Registration Dossier

Diss Factsheets

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because (i) the substance is of low toxicological activity (no evidence of toxicity seen in any of the tests available), (ii) it can be proven from toxicokinetic data that no systemic absorption occurs via relevant routes of exposure (e.g. plasma/blood concentrations below detection limit using a sensitive method and absence of the substance and of metabolites of the substance in urine, bile or exhaled air) and (iii) there is no or no significant human exposure
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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.
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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.
Reason / purpose for cross-reference:
data waiving: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-22 to 2015-08-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008-10-03
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
GLP certificate signed 2014-05-14
Limit test:
yes
Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
Rats were selected because of its proven suitability in toxicology studies and to comply with regulatory requirements for testing in a rodent animal species.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 55 days; females: 62 days
- Weight at study initiation: males: 291.6 g - 323.2 g; females: 222.6 g - 257.9 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 23 cm and a height of approx. 18 cm; bedding material: Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): Commercial ssniff® R/M-H V1530 diet (ssniff Spezialdiäten GmbG, 59494 Soest, Germany)
- Water (ad libitum): tap water
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C
- Humidity: 55 % ± 15 %
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
The route of administration was selected according to the expected route of exposure
Vehicle:
other: 0.8 % aqueous hydroxyl propyl methylcellulose gel
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was suspended in the vehicle to the appropriate concentration. The administration formulation was continuously agitated by stirring throughout the entire administration procedure. The administration formulation was freshly prepared every day. Administration volume: 10 mL/kg b.w./day. The amount of the test item was adjusted to each animal's current body weight daily.

VEHICLE
- Source: Fagron GmbH & Co. KG, 22885 Barsbüttel, Germany
- Batch no.: 13D03-N03
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For each test item that was mixed with a vehicle, tests by ICP-OES were conducted to determine the concentration, stability and homogeneity of the test item in the formulations (Fraunhofer IME, report no. EBR-214/6-27). For the analysis of the test item-vehicle mixtures, samples of approximately 10 mL were taken at the following times and stored at ≤-20°C until dispatch:
1) At study initiation:
- analysis of stability and concentration: Immediately after preparation of the administration formulation as well as after 8 and 24 hours storage at room temperature (number of samples: 3).
- homogeneity: At the start of administration, during (middle) administration and before administration to the last animal of group 2 (number of samples: 3).
2) at study termination:
- analysis of concentration: During treatment always before administration to the last animal of group 2 (number of samples: 1- sum of all samples: 7).

The samples were digested as follows:

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

Then the digested samples were measured by ICP-OES.

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

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

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

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

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

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

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

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

Anaylsis of concentration (1 sample):
Recovery [%]:
Cd:. 111
Zr: 87.7
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males/ 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dose selection rationale: In agreement with the Sponsor and based on available toxicity data a limit test was performed.
Positive control:
none
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule (clinical signs): before and after dosing at each time of dosing as well as regularly throughout the working day from 7:30 a.m. to 4.30 p.m. and on Saturdays and Sundays from 8.00 a.m. to 12.00 noon with a final check performed at approximately 4.00 p.m.
- Time schedule (mortality): early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays with a final check at approximately 4.00 p.m.
- Cage side observations checked: clinical signs and mortality

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

BODY WEIGHT: Yes
- Time schedule for examinations: at the time of group allocation, on the day of commencement of treatment and once a week thereafter, always on the same day of the week

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The quantity of food left by individual animals was recorded on a weekly basis throughout the experimental period. Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment week. The relative food consumption (in g/kg b.w./day) was determined
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily

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

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

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters examined: albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), urea (in blood), calcium, chloride, potassium, sodium, alanine amino-transferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in test week 4 approx. 1 to 2 hours after dosing and before any blood sampling
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity / grip strength / motor activity
1) Observational screening: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, pilo-erection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind-leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function
2) Functional tests: grip strength and locomotor activity

IMMUNOLOGY: No

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

HISTOPATHOLOGY: Yes

On test day 29 (approximately one day after the last administration), the animals were dissected following a randomisation scheme. The animals were sacrificed by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically
under the direction of a pathologist. All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart. The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded. The weights of the following organs of all animals were determined before fixation: Adrenal gland (2), Brain, Epididymis (2), Heart, Kidney (2), Liver, Ovary (2), Spleen, Testicle (2), Thymus, As a whole: Prostate and seminal vesicles with coagulating glands. Paired organs were weighed individually and identified as left or right.

The following organs or parts of organs of all animals were fixed in 7% buffered formalin. The eyes were fixed in Davidson's solution and the testes in Bouin's solution for optimum fixation:
Adrenal gland (2), Bone (os femoris with joint), Bone marrow (os femoris) Brain (3 levels: cerebrum, cerebellum, medulla/pons), Epididymis (2), Eye with optic nerve (2), Gross lesions observed, Heart (3 levels: right and left ventricle, septum), Intestine, large (colon, rectum), Intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches), Swiss roll method, Kidney and ureter (2), Liver, Lungs (with mainstem bronchi and bronchioles (preserved by inflation with fixative and then immersion)), Lymph node (1, cervical), Lymph node (1, mesenteric), Mammary gland, Muscle (skeletal, leg), Nerve (sciatic), Ovary (2), Pituitary, Prostate and seminal vesicles with coagulating glands, Spinal cord (3 sections), Spleen, Stomach, Testicle (2), Thymus, Thyroid (2) (incl. parathyroids), Tissue masses or tumours (incl. regional lymph nodes), Trachea (incl. larynx), Urinary bladder, Uterus (incl. cervix and oviducts), Vagina

The above-listed organs of all animals were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made, stained with Oil Red O and examined microscopically. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they were noted as grossly enlarged.
Statistics:
The test item-treated Group 2 was compared with the control Group 1.
The following statistical methods were used:
1) STUDENT's t-test All numerical functional tests / Body weight / Food consumption / Haematology and coagulation / Clinical biochemistry / Relative and absolute organ weights (p ≤ 0.05 and p ≤ 0.01)
The following limits were used:
p = 0.05/0.01 ^ t = 2.3060/3.3554 (for 8 degrees of freedom)

2) Exact test of R. A. FISHER Histology (p ≤ 0.05 and p ≤ 0.01)
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Details on results:
CLINICAL SIGNS:
- No adverse changes in behaviour, external appearance or faeces were noted for the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day or for the animals treated with the vehicle control once daily for 28 days.
- all male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day revealed reddish discoloured faeces as of test day 2. This finding is considered to be due to the test item per se being a neonred powder and, hence, is not an adverse effect.

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

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

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

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

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

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

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

BEHAVIOUR (FUNCTIONAL FINDINGS)
- The neurological screening performed at the end of the treatment period in test week 4 approximately 1 to 2 hours after dosing did not reveal any test item-related influence in the male and female rats treated with 1000 mg Silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day once daily for 28 days, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility. The examination results of the animals treated with the vehicle control were also in the normal range.

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

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

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

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

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

Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
NOAEL (oral; rats) > 1000 mg silicic acid, zirconium salt, cadmium pigment encapsulated/kg b.w./day

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

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

Data source

Materials and methods

Results and discussion

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion