Registration Dossier

Diss Factsheets

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

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
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)
Remarks:
signed 2014-05-14
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
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
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
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)

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

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 %

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..

Endpoint:
basic toxicokinetics, other
Remarks:
mass balance
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-01-18 to 2015-01-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Objective of study:
other: mass balance
Qualifier:
according to guideline
Guideline:
OECD Guideline 417 (Toxicokinetics)
Version / remarks:
2010-07-22
Deviations:
no
Principles of method if other than guideline:
Groups of 5 male and 5 female Crl:CD(SD) rats were dosed orally with a singel administration of cobalt zinc aluminate blue spinel, vehicle (0.8 % aqueous hydroxyl propyl methylcellulose gel), or reference item (mixure composed of aluminium sulfate, cobalt chloride, and zinc sulfate) via gavage. Clinical signs, mortality and body weight were recorded. Furthermore, urine and faeces of all animals were collected in metabolic cages after administration (sampling period: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours). Lastly, gross pathology was conducted and organ weights were determined.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2014-05-14
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
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 Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at dosing: males: 48 days; females: 56 days
- Weight at dosing: males: 228.1 - 254.6 g; females: 201.2 - 221.5 g
- Housing (exception: sampling period): 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): commercial ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany)
- Water (ad libitum): drinking water
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 3°C (maximum range)
- Relative humidity: 55% ± 15% (maximum range).
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: cobalt zinc aluminate blue spinel: 0.8 % aqueous hydroxyl propyl methylcellulose gel; reference item (mixture of Al2(SO4)3; CoCl2; ZnSO4): water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
1) Cobalt zinc aluminate blue spinel:
The test item formulation was freshly prepared on the administration day by dissolving the test item in the vehicle to the appropriate concentration. During and after preparation the formulation was stirred and the homogeneity of the suspension was checked by visual appraisal.
Administration volume: 10 mL/kg bw
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount of the test item was adjusted to the animal's current body weight 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:
aluminium sulfate: 360 mg/kg bw
zinc sulfate: 101 mg/kg bw
cobalt chloride: 67 mg/kg bw
The reference item formulation was freshly prepared on the administration day by dissolving the reference item in the vehicle to the appropriate concentration. During and after preparation the formulation was stirred and the homogeneity of the suspension was checked by visual appraisal.
Administration volume: 10 mL/kg bw
The administration formulations were continuously agitated by stirring throughout the entire administration procedure.
The amount 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:
yes, concurrent vehicle
Positive control reference chemical:
none
Details on study design:
- Dose selection rationale: the dose levels for this study have 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, oral bioavailabilities of soluble Al, Co and Zn substances are given in the public domain as <1% (Al), 13 - 34% (Co) and approx. 40 - 50% (Zn), respectively.

The oral test item 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 Cobalt zinc aluminate blue spinel/kg bw 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)
As the intended dose of cobalt chloride is in the range of the oral LD50 value, these doses were reduced to 20% of these initial values as follows:
1) aluminium sulfate: 360 mg/kg bw
2) cobalt chloride: 67 mg/kg bw
3) zinc sulfate: 101 mg/kg bw

The dose level for the reference item will be confirmed in a preliminary experiment employing two animals (please also refere to IUCLID Section 7.1.1: k_Leuschner_2017_ in vivo). In this preliminary experiment, both animals were treated once orally with a mixture composed of 360 mg Al2(SO4)3/kg bw, 67 mg CoCl2/kg bw and 101 mg ZnSO4/kg bw and revealed slight pilo-erection starting approx.15 minutes p.a. lasting for 4 hours.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine and faeces
- Time and frequency of sampling: all animals of the test item, vehicle and reference item groups were scheduled for urine and faeces sampling. After the single administration, the animals were kept in metabolism cages. Urine and faeces were collected in 3 fractions/animal (sampling periods: 0 - 24 hours, 24 - 48 hours, and 48 - 72 hours).
The urine and faeces weight per collection fraction and animal were determined upon removal of the sample fraction.
All samples were frozen at -20°C or colder and stored at this temperature until analysis.

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 PATHLOLOHY / 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 was removed to allow observation of the brain, pituitary gland, and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole. The stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intraabdominal lymph nodes, and accessory reproductive organs were recorded.

The weight of the following organs was determined: adrenal gland (2), brain, heart, kidney (2), liver, lungs, lymph nodes (cervical (1), mesenteric (1)), ovary (2), pituitary, prostate, spleen, testicle (2), thymus, and thyroid (1) (including parathyroids).
Paired organs were weighed individually and identified as left or right.

TEST ITEM FORMULATION ANALYSIS
Remaining administration formulations (approx. 5 mL) of each test and reference item that were mixed with a vehicle were stored at ≤-20°C until analysis (number of samples: 2).
Statistics:
The test item-treated and reference item-treated groups were compared statistically to the vehicle control group.
The following statistical method was used:
Multiple t-test based on DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics, 482-491 (Sept 1964): body weight / relative and absolute organ weights (p ≤ 0.01 and p ≤ 0.05)
Preliminary studies:
Please refer to the field "Details on study design" above.
Type:
absorption
Results:
Absorption rate - oral: 0.01 %
Details on absorption:
Urinary excretion for the elements Co, Zn and Al was negligible and below 0.01% for Zn, <0.006% for Co and 0% for Al. Absorption rate - oral: 0.01 %
Details on excretion:
Animals that received 1000 mg pigment /kg bw excreted 97.4 % Co, 105% Al and 100% Zn 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 93% of Al, 90% of Co and 91% of Zn were excreted via faeces as largest fraction. Further 10.3% and 1.7% (Al), 7.6% and 0.22%(Co) as well as 8.3% and 1.1% (Zn) were excreted via faeces on the second and third day.
Urinary excretion for all three elements was negligible and below 0.01% for Zn, <0.0036% for Co and 0% for Al.

CLINICAL SIGNS, MORTALITY, BODY WEIGHT, GROSS PATHOLOGY

Vehicle control group:

- no signs of systemic intolerance was observed

- faeces of control animals were normally formed.

- none of the rats died prematurely.

- individual body weights ranged from 228.1 to 247.3 g for the males and from 210.1 g to 221.5 g for the females on test day 1 and were within the expected range.

- no influence was noted on the body weight at autopsy.

- no pathological findings were recorded

Cobalt zinc aluminate blue spinel:

- none of the rats treated with the test item showed any changes in behaviour or external appearance.

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

- none of the rats died prematurely.

- individual body weights ranged from 239.1 to 254.6 g for the males and from 201.2 g to 216.1 g for the females on test day 1 and were within the expected range.

- no influence was noted on the body weight at autopsy.

- no test-item-related changes were noted for the animals treated with the test item at macroscopic inspection at necropsy.

- no test-item-related changes in relative and absolute organ weights were noted for the animals treated with the test item.

- statistically significant differences in organ weights compared to vehicle control group which are not considered to be test item-related are as follows:

females (test day 4): decreased relative brain weight (p ≤ 0.05)

Reference item:

- none of the rats treated with the reference item showed any changes in behaviour or external appearance.

- none of the rats died prematurely.

- individual body weights ranged from 235.1 to 245.3 g for the males and from 203.3 g to 220.4 g for the females on test day 1 and were within the expected range.

- no influence was noted on the body weight at autopsy.

- no reference item-related changes were noted for the animals treated with the reference item at macroscopic inspection at necropsy.

- an enlarged cervical lymph node was observed for 1/5 female animals treated with the reference item (coincidental finding).

- no reference item-related changes in relative and absolute organ weights were noted for the animals treated with the reference item.

- statistically significant differences in organ weights compared to vehicle control group which are not considered to be reference item-related are as follows:

females (test day 4): decreased relative brain weight and increased mesenteric absolute lymph node weight (p≤ 0.05)

Conclusions:
The mass balances for Co, Al and Zn are essentially complete and indicate that the elements contained in the pigment "cobalt zinc aluminate blue spinel", present as Co2+, Al3+and Zn2+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.

For cobalt only a minor fraction of 2.6% remains unaccounted. It may be considered that delayed excretion of the whole dose is reasonable since at 72h still ~0.22% of the cobalt dose (mean, mainly female animals) was excreted via faeces. Further it may be considered that the actually received dose did not fully correspond to the nominal dose as calculated. These aspects were not further addressed within the context of this study.

Zinc is part of the diet (ca. 90mg/kg diet) and is added therein as bioavailable/soluble zinc. It is questionable whether the determination of ca. 0.01% Zn in urine during the first day of exposure is a result of zinc absorbed from the pigment or is presumably caused by bioavailable zinc from the diet. This assumption is supported by the urine measurement results for zinc for the first 24 hours. The control group excreted a mean of ca. 590 µg Zn/L within the first 24 hours whereas the dosing group (1000 mg pigment/kg bw/day) excreted a nearly similar mean of 607 µg Zn/L which indicates that there is no difference in zinc uptake between dosing and control group.
Executive summary:

Animals that received 1000 mg pigment /kg bw excreted 97.4 % Co, 105% Al and 100% Zn 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 93% of Al, 90% of Co and 91% of Zn were excreted via faeces as largest fraction. Further 10.3% and 1.7% (Al), 7.6% and 0.22%(Co) as well as 8.3% and 1.1% (Zn) were excreted via faeces on the second and third day.

Urinary excretion for all three elements was negligible and below 0.01% for Zn, <0.006% for Co and 0% for Al.

The mass balances for Co, Al and Zn are essentially complete and indicate that the elements contained in the pigment "cobalt zinc aluminate blue spinel", present as Co2+, Al3+and Zn2+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Objective of study:
other: Bioaccessibility
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 2 h and 24 h; determination of Co, Al and Zn concentrations after filtration by AAS-GF and ICP-OES.
Principles of method if other than guideline:
Solubility of test item in simulated human fluids. Principle of test is similar to Transformation/Dissolution testing according to OECD Series 29 (2001)
GLP compliance:
no
Species:
other: in vitro (simulated human body fluids)
Details on exposure:
The test item was exposed to five different test media at a pH range from 1.7 to 7.4. The following synthetic biological fluids were used:
• Gamble’s solution (GMB, pH 7.4) which mimics the interstitial fluid within the deep lung under normal health conditions (de Meringo et. Al. 1994).
• Phosphate-buffered saline (PBS, pH 7.2), which is a standard physiological solution that mimics the ionic strength of human blood serum. It is widely used in the research (e.g. Norlin et al, 2002) and medical health care community (e.g. Hanawa et al, 2004, Okazaki and Gotoh, 2005) as a reference test solution for comparison of data under simulated physiological conditions.
• 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. The fluid is recommended in the available standard for testing of nickel release from nickel containing products (EN 1811, 1998).
• Artificial lysosomal fluid (ALF, pH 4.5), which simulates intracellular conditions in lung cells occurring in conjunction with phagocytosis and represents relatively harsh conditions (Moss 1979).
• Artificial gastric fluid (GST, pH 1.7), which mimics the very harsh digestion milieu of high acidity in the stomach (Hamel et al, 1998; ASTM, 2003).

The test media were selected in order to simulate human exposure as far as possible, e.g. skin contact. Ingestion to the gastro-intestinal tract can either be direct, or previously inhaled particles can be translocated from the respiratory tract to the gastro-intestinal tract by mucociliary clearance. It should be stressed though, that the different test media only simulate physiological conditions to a limited extent, as the complexity and function of the real body fluids are difficult to simulate. However, in vitro results in such synthetic biological media can, in a simple way, provide information that could be relevant for a real situation.

The test solutions were prepared using ultra-pure water and chemicals of analytical grades.

The pH of ALF and GMB was adjusted using 50 % NaOH and 25 % HCl, respectively. The pH of ASW and PBS was adjusted with 1 % ammonia solution and 50 % NaOH, respectively.

Artificial gastric fluid, pH 1.6, was prepared according to the ASTM standard using 4 g of 25% HCl solution diluted with ultra-pure water to 1 L (ASTM D5517, 2003).
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:
Experimental Procedure
Triplicate samples were prepared for exposure in different test media, each for two different time periods. In addition, one blank sample (without addition of test item) containing only the test solution was incubated together with the triplicate samples for each time period. 5 ± 0.5 mg of the test item was weighed using a Mettler AT20 balance with readability of 2 μg, and placed in a PMP Nalgene® jar. 50 mL of the test solution (no adjustment of solution volume to powder mass was made) was then added to the Nalgene® jar containing the test item, before incubated in a Platform Rocker incubator SI 80 regulated at 37 ± 2 °C. The solution was gently shaken (bi-linearly) with an intensity of 25 cycles per minute for 2 and 24 hours, respectively.
Details on dosing and sampling:
A “standard loading” of 0.1 g/L was selected, which has some physiological relevance. It further allows a comparison of the generated data with results from the OECD Transformation/Dissolution test (OECD, 2001) and similar bioaccessibility tests conducted with other materials under the same conditions .

The time periods for exposure of the test item were selected to have some relevance to the inhalation/ingestion scenario and to enable comparison with other reported metal release/dissolution data generated for similar time periods. The approximate time for the gastric phase of digestion is about 2 hours, and therefore this exposure time period was considered relevant for testing in artificial gastric fluid (Hamel et al, 1998). The
24 hour exposure was selected as a standard time duration that is relatively easy to compare with existing metal release/dissolution data as well as toxicity data for further evaluation of the bioaccessibility of released metals. Moreover, it can be assumed that human exposure to particles last no longer than 24 hours at ambient conditions.

After exposure, the samples were allowed to cool to ambient room temperature before the final pH of the test solution was measured. The test medium was then separated from the powder particles by centrifugation at 10000 rpm for 10 minutes, resulting in a visually clear supernatant with remaining particles in the bottom of the centrifuging tube. Dynamic light scattering, (Malvern Zetasizer nano ZS instrument) was used to confirm the successful removal of all pigment particles. The supernatant solution was decanted into a polypropylene storage flask and acidified to a pH less than 2 (not needed in the case of artificial gastric fluid) with 65 % pure HNO3 prior to solution analysis
Type:
other: Bioaccessibility
Results:
Highest dissolution at a loading of 0.1 g/L after 24 hours: Al: 120 ± 6.5 µg/L (GST), Co: 10.7 ± 0.5 µg/L (GST), Zn: 28.3 ± 2.2 µg/L (GST); after 2 hours: Al: 78.1 ± 10.9 µg/L (GST); Co: 14 ± 4 µg/L (ALF); Zn: 19.9 ± 2.9 µg/L (ASW)

BET-analysis:

The specific surface area, measured by BET-analysis is 5.92 m²/g. It should be underlined that this specific surface area is measured by nitrogen absorption and includes also the surface of surface pores.

Abbreviations:

GMB: Gamble´s solution, PBS: phosphate buffered saline, ASW: artificial sweat, ALF: artificial lysosomal fluid, GST: gastric fluid.

Average total concentration of released elements [μg/L] and the standard deviation of triplicate samples in the different media. Blank values for each individual media and exposure period have been subtracted

(test item) Exposure  GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
AlCoZn 2 h 14.8 ± 4.6 13.3 ± 3.8 39.7 ± 13.3 58.5 ± 7.  78.1 ± 10.9
Al release 24 h 22.8 ± 4.8 16.7 ± 2.8 17.4 ± 5.5 74.0 ± 8.0 120 ± 6.5
AlCoZn 2 h 10.6 ± 11.0 2.7 ± 2.3 7.1 ± 2.5 14.1 ± 4 7.6 ± 0.6
Co release 24 h 0.7 ± 0.7 1.5 ± 1.2 4.5 ± 1.0 5.2 ± 0.2 10.7 ± 0.5
AlCoZn 2 h 6.4 ± 3.6 10.0 ± 3.7 19.9 ± 2.9 18.6 ± 2.9 18.2 ± 2.8
Zn release 24 h 4.9 ± 6.4 9.1 ± 3.0 20.8 ± 8.2 20.5 ± 1.1 28.3 ± 2.2

Release rate of elements given by the BET surface area [μg/cm².h]

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
AlCoZn 2 h 0.0013 ± 0.0004 0.0012 ± 0.0004 0.0035 ± 0.0012 0.0050 ± 0.0008 0.0067 ± 0.001
Al release 24 h 0.0002 ± 0.00004 0.0001 ± 0.00002  0.0001 ± 0.00004 0.0005 ± 0.0001 0.0008 ± 0.00002
AlCoZn 2 h 0.0009 ± 0.001 0.0002 ± 0.0002 0.0006 ± 0.0002 0.0012 ± 0.0004 0.0006 ± 0.00004
Co release 24 h 0.00001 ± 0.00001 0.00001 ± 0.00001 0.00003 ± 0.00001 0.00004 ± 0.00000 0.00007 ± 0.00000
AlCoZn 2 h 0.0006 ± 0.0003 0.0009 ± 0.0003 0.0017 ± 0.0003 0.0016 ± 0.0002 0.0034 ± 0.0032
Zn release 24 h 0.00004 ± 0.00005 0.0001 ± 0.00002 0.0001 ± 0.00006 0.0002 ± 0.00001 0.0002 ± 0.00002

Released/dissolved amount of elements per total amount of loaded material [μg/μg]

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
AlCoZn 2 h 0.0002 ± 0.00005 0.0001 ± 0.00004 0.0004 ± 0.0001 0.0006 ± 0.00009 0.0008 ± 0.0001
Al release 24 h 0.0002 ± 0.00005 0.0002 ± 0.00003 0.0002 ± 0.00005 0.0008 ± 0.00008 0.0011 ± 0.00003
AlCoZn 2 h 0.0001 ± 0.0001 0.00003 ± 0.00002 0.00007 ± 0.00003 0.0001 ± 0.00004 0.00008 ± 0.000005
Co release 24 h 0.00001 ± 0.00001 0.00002 ± 0.00001 0.00004 ± 0.00001 0.00005 ± 0.000002 0.0001 ± 0.000003
AlCoZn 2 h 0.00007 ± 0.00004 0.0001 ± 0.00004 0.0002 ± 0.00003 0.0002 ± 0.00002 0.0004 ± 0.0004
Zn release 24 h 0.00005 ± 0.00007 0.0001 ± 0.00003 0.000 ± 0.00008 0.0002 ± 0.00001 0.0003 ± 0.00003

Elements transformed [mass %], equivalent to their percentage of the elemental content of the total amount of particles loaded; shown as average of triplicate samples in the different media. Blank values for each individual media and exposure period have been subtracted

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
AlCoZn 2 h 0.015 ± 0.0050 0.014 ± 0.0042 0.041 ± 0.014 0.059 ± 0.0093 0.077 ± 0.0082
Al release 24 h 0.024 ± 0.0053 0.018 ± 0.0030 0.017 ± 0.0051 0.071 ± 0.0082 0.27 ± 0.0073
AlCoZn 2 h 0.010 ± 0.011 0.0028 ± 0.0024 0.0073 ± 0.0026 0.014 ± 0.0043 0.0076 ± 0.0005 
Co release 24 h 0.0007 ± 0.0007 0.0016 ± 0.0013 0.0043 ± 0.0009 0.0054 ± 0.0002 0.011 ± 0.0003
AlCoZn 2 h 0.0066 ± 0.0037 0.010 ± 0.0040 0.021 ± 0.0030 0.019 ± 0.0021 0.040 ± 0.038
Zn release 24 h 0.0052 ± 0.0068 0.0096 ± 0.0032 0.020 ± 0.0079 0.021 ± 0.0010 0.028 ± 0.0025

Total released/dissolved amount of elements per total amount of loaded material [μg/μg] in %.

   Exposure GMB PBS ASW ALF GST
Test item time pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
AlCoZn 24 h 0.030 ± 0.013 0.029 ± 0.008 0.041 ± 0.010 0.10 ± 0.01 0.16 ± 0.006

Elements transformed [mass %], equivalent to the percentage of the released element compared to its amount within the amount of particles loaded.

(test item) Exposure GMB PBS ASW ALF GST
Material period pH 7.4 pH 7.2 pH 6.5 pH 4.5 pH 1.7
AlCoZn 2 h 0.054 ± 0.017 0.048 ± 0.015 0.14 ± 0.049 0.21 ± 0.033 0.28 ± 0.038
Al release 24 h 0.084 ± 0.019 0.062 ± 0.011 0.059 ± 0.018 0.27 ± 0.029 0.41 ± 0.0011
AlCoZn 2 h 0.072 ± 0.074 0.019 ± 0.016 0.048 ± 0.017 0.094 ± 0.029 0.050 ± 0.0030
Co release 24 h 0.0047 ± 0.0049 0.011 ± 0.0086 0.0028 ± 0.0058 0.035 ± 0.0015 0.069 ± 0.0018
AlCoZn 2 h 0.032 ± 0.018 0.051 ± 0.019 0.10 ± 0.015 0.092 ± 0.010 0.20 ± 0.18
Zn release 24 h 0.025 ± 0.033 0.047 ± 0.015 0.097 ± 0.039 0.10 ± 0.005 0.14 ± 0.012
Conclusions:
As dissolved Co, Zn and Al concentrations were below 14 µg/L, 120 μg/L and 28 µg/L, respectively, even at the highest loading of 0.1 g/L, corresponding to a solubility of 0.014 %, 0.12 % and 0.028 %, respectively, this pigment may reasonably be considered biologically inert.
Executive summary:

The bioaccessibility of Cobalt zinc aluminate blue spinel 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.

The dissolution of cobalt of the test item cobalt zinc aluminate blue spinel at a loading of 0.1 g/L is in a range of 2.7 µg/L (PBS) and 14 µg/L (ALF) after 2 hours and a range of 0.7 µg/L (GMB) and 10.7 µg/L (GST) after 24 hours.

The dissolution of aluminium of the test item cobalt zinc aluminate blue spinel is at a loading of 0.1 g/L is in a range of 13.3 µg/L (PBS) and 78.1 µg/L (GST) after 2 hours and a range of 16.7 µg/L (PBS) and 120 µg/L (GST) after 24 hours.

The dissolution of zinc of the test item cobalt zinc aluminate blue spinel is at a loading of 0.1 g/L is in a range of 6.4 µg/L (GMB) and 19.9 µg/L (ASW) after 2 hours and a range of 4.9 µg/L (GMB) and 28.3 µg/L (GST) after 24 hours.

In conclusion, since the dissolved Co, Zn and Al concentrations from this pigment were below 120 μg/L even at the highest loading of 0.1 g/L, corresponding to a solubility of less than 0.2 %, this pigment may reasonably be considered biologically inert.

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 Co, Al and Zn contained in the pigment.

(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Co, Zn and Al concentrations from this pigment were below 120 μg/L even at the highest loading of 0.1 g/L, corresponding to a solubility of less than 0.2 %.

(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Al and Zn plasma and urine concentrations and only a small increase in Co plasma and urine concentrations was 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 < 0.003 % (m/f) were found in the terminal 24-h urine collection period.

(3)   In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 105 % Al, 97.35 % Co and 100.16 % Zn of the dose were excreted via faeces within 3 days, with only <0.01 % of the dose being excreted via urine at the same time.

(4)   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..

Comparing the findings of in-vitro dissolution testing (1) with in-vivo results (2-4), the in-vivo data consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding that in-vitro experiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.

In conclusion, the oral relative bioavailability of the pigment "Cobalt zinc aluminate blue spinel" 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 <0.01 % 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 (<<0.003 % for all three metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.01 % for Zn, <0.0036 % for Co and 0 % for Al).

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential
Absorption rate - oral (%):
0.01

Additional information

The toxicity data in this registration dossier refer explicitly to the pigment cobalt zinc aluminate blue spinel 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 (Herting, Wallinder, 2010)


The chemical and physiological properties of the pigment cobalt zinc aluminate blue spinel 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. This manufacturing process leads to a very low bioaccessibility of the elements contained in the pigment. This 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:


 


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


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


3.) 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,


4.) 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


5.) Artificial gastric fluid (GST, pH 1.7), which mimics the very harsh digestion milieu of high acidity in the stomach.


 


The dissolution of cobalt of the test item cobalt zinc aluminate blue spinel is at a loading of 0.1 g/L in a range of <3 (pH 7.2) and 7.6 µg/L (pH 1.7) after 2 hours and below 11 µg/L after 24 hours. A value of 10.6 µg/L was measured at pH 7.4 after 2 hours. The dissolution of aluminium of the test item cobalt zinc aluminate blue spinel is at a loading of 0.1 g/L in a range of <15 (pH 7.2 / 7.4) and 79 µg/L (pH 1.7) after 2 hours and below 120 µg/L after 24 hours. The dissolution of zinc of the test item cobalt zinc aluminate blue spinel is at a loading of 0.1 g/L in a range of <7 (pH 7.4) and 18.2 µg/L (pH 1.7) after 2 hours and below 29 µg/L after 24 hours.


In conclusion, since the dissolved Co, Zn and Al concentrations from this pigment were below 120 μg/L even at the highest loading of 0.1 g/L, corresponding to a solubility of less than 0.2 %, this pigment may reasonably be considered biologically inert.


 


  


Toxicokinetic screening data from a 28 day repeated dose oral toxicity study (Leuschner, 2018)


In a 28 day repeated dose toxicity study, male and female rats were given a daily dose of the pigment "Cobalt zinc aluminate blue spinel" 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 cobalt, aluminium and zinc content.


The uptake of cobalt, aluminium and zinc during a 24 hour urine and plasma sampling period was demonstrated to be negligible considering that <<0.003 % of the dose was excreted via urine for all three metals, mirrored by either minimal or no increases in blood plasma concentrations.


Only a slight increase in cobalt urine levels form treated animals was observed. The cobalt, zinc and aluminium concentration of the 24 h-urine samples, collected during the day before final sacrifice, ranged from: 3.75 - 8.06 μg/L urine (mean: 5.45 ± 1.78) and 5.59 - 9.03 μg/L urine (mean: 7.33 ± 1.25) for Co, from < LOQ - 38.9 μg/L urine (mean: 11.3 ± 18.4) and 2.78 - 17.6 μg/L urine (mean: 6.11 ± 6.46) for Al and from 146 - 251 μg/L urine (mean: 204 ± 42.6) and 199 - 581 μg/L urine (mean: 347 ± 143) for the male and female animals of the control group, respectively.


For the dosed group, the concentrations were 94.6 - 173 μg/L urine (mean: 122 ± 32.4) and 61.3 - 169 μg/L urine (mean: 108 ± 46.1) for Co, 2.07 - 11.1 μg/L urine (mean: 4.48 ± 3.77) and 2.59 - 6.50 μg/L urine (mean: 4.34 ± 1.55) for Al and 130 - 283 μg/L urine (mean: 208 ± 64.5) and 184 - 396 μg/L urine (mean: 261 ± 79.7) for Zn for the male and female animals, respectively.


Following a subtraction of the background urinary element excretion (control group), and taking into account the excreted urine volume (mean 13.8 mL (m) and 8.6 mL (f)) and the body weight of the animals at the end of the study (mean 345 g (m) and 214 g (f)), the following conclusion can be made:


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 0.0026 % (m) or 0.0027 % (f) were found in the terminal 24-h urine collection period.


 


Summary of comparative Mass-Balance Study (Leuschner 2018a):


In a comparative mass balance study involving oral dosing of (i) the inorganic pigment "cobalt zinc aluminate blue spinel" and (ii) soluble salts of the elements contained therein (Co2+, Al3+and Zn2+), the gastrointestinal absorption as well as urinary and faecal excretion were compared, plus consideration of dietary „background“ intake/excretion via a vehicle-dosed control. 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 360 mg Al2(SO4)3, 67 mg CoCl2 and 101 mg ZnSO4/kg(corresponding to 56.88 mg/kg Al, 30.42 mg/kg Co and 40.90 mg/kg zinc). 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 aluminium, cobalt and zinc. 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 a mixture of 30.4 mg Co /kg bw (administered as CoCl2), 56.9 mg Al/Kg bw (administered as Al2(SO4)3 and 40.9 mg Zn/Kg bw (administered as ZnSO4) excreted 70.3 % (Al), 91.2 % (Co) and 95.6 % (Zn) of the administered dose (as mean, male and female animals) via urine and faeces during the first three days after exposure.


The largest fraction (46.4 % for Al, 77.9 % for Co and 77.3 % for Zn) was excreted via faeces and urine (4 % for Co and 0.03 % for Zn) already within the first 24 h.


Approximately 30 % of the administered dose for Al remain unaccounted for in this group. Due to the fact that Al3+ has a very low absorbance ability within the g.i. tract (~0.9 %) a hypothesis that this fraction was contained in the exsanguinated blood of the animals or has distributed to the organs seems not appropriate. It may be considered that delayed excretion of the whole dose is reasonable since at 72 h still ~4 % of the Al dose (mean, mainly female animals) was excreted.


Further it may be considered that the actually received dose did not fully correspond to the nominal dose as calculated. These aspects were not further addressed within the context of this study.


Animals that received 1000 mg pigment /kg bw excreted 97.4 % Co, 105 % Al and 100 % Zn 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 93 % of Al, 90 % of Co and 91 % of Zn were excreted via faeces as largest fraction. Further 10.3 % and 1.7 % (Al), 7.6 % and 0.22 % (Co) as well as 8.3 % and 1.1 % (Zn) were excreted via faeces on the second and third day.


Urinary excretion for all three elements was negligible and below 0.01 % for Zn, <0.006 % for Co and 0 % for Al.


The mass balances for Co, Al and Zn are essentially complete and indicate that the elements contained in the pigment "cobalt zinc aluminate blue spinel", present as Co2+, Al3+ and Zn2+, are not absorbed in the gastrointestinal tracts to any significant extent, but pass the animal effectively unchanged.


 


Summary of relative bioavailability study (Leuschner 2018b):


A relative bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble Al, Co and Zn reference substances (aluminium sulfate, cobalt chloride and zinc sulfate) 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) a mixture of 1.8 mg/kg Al2(SO4)3, 0.34 mg/kg bw CoCl2 and 0.5 mg/kg bw ZnSO4 intravenously, (2) a mixture of 360 mg/kg Al2(SO4)3, 67 mg/kg bw CoCl2 and 101 mg/kg bw ZnSO4 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 elements Al, Co and Zn.


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 (tmax as mean m/f) after intravenous administration of a mixture of 1.8 mg/kg Al2(SO4)3, 0.34 mg/kg bw CoCl2 and 0.5 mg/kg bw ZnSO4 for 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 CoCl2 and 101 mg/kg bw ZnSO4 for 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.


A relative bioavailability of 24.1 / 12.2 % (m/f) was calculated for Co from soluble CoCl2 following oral administration compared to intravenous administration, and of approximately 0.009 % for Co present in the pigment.


For Al a relative bioavailability of 0.38 / 0.47 % (m/f) calculated from soluble Al2(SO4)3 following oral administration compared to intravenous administration, and of approximately 0.173 / 0.311 % (m/f) for Al present in the pigment.


Relative bioavailability of 0.45 / 0.41 % (m/f) was calculated for Zn from soluble ZnSO4 following oral administration compared to intravenous administration, and of approximately 0.09 / 0.07 % (m/f) for Zn present in the pigment.


In sum, experimentally determined plasma kinetic values for Co and Zn are very consistent whereas the values measured for Al are slightly inconsistent which is presumably caused by the very low bioavailability of Al by administration via the oral route. Since Co is the most relevant element contained in the pigment regarding human health hazards, the very low relative bioavailability of Co from the pigment compared to the relative availability from soluble Co substances (factor of ~2,000) demonstrates that the pigment can be considered inert without any systemic hazard potential for human health.


 


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 Co, Al and Zn contained in the pigment.


(1)   In in-vitro dissolution experiments in five different artificial physiological media, dissolved Co, Zn and Al concentrations from this pigment were below 160 μg/L even at the highest loading of 0.1g/L, corresponding to a solubility of less than 0.2 %.


(2)   In a 28-day oral toxicity study with 1,000 mg/kg pigment no increase in Al and Zn plasma and urine concentrations and only a small increase in Co plasma and urine concentrations was 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 < 0.003 % (m/f) were found in the terminal 24-h urine collection period.


(3)   In a mass balance study with a single oral dose of 1,000 mg/kg of the pigment, 105 % Al, 97.35 % Co and 100.16 % Zn of the dose were excreted via faeces within 3 days, with only <0.01 % of the dose being excreted via urine at the same time.


(4)   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..


Comparing the findings of in-vitro dissolution testing (1) with in-vivo results (2-4), the in-vivo data consistently demonstrates slightly lower bioavailability. This is in agreement with the general understanding that in-vitro experiments in simulated gastric juice provide a conservative estimate of actual (in-vivo) bioavailability.


In conclusion, the oral relative bioavailability of the pigment "Cobalt zinc aluminate blue spinel" 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 <0.01 % 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 (<<0.003 % for all three metals) and (ii) a mass balance study involving a single dose of 1,000 mg pigment/kg bw (0.01 % for Zn, <0.0036 % for Co and 0 % for Al).


 


Absorption rate - oral: 0.01 %