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Toxicological information

Basic toxicokinetics

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Administrative data

Endpoint:
basic toxicokinetics, other
Remarks:
90 day repeated dose oral toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014-10-09 to 2015-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Objective of study:
distribution
Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998-09-21
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
signed 2014-05-14

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): Cobalt dichloride hexahydrate
- State of aggregation: solid, lilac crystals
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: to be stored cool and well-ventilated in a closed container, preferably under inert atmosphere.
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: CD
Details on species / strain selection:
The rat was 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 and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at first dosing: males: 52 days; females: 65 days
- Weight at first dosing: males: 244.9 - 295.5 g; females: 204.1 - 246.5 g
- Housing: animals were 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. Granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material.
- Diet (ad libitum): commercial ssniff®-R/M-H V1534 (ssniff® Spezialdiäten GmbH, 59494 Soest, Germany); food residue was removed and weighed.
- Water (ad libitum): drinking water
- Acclimation period: 8 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:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The administration formulations were freshly prepared every day by dissolving the test item in the vehicle to the appropriate concentrations.
Administration volume: 2 mL/kg bw/day
The dose of the test item was adapted to the animal's body weight daily up to and including test week 6, and weekly thereafter.
The control animals received the vehicle at a constant volume of 2 mL/kg bw/day orally once daily in the same way.
Duration and frequency of treatment / exposure:
90 days, once daily
Doses / concentrations
Dose / conc.:
30 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males/10 females
Control animals:
yes, concurrent vehicle
Positive control:
none
Details on study design:
- Dose selection rationale: the dose levels for this study have been selected based on available data.
Details on dosing and sampling:
TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled: urine, faeces and organs (reproductive organs (ovary, prostate, uterus, testis), brain (if possible distinguish regions), kidney, bone marrow, adrenal medulla, pancreas, liver, intestine and lungs)
- Time and frequency of sampling: five animals/sex of the control group and the 30 mg/kg bw/day dose group were scheduled for urine and faeces sampling in test week 13. The animals were kept in metabolism cages. Urine and faeces were collected over 24 hours (one 24-hour fraction per animal and sample type).
The weight of each urine and faeces fraction per animal was determined upon removal of the sample fraction.
All samples were stored frozen at ≤ -20 °C.

Samples of the organs were taken from five animals/sex of the control and 30 mg/kg bw/day dose group. The organ samples were placed in aluminium vials and stored at ≤-20°C until analysis.

The different biological matrices (urine, faeces, organs) were handled differently for the analytical measurements using ICP-MS. Urine was directly analysed by ICP-MS without any pre-treatment except for a dilution of samples (according to concentration of cobalt in samples). Faeces were initially freeze-dried (lyophilisation), followed by homogenisation and microwave digestion, and quantification of total cobalt concentrations by ICP-MS. Organs were visually checked before further treatment. The study director decided which organs were lyophilized and homogenized and which organs were directly used for microwave digestion. This was decided based on whether there was sufficient sample material to obtain enough dry material for the microwave procedure after the pre-treatment. If this was not the case the samples were directly used for the microwave digestion, so for these samples only wet weight concentration are given.

The ICP-MS measurements were performed using an Agilent 7700 ICP-MS. For the determination of cobalt the isotope 59Co was measured (cobalt is a monoisotopic element). Depending on the concentration range of the samples, the following standard solutions were used for the calibrations: blank, 0.1 μg/L, 0.25 μg/L, 0.5 μg/L, 0.75 μg/L, 1.0 μg/L, 2.5 μg/L, 5.0 μg/L, 7.5 μg/L, 10.0 μg/L, 12.5 μg/L, 15 μg/L, 17.5 μg/L and 20 μg/L. Calibrations were performed before each measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-MS instrument software. The respective isotope and measurement mode (noGas, Helium mode or HiHelium mode) for interference-free measurement with the best recoveries for the validation samples (certified reference material, quality control standards, recalibration standards and fortifications) in the measurement series and a correlation coefficient with at least 0.995 were used for calculating concentrations. Correlation factors (r) were at least 0.999572 for all measurement series. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.

Instrumental and analytical set-up for the ICP-MS instrument:
Agilent 7700 ICP-MS, Agilent Technologies, Waldbronn Germany
Nebulizer: Concentric nebulizer, from Agilent
Spray chamber: Scott Type spray chamber, from Agilent
Carrier gas flow: 0.93 L/min
Make-up/Dilution Gas gas flow: 0.1 L/min
RF power: 1500 W
Isotopes: 59Co and 103Rh (internal standard)

The applied LOD/LOQs were calculated as follows:
LOD: 3 x standard deviation of calibration blank divided by the slope of calibration line;
LOQ: 3 x LOD.
The data for the LODs were read directly from the Agilent 7700 ICP-MS instrument output (data calculated by internal algorithms of the instrument software).

The resulting LODs/LOQs are as follows:
- LOD: 0.001 µg/L (faeces); 0.002 (urine); 0.001 - 0.006 µg/L (organs)
- LOQ: 0.002 - 0.003 µg/L (faeces); 0.006 (urine); 0.002 - 0.017 µg/L (organs)
- correlation coefficient: 0.999838 - 0.999957 (faeces); 0.999961 (urine); 0.999572 - 0.999961 (organs)

The certified reference materials as well as quality control standards and recalibration standards were analysed as quality assurance samples along with the test samples. To meet quality assurance requirements recovery needs to be in the range of ± 15 % of the respective certified value.
Selected samples were fortified with a known amount of cobalt (by standard addition of commercial standards) to determine the standard recovery of cobalt. For fortified urine rat samples, recoveries were in the range of 99.7 - 103%, for faeces sample 99.7 - 101%, and for organ samples 93.9 - 106%.
Statistics:
Please refer to the field "Details on dosing and sampling" above.

Results and discussion

Preliminary studies:
not specified
Main ADME resultsopen allclose all
Type:
distribution
Results:
As was seen in the mass balance study by Leuschner (2018) cobalt is mostly excrected via faeces and only some urinary excretion of cobalt occurred.
Results:
After administration of test item, the absorbed Co was mainly found in the kidney of male & female rats followed by the liver, adrenal gland & pancreas.
Results:
Co was also found in the ovaries of the treated females, but to a lesser extend as in the aforementioned organs.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
not specified
Details on distribution in tissues:
1) Ovary
The following Co concentrations were detected in ovary (mean values ± SD):
Control group: 0.026 ± 0.010 µg/g
30 mg/kg bw/day: 0.58314 ± 0.160 µg/g

2) Prostate
The following Co concentrations were detected in prostate (mean values ± SD):
Control group: 0.0049 ± 0.0012 µg/g
30 mg/kg bw/day: 0.292 ± 0.035 µg/g

3) Uterus
The following Co concentrations were detected in uterus (mean values ± SD):
Control group: 0.021 ± 0.016 µg/g
30 mg/kg bw/day: 0.32499 ± 0.114 µg/g

4) Testes
The following Co concentrations were detected in testes (mean values ± SD):
Control group: 0.00745 ± 0.0039 µg/g
30 mg/kg bw/day: 0.133 ± 0.016 µg/g

5) Brain
The following Co concentrations were detected in brain (mean values ± SD):
Control group: males: 0.0064 ± 0.0016 µg/g; females: 0.006 ± 0.000 µg/g; combined sexes: 0.00604 ± 0.002 µg/g
30 mg/kg bw/day: males: 0.205 ± 0.0167 µg/g; females: 0.2447 ± 0.037 µg/g; combined sexes: 0.225 ± 0.0343 µg/g

6) Kidney
The following Co concentrations were detected in kidney (mean values ± SD):
Control group: males: 0.262 ± 0.033 µg/g; females: 0.354 ± 0.088 µg/g; combined sexes: 0.30774 ± 0.079 µg/g
30 mg/kg bw/day: males: 3.220 ± 0.54 µg/g; females: 4.3 ± 0.460 µg/g; combined sexes: 3.757 ± 0.7396 µg/g

7) Adrenal gland
The following Co concentrations were detected in adrenal gland (mean values ± SD):
Control group: males: 0.0748 ± 0.0183 µg/g; females: 0.076 ± 0.042 µg/g; combined sexes: 0.07533 ± 0.031 µg/g
30 mg/kg bw/day: males: 1.492 ± 0.49 µg/g; females: 1.7827 ± 0.598 µg/g; combined sexes: 1.637 ± 0.5375 µg/g

8) Pancreas
The following Co concentrations were detected in pancreas (mean values ± SD):
Control group: males: 0.0185 ± 0.0101 µg/g; females: 0.064 ± 0.055 µg/g; combined sexes: 0.04121 ± 0.044 µg/g
30 mg/kg bw/day: males: 0.740 ± 0.341 µg/g; females: 0.8929 ± 0.166 µg/g; combined sexes: 0.816 ± 0.2657 µg/g

9) Lung
The following Co concentrations were detected in lung (mean values ± SD):
Control group: males: 0.00516 ± 0.0026 µg/g; females: 0.013 ± 0.008 µg/g; combined sexes: 0.00912 ± 0.007 µg/g
30 mg/kg bw/day: males: 0.204 ± 0.039 µg/g; females: 0.23589 ± 0.052 µg/g; combined sexes: 0.220 ± 0.0467 µg/g

10) Liver
The following Co concentrations were detected in liver (mean values ± SD):
Control group: males: 0.0164 ± 0.0020 µg/g; females: 0.125 ± 0.112 µg/g; combined sexes: 0.0708 ± 0.094 µg/g
30 mg/kg bw/day: males: 2.348 ± 0.292 µg/g; females: 1.85652 ± 0.266 µg/g; combined sexes: 2.102 ± 0.3692 µg/g

11) Bone plus bone marrow
The following Co concentrations were detected in bone plus bone marrow (mean values ± SD):
Control group: males: 0.0042 ± 0.0004 µg/g; females: 0.008 ± 0.005 µg/g; combined sexes: 0.0061 ± 0.004 µg/g
30 mg/kg bw/day: males: 0.687 ± 0.064 µg/g; females: 0.7016 ± 0.144 µg/g; combined sexes: 0.694 ± 0.1053 µg/g

Please also refer to the field "Attached background material" below.
Details on excretion:
1) Urine
The following Co concentrations were detected in urine (mean values ± SD):
Control group: males: 0.07672 ± 0.0540 µg/mL; females: 0.243 ± 0.193 µg/mL; combined sexes: 0.15981 ± 0.160 µg/mL
30 mg/kg bw/day: males: 9.14 ± 1.668 µg/mL; females: 9.6683 ± 3.978 µg/mL; combined sexes: 9.40 ± 2.8895 µg/mL

2) Faeces
The following Co concentrations were detected in faeces (mean values ± SD):
Control group: males: 0.53286 ± 0.1701 µg/g; females: 0.511 ± 0.111 µg/g; combined sexes: 0.52199 ± 0.136 µg/g
30 mg/kg bw/day: males: 602.46 ± 127.4 µg/g; females: 424.027 ± 239.899 µg/g; combined sexes: 513.24 ± 204.04 µg/g

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
not measured

Bioaccessibility

Bioaccessibility testing results:
not specified

Applicant's summary and conclusion

Conclusions:
The study conducted by Hansen (2015) investigated the tissue distribution of cobalt in male and female CD rats. Cobalt dichloride hexahydrate in water (30 mg/kg bw/day) was given to a group of 10 male and 10 female rats via oral administration (gavage) daily for a duration of 90 days. A vehicle control group was run concurrently. In addition, the animals were placed individually in metabolic cages one day before scheduled sacrifice for 24 hours to collect urine and faeces for cobalt analysis. The analysis of the urine and faeces samples confirms the findings of the mass balance study after bolus oral exposure, that after 90-days, cobalt is mostly excreted via faeces and only a small portion of the cobalt is excreted via urine. Samples of organs (ovary, prostate, uterus, testis, brain, kidney, bone marrow, adrenal medulla, pancreas, liver, intestine, lungs) were obtained after 13 weeks of administration and analysed for cobalt content using ICP-MS. After administration of cobalt dichloride hexahydrate for 90-days, the absorbed cobalt was found primarily in organs relevant for the elimination, the kidney and liver of male and female rats. Elevated cobalt concentrations were measured in the adrenal gland and the pancreas. Cobalt was also found in the ovaries of the treated females, but to a lesser extend as in the aforementioned organs.