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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-07-13 to 2015-07-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Objective of study:
toxicokinetics
Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
2010-07-22
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14

Test material

Constituent 1
Chemical structure
Reference substance name:
Cobalt zinc aluminate blue spinel
EC Number:
269-049-5
EC Name:
Cobalt zinc aluminate blue spinel
Cas Number:
68186-87-8
Molecular formula:
Co(x)Zn(1-x)Al2O4 0,1≤x≤0,9
IUPAC Name:
Cobalt zinc aluminate blue spinel
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Cobalt zinc aluminate blue spinel
- Physical state: Solid, blue powder, odourless
- Structure: spinel
- Storage condition of test material: Kept dry in closed containers
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, kept dry, and stored in a tightly closed container
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Details on species / strain selection:
The species was selected for this study design because 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: 53 days; females: 67 days
- Weight at administration: males: 260 - 284 g; females: 233 - 268 g
- Housing: kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm × 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany)
- Diet (ad libitum): certified 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)
- Relative humidity: 55% ± 15% (maximum range).
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
other: cobalt zinc aluminate blue spinel: oral (gavage); reference item (mixture of Al2(SO4)3; CoCl2; ZnSO4): oral (gavage) and intravenously injected
Vehicle:
other: cobalt zinc aluminate blue spinel: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (mixture of Al2(SO4)3; CoCl2; ZnSO4): water (oral administration) or 0.9 % NaCl solution (intravenous administration)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Cobalt zinc aluminate blue spinel:
The test item was suspended in the vehicle to the appropriate concentration freshly on the administration day.

2) Reference item (mixture of aluminium sulfate (purity: 99.99 %), zinc sulfate (purity: >99%), and cobalt chloride (purity: >98%)):
The components of the reference item were combined as follows:
a) oral administration:
aluminium sulfate: 360 mg/kg bw
zinc sulfate: 101 mg/kg bw
cobalt chloride: 67 mg/kg bw
b) intravenous administration
aluminium sulfate: 1.8 mg/kg bw
zinc sulfate: 0.5 mg/kg bw
cobalt chloride: 0.34 mg/kg bw
The mixture was dissolved in the respective vehicle for oral or intravenous injection.

The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
Administration volume (oral administration / intravenous administration): 10 mL/kg bw

Injection speed (intravenous adminsitration): dose per approx. 15 seconds

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


Duration and frequency of treatment / exposure:
single administration
Doses / concentrations
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose / concentration:
5 males / 5 females
Control animals:
no
Positive control reference chemical:
none
Details on study design:
- Dose selection rationale: the dose levels for this preliminary study had been selected after consultation with the Sponsor based on available toxicity data:
The oral LD50 values for the reference items were as follows:
1) aluminium sulfate: >2000 to < 6200 mg/kg bw
2) cobalt chloride: 293 to 418 mg/kg bw
3) zinc sulfate: 1710 mg/kg bw
Furthermore, the oral bioavailabilities of soluble Al, Co and Zn substances are given in the public domain with <1% (Al), 13 to 34% (Co) and approx. 40 to 50% (Zn), respectively.

The test item oral dose of 1000 mg/kg bw corresponded to the limit dose used in a separate 28-day oral toxicity study, and was considered the maximum feasible dose. Based on the chemical composition of the test item, a dose of 1000 mg/kg bw of cobalt zinc aluminate blue spinel equates to the following doses:
1) Al: 284 mg/kg bw (corresponding to 1802 mg aluminium sulfate/kg bw)
2) Co:152 mg/kg bw (corresponding to 336 mg cobalt chloride/kg bw)
3) Zn: 204 mg/kg bw corresponding to 505 mg zinc sulfate/kg bw)
In view of proximity of this dosing to the oral LD50 for cobalt chloride, these doses were reduced to 20% of the initial values, i.e. 360 mg aluminium sulfate/kg bw, 67 mg cobalt chloride/kg bw and 101 mg zinc sulfate/kg bw, respectively.

The dosage for the reference item administered by intravenous injection was set 10% 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 doses as follows:
Al: 28 mg/kg bw (corresponding to 180 mg aluminium sulfate/kg bw)
Co: 15 mg/kg bw (corresponding to 34 mg cobalt chloride/kg bw)
Zn: 21 mg/kg b.w (corresponding to 51 mg zinc sulfate/kg bw)

The dose levels for the reference item were confirmed in two preliminary experiments each employing two animals per group:
1) First preliminary experiment:
Both animals treated once orally with a mixture composed of 360 mg aluminium sulfate, 67 mg cobalt chloride and 101 mg zinc sulfate/kg bw revealed slight pilo-erection starting approx. 15 minutes after administration lasting for 4 hours. Further, both animals treated once intravenously with a mixture composed of 180 mg aluminium sulfate, 34 mg cobalt chloride and 51 mg zinc sulfate/kg bw died during the injection.

2) Second preliminary experiment
Both animals treated once intravenously with 63.6 mg aluminium sulfate/kg bw revealed convulsions, lateral position and laboured breathing, and died within 2 minutes of the injection.
For both animals treated once with 22 mg cobalt chloride/kg bw lateral position, laboured breathing and convulsions were observed starting immediately after the intravenous injection. The male animal died 3 minutes and the female animal died 10 minutes after injection.
Both animals treated once intravenously with 24.7 mg Zinc sulfate/kg b.w. revealed convulsions and died immediately after injection.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: plasma
- Time and frequency of sampling: blood was collected 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 plasma was frozen at -80°C, and stored at this temperature until analysis.
In addition, 4 mL pooled blank plasma (approx. 2 mL per sex) were obtained from spare animals.

A toxicokinetic evaluation of the data on cobalt zinc aluminate blue spinel plasma levels will be performed. A non-compartment model will be employed. The following parameters will be determined:
AUC0-inf = extrapolated area from zero to infinity
AUC0-t last = extrapolated area from time zero to the last quantifiable plasma concentration >LLOQ
Kel = elimination rate constant
t½ = elimination half-life

Cmax values will be the highest measured plasma concentrations, and tmax values will be the time points of highest plasma concentrations.

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

Area under the curve (AUC) values will be 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 will be taken to be those at the first blood sampling time.

Furthermore, AUC0-t last-values will be calculated according to the linear trapezoidal rule. Values below or at the lower limit of quantification (LLOQ) will be excluded from the calculation.

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 of each test and reference item that was mixed with a vehicle were stored at ≤- 20°C (approximately 5 mL per formulation, in total 3 samples).
Statistics:
The reference item-treated group (oral administration) was compared to the test item treated group.
The following statistical method was used:
STUDENT's t-test: body weight (p ≤ 0.01 and p ≤ 0.05)
The following limits were used:
p = 0.05 / 0.01 about t = 2.3060 / 3.3554
(for 8 degrees of freedom)

Results and discussion

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

Toxicokinetic / pharmacokinetic studies

Toxicokinetic parameters
Toxicokinetic parameters:
other: bioavailability
Remarks:
relative bioavailability for Co: approximately 0.009% for Co present in the pigment. relative bioavailability for Al: 0.171/0.104% (m/f) for Al present in the pigment. relative bioavailability for Zn: 0.09/0.06% (m/f) for Zn present in the pigment.

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:
An absolute bioavailability of 23.4/11.8 % (m/f) was calculated for Co from soluble CoCl2 following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.009 % for Co present in the pigment.

For Al an absolute bioavailability of 0.37/0.47% (m/f) calculated from soluble Al2(SO4)3 following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.171/0.104% (m/f) for Al present in the pigment.

An absolute bioavailability of 0.45/0.40 % (m/f) was calculated for Zn from soluble ZnSO4 following oral administration compared to intravenous administration, and a relative bioavailability of approximately 0.09/0.06% (m/f) for Zn present in the pigment.

Any other information on results incl. tables

LOCAL TOLERANCE (REFERENCE ITEM; INTRAVENOUS ADMINISTRATION)

None of the animals treated with a single intravenous dose of the reference item revealed any signs of local intolerance reactions at the injection sites as observed during daily visual inspections.

CLINICAL SIGNS, MORTALITY, AND BODY WEIGHT

Cobalt zinc aluminate blue spinel:

- no abnormalities were observed in behaviour or external appearance for any animal treated

- faeces of all animals were normally formed. A bluish discolouration was noted for the faeces of 3/5 animals per sex treated with the test item approx. 8 hours after administration. The discolouration of the faeces is related to the deep blue colour of the test item per se which is due to its cobalt salt compound and is not an adverse effect.

- no deaths were noted.

- body weight of all animals was in the expected range on test day 1.

Reference item (oral administration):

- all animals treated revealed slightly reduced motility starting approx. 10 minutes after administration lasting for approximately 30 minutes. This effect is considered to be related to the reference item.

- faeces of all animals were normally formed.

- no deaths were noted.

- body weight of all animals was in the expected range on test day 1.

Reference item (intravenous administration):

- no abnormalities were observed in behaviour or external appearance for any animal treated

- faeces of all animals were normally formed.

- no deaths were noted.

- body weight of all animals was in the expected range on test day 1.

Cmax-levels in plasma of 2.29 µg Al/g and 1.31 µg Al/g, 0.29 µg Co/g and 0.27 µg Co/g and 4.72 µg Zn/g and 2.48 µg Zn/g were noted 1 or 2 hours (tmaxas mean m/f) after intravenous administration of a mixture of 1.8 mg/kg Al2(SO4)3, 0.34 mg/kg bw CoCl2and 0.5 mg/kg bw ZnSO4for the male and female rats on test day 1, respectively.

Furthermore, Cmax-levels of 1.12 µg Al/g and 1.25 µg Al/g, 2.12 µg Co/g and 1.48 µg Co/g and 4.76 µg Zn/g and 6.19 µg Zn/g were noted 2, 4 or 2 hours (tmaxas mean m/f) after oral administration of a mixture of 360 mg/kg Al2(SO4)3, 67 mg/kg bw CoCl2and 101 mg/kg bw ZnSO4for the male and female rats on test day 1, respectively.

Lastly, Cmax-levels of 2.51 µg Al/g and 4.88 µg Al/g, 0.016 µg Co/g and 0.018 µg Co/g and 4.23 µg Zn/g and 1.93 µg Zn/g were noted 6, 1 and 3 hours (tmax as mean m/f) after oral administration of 1000 mg pigment/kg for the male and female rats on test day 1, respectively. For comparison, the average (n=30) concentration of cobalt in plasma taken before exposure at t=0 h was 0.0206 µg Co/g, for aluminium the mean blank plasma concentration was 1.41 µg Al/g and for zinc the mean blank plasma concentration was 1.96 µg Zn/g plasma.

The plasma concentrations declined post dosing with an elimination half-life ranging from 4.3 to 47 hours for Al, 4.4 to 41 hours for Co and from 6.1 to 43.8 hours for Zn.

TEST ITEM FORMULATION ANALYSIS

Concentration of aluminium, cobalt and zinc in application solution:

1) aluminium:

Nominal concentration: 383 mg

Actual concentration: 346 mg

Recovery [%]: 90.2 %

2) cobalt:

Nominal concentration: 206 mg

Actual concentration: 182 mg

Recovery [%]: 88.6 %

3) zinc:

Nominal concentration: 276 mg

Actual concentration: 241 mg

Recovery [%]: 87.3 %

Applicant's summary and conclusion

Conclusions:
In a relative bioavailability study, the relative bioavailability of orally administered pigment was calculated at 0.009% (Co), 0.27% (Al) and 0.09% (Zn) in relation to a mixture of soluble Al3+, Co2+ and Zn2+ compounds (Al2(SO4)3, CoCl2 and ZnSO4) injected i.v..
Executive summary:

In conclusion, the relative bioavailability of the pigment "Cobalt zinc aluminate blue spinel" (oral route) can be assumed to be negligible. The relative bioavailability of orally administered pigment was calculated at 0.009% (Co), 0.27% (Al) and 0.09% (Zn) in relation to a mixture of soluble Al3+, Co2+ and Zn2+ compounds (Al2(SO4)3, CoCl2 and ZnSO4) injected i.v..