information

The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Zr, Cd and Se contained in the pigment.
(1) The bioaccessibility of Zirconium zircon with encapsulated cadmium selenium sulphide has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation). Dissolved Cd, Zr and Se concentrations were below 6.13 µg/L, 1.25 µg/L and 0.82 µg/L, respectively, even at the highest loading of 0.1 g/L, representing a solubility of 0.006 %, 0.001 % and 0.0008 %, respectively. Therefore, Zirconium zircon with encapsulated cadmium selenium sulphide may reasonably be considered not bioaccessible.
(2) In a 28-day oral toxicity study with 1,000 mg/kg pigment 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 Zirconium zircon with encapsulated cadmium selenium sulphide.
(3) In a bioavailability study, a relative bioavailabilities of 0.032% or 0.033% (males or females) were calculated for cadmium within the test item Zirconium zircon with encapsulated cadmium selenium sulphide administered orally compared to intravenous administration of the reference item cadmium chloride.


4) In a mass balance study with a single dose of 1000 mg/kg of the pigment, 86.3 % cadmium (Cd) of the dose was excreted via urine and faeces during the first three days after exposure (mean for 10 animals), with only <0.000001 % of the dose being excreted via urine.


In conclusion, the oral relative bioavailability of the pigment "Zirconium zircon with encapsulated cadmium selenium sulphide" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by an in-vitro dissolution experiment in five different artificial physiological media.



A rounded value of <<0.05 % for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.05 % for Cd, Zr and Se) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.000001 % for Cd).


Absorption rate - oral: 0.05 %

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
0.05

Additional information

The toxicity data in this registration dossier refer explicitly to the pigment Zirconium zircon with encapsulated cadmium selenium sulphide and document its negligible bioavailability and the complete lack of any human health hazard. Experiments on the bioavailability of the pigment are summarised and discussed in this section.


 


Summary of in-vitro bioaccessibility experiments (Knopf, 2013)


The chemical and physiological properties of the pigment Zirconium zircon with encapsulated cadmium selenium sulphide are characterised by inertness because of the specific synthetic process (calcination at high temperatures, approximately 1000 °C), rendering the substance to be of a unique, stable crystalline structure in which all atoms are tightly bound and not prone to dissolution in environmental and physiological media. The bioaccessibility of Zirconium zircon with encapsulated cadmium selenium sulphide has been investigated experimentally in vitro by simulating dissolution under physiological conditions considered to mimic the most relevant exposure routes (oral, dermal and inhalation), as follows:


- Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions,


- Phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum,


- Artificial sweat (ASW, pH 6.5) which simulates the hypoosmolar fluid, linked to hyponatraemia (loss of Na+ from blood), which is excreted from the body upon sweating,


- Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions and


- Artificial gastric fluid (GST, pH 1.5), which mimics the very harsh digestion milieu of high acidity in the stomach.


Under the conditions of this test dissolved Cd, Zr and Se concentrations were below 6.13 µg/L, 1.25 µg/L and 0.82 µg/L, respectively, even at the highest loading of 0.1 g/L, representing a solubility of 0.006 %, 0.001 % and 0.0008 %, respectively. Therefore, Zirconium zircon with encapsulated cadmium selenium sulphide may reasonably be considered not bioaccessible.


Toxicokinetic screening data from a 28 day repeated dose oral toxicity study (Leuschner, 2021)


In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of the pigment "Zirconium zircon with encapsulated cadmium selenium sulphide" of 1,000 mg/kg bw/day via gavage. Individual urine samples were collected from all animals prior to sacrifice in one cumulated 24-h fraction/animal after the last oral application, and blood samples were collected from each animal upon sacrifice. The plasma and urine samples were analysed for total cadmiun, zirconium and selenium content.


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.


The cadmium, zirconium, and selenium concentrations of the 24h-urine samples, collected during the day before final sacrifice, ranged from:


Cd: 0.054 – 0.165 μg/L urine (mean: 0.099 μg/L ± 0.044 ) and 0.186 – 0.346 μg/L urine (mean: 0.226 μg/L ± 0.077), for the male and female animals of the control group, respectively, and from 0.085 – 0.147 μg/L urine (mean: 0.121 μg/L ± 0.024) and 0.106 – 0.305 μg/L urine (mean: 0.214 μg/L ± 0.099) for the male and female animals of the dose group (1000 mg Zirconium zircon with encapsulated cadmium selenium sulphide/kg b.w./day), respectively.


Zr: 0.217 – 0.878 μg/L urine (mean: 0.417 μg/L ± 0.271) and 0.384 – 2.88 μg/L urine (mean: 1.57 μg/L ± 1.14) for the male and female animals of the control group, respectively, and from 0.231 – 1.12 μg/L urine (mean: 0.601 μg/L ± 0.375) and 0.326 – 1.97 μg/L urine (mean: 1.01 μg/L ± 0.717) for the male and female animals of the dose group (1000 mg Zirconium zircon with encapsulated cadmium selenium sulphide /kg bw/day), respectively.


Se: 108 – 188 μg/L urine (mean: 149 μg/L ± 36.6) and 153 – 257 μg/L urine (mean: 212 μg/L ± 37.3), for the male and female animals of the control group, respectively, and from 177 – 318 μg/L urine (mean: 249 μg/L ± 69.6) and 280 – 455 μg/L urine (mean: 372 μg/L ± 74.9) for the male and female animals of the dose group (1000 mg Zirconium zircon with encapsulated cadmium selenium sulphide /kg bw/day), respectively.


The cadmium, zirconium, and selenium concentrations of plasma samples, collected from control and dose group animals at the day of sacrifice, were measured as follows:


Cd: 0.002 μg/L plasma (mean: 0.002 μg/L) and 0.002 μg/L plasma (mean: 0.002 μg/L), for the male and female animals of the control group, respectively, and from 0.002 – 0.002 μg/L plasma (mean: 0.002 μg/L) and 0.002 – 0.093 μg/L plasma
(mean: 0.02 μg/L) for the male and female animals of the dose group (1000 mg Zirconium zircon with encapsulated cadmium selenium sulphide /kg bw/day),
respectively.


Zr: 0.003 – 0.025 μg/L plasma (mean: 0.011 μg/L) and 0.003 – 0.008 μg/L plasma (mean: 0.005 μg/L) for the male and female animals of the control
group, respectively, and from 0.003 – 0.008 μg/L plasma (mean: 0.006 μg/L) and 0.003 – 0.014 μg/L plasma (mean: 0.009 μg/L) for the male and female
animals of the dose group (1000 mg Zirconium zircon with encapsulated cadmium selenium sulphide/day), respectively.


Se: 4.92 – 6.68 μg/L plasma (mean: 5.80 μg/L) and 4.32 – 7.76 μg/L plasma (mean: 5.33 μg/L), for the male and female animals of the control group,
respectively, and from 4.31 – 5.86 μg/L plasma (mean: 5.07 μg/L) and 4.63 – 6.02 μg/L plasma (mean: 5.21 μg/L) for the male and female animals of the dose group (1000 mg Zirconium zircon with encapsulated cadmium selenium sulphide/day), respectively.


From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of metals were found in the terminal 24-h urine collection period, as follows:


Cd: 0.00000016 % (males) and 0.0000016 % (females)


Zr: 0.0000007 % (males) and -0.0000029 % (females)


Se: 0.0155 % (males) and 0.0462 % (females)


Summary of relative bioavailability study (Leuschner 2021):


A absolute bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble Cd reference substance (cadmium chloride) compared to single oral doses of the same substances and the pigment was performed. For details, please refer to the corresponding robust study summary. In brief, 10 animals (5m/5f) per group received single doses of (1) 0.4 mg/kg cadmium chloride intravenously, (2) 38 mg/kg cadmium chloride, via oral gavage, and (3) 1000 mg/kg of the pigment via oral gavage.


Blood samples were taken at 0, 1, 2, 4, 8, 12, 24, 48 and 72 hours post exposure and blood plasma samples were prepared and analysed for the element Cd.


Cmax-levels in plasma of 0.0224 µg Cd/g and 0.0204 µg Cd/g, were noted 1 hour (tmaxas mean m/f) after intravenous administration of a mixture of 0.4 mg/kg cadmium chloride for the male and female rats on test day 1, respectively.


Furthermore, Cmax-levels of 0.1318 µg Cd/g and 0.1564 µg Cd/g were noted 1.2 or 1.0 hours (tmaxas mean m/f) after oral administration of 38 mg/kg cadmium chloride for the male and female rats on test day 1, respectively.


Lastly, Cmax-levels of 0.00046 µg Cd/g and 0.00044 µg Cd/g, were noted 0 - 1 hour (tmaxas mean m/f) after oral administration of 1000 mg pigment/kg for the male and female rats on test day 1, respectively.


The plasma concentrations declined post dosing with an elimination half-life ranging from 13.8 to 64.3 hours for Cd.


For Cd an absolute bioavailability of 4.81/ 4.89 % (m/f) calculated from soluble cadmium chloride following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.032/0.033 % (m/f) for Cd present in the pigment could be calculated.


In sum, experimentally determined plasma kinetic values for Cd are very consistent and the very low relative bioavailabilities of Cd from the pigment demonstrates that the pigment can be considered inert without any systemic hazard potential for human health.


Summary of comparative Mass-Balance Study (Leuschner, 2021)


In a comparative mass balance study involving oral dosing of (i) the inorganic pigment "Zirconium zircon with encapsulated cadmium selenium sulphide" and (ii) soluble salts of the elements contained therein (Cd2+), the gastrointestinal absorption as well as urinary and faecal excretion were compared. For details, please refer to the corresponding robust study summary.


In brief, 10 (5m/5f) animals per group received a single oral dose of 1000 mg/kg of the pigment or 38 mg/kg bw cadmium chloride (CdCl2; corresponding to 23.3 mg/kg Cd). A third group served as vehicle treated control. Animals were individually housed in metabolic cages and daily samples of urine and faeces were collected for three days. All samples were analysed for cadmium. The averaged “background” excretion via urine and faeces of the control animals was subtracted from the amounts excreted by the dosed animals, and a mass balance was calculated.


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.


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 cadmium (Cd) is essentially complete and indicates that Cd contained in the pigment " Zirconium zircon with encapsulated cadmium selenium sulphide", present as Cd2+ is not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.


Overall conclusion:


The in-vitro and in-vivo experiments described above are in very good agreement with regards to the negligible level of bioavailability of the elements Cd, Zr and Se contained in the pigment.


(1)   In in-vitro dissolution experiments in artificial gastric fluid,  dissolved Cd, Zr and Se concentrations were below 6.13 µg/L, 1.25 µg/L and 0.82 µg/L, respectively, even at the highest loading of 0.1 g/L, representing a solubility of 0.006 %, 0.001 % and 0.0008 %, respectively. Therefore, Zirconium zircon with encapsulated cadmium selenium sulphide may reasonably be considered not bioaccessible.


(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Cd, Zr and Se plasma and urine concentrations were observed when sampled at the end of the 28-day exposure period. From a final dose of 1,000 mg/kg of the pigment that the animals received on the last day of the study, only cumulated relative amounts of << 0.05 % (m/f) were found in the terminal 24-h urine collection period.


(3)   In a bioavailability study, absolute bioavailabilities of 0.032% or 0.033% (males or females) were calculated for cadmium within the test item Zirconium zircon with encapsulated cadmium selenium sulphide administered orally compared to intravenous administration of the reference item cadmium chloride.


(4) In a mass balance study with a single dose of 1000 mg/kg of the pigment, 86.3 % cadmium (Cd) of the dose was excreted via urine and faeces during the first three days after exposure (mean for 10 animals), with only <0.000001 % of the dose being excreted via urine.


In conclusion, the oral relative bioavailability of the pigment "Zirconium zircon with encapsulated cadmium selenium sulphide" can be assumed to be negligible, as demonstrated in three independent in-vivo studies in rats yielding very comparably results supported by an in-vitro dissolution experiment in five different artificial physiological media.


A rounded value of <<0.05 % for oral absorption can be taken forward from (i) terminal urine/plasma sampling in a study involving 28 repeated oral doses of 1,000 mg pigment/kg bw/d (<<0.05 % for Cd, Zr and Se) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.000001 % for Cd).


Absorption rate - oral: 0.05 %