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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:
2015-04-27 to 2015-05-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: cobalt dichloride hexahydrate: crystals; tricobalt tetraoxide: powder; cobalt 9.5 % stearate: pellets
Details on test material:
1) cobalt dichloride hexahydrate- State of aggregation: solid, lilac crystals- Water content: 45.2 %2) tricobalt tetraoxide- State of aggregation: grey-black powder- Particle size distribution: D50: 1.6 µm; D90: 5.4 µm; D95: 7.0 µm- Surface area (BET): 3.3 m²/g3) cobalt 9.5 % stearate- State of aggregation: purple pellets
Specific details on test material used for the study:
1) cobalt dichloride hexahydrateSTABILITY 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
2) tricobalt tetraoxideSTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: to be stored cool and well-ventilated in closed container
3) cobalt 9.5 % stearateSTABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: at room temperature
Radiolabelling:
no

Test animals

Species:
rat
Strain:
other: CD®/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 and environmental conditions:
TEST ANIMALS - Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany - Age at dosing: males: 53 days; females: 44 days - Weight at dosing: males: 265.0 - 315.2 g; females: 149.5 - 183.2 g - Housing: singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 x 23 cm and a height of approx. 18 cm; granulated textured wood was used as bedding material for the cages. - Diet (ad libitum): commercial ssniff R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) - Water (ad libitum): drinking water - Acclimation period: males: 5 adaptation days; females: 6 adaptation 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: CoCl2 6H2O: intravenous & oral: gavage; Co3O4 & (C18H35O2)2Co: oral: gavage
Vehicle:
other: CoCl2 6H2O: 0.9 % NaCl solution (i.v.) & 0.5% hydroxyl-propyl methylcellulose (gavage); Co3O4 & (C18H35O2)2Co: 0.5% hydroxyl-propyl methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:- test items were suspended or dissolved in the respective vehicles to the appropriate concentrations freshly on the day of administration.- administration volume: 5 mL/kg bw- amount of the test items was adjusted to the animal's current body weight on the administration day.
Duration and frequency of treatment / exposure:
single dose on one day
Doses / concentrationsopen allclose all
Dose / conc.:
0.1 mg/kg bw (total dose)
Remarks:
cobalt dichloride hexahydrate; administration route: intravenous
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Remarks:
cobalt dichloride hexahydrate
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
tricobalt tetraoxide
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
cobalt stearate
No. of animals per sex per dose:
10 males / 10 females
Control animals:
no
Positive control:
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 dose level for the test item cobalt dichloride hexahydrate (administration: intravenous) has been confirmed in a preliminary experiment (non-GLP) employing two male or female animals. No toxicity was observed.
Details on dosing and sampling:
OBSERVATIONS- clinical signs: animals were observed individually before and after dosing at each time of dosing for any signs of behavioural changes, reaction to treatment or illness. In addition, animals were checked regularly by cageside observations throughout the working day as well as on saturdays and sundays. - mortality: checks were made early in the morning of each working day and again in the afternoon to identify dead or moribund animals. - body weight: weight of each rat was recorded at the time of group allocation and on the day of administration.TOXICOKINETIC / PHARMACOKINETIC STUDY (Absorption, distribution, excretion)- Tissues and body fluids sampled: blood- Time and frequency of sampling:In order to obtain approximately 100 μL citrate plasma per animal and sampling time, a sufficient volume of blood was collected of all animals under isoflurane anaesthesia. The blood was withdrawn at the following sampling times:- 0 (predose), 1, 4, 24, and 72 hours after dosing: 5 males and 5 females/group- 0.5, 2, 8, and 12 hours after dosing: 5 males and 5 females/groupThe whole blood samples were centrifuged and plasma was obtained.4 mL pooled blank plasma (approximately 2 mL per sex) were obtained from spare animals. For plasma, a pretreatment by a microwave digestion with HNO3 was necessary to digest the proteins in plasma. Afterwards cobalt in digested samples was measured by ICP-MS.- Toxicokinetic data evaluation:Pharmacokinetic evaluation of the plasma data (cobalt in plasma) was performed using WinNonlin version 6.4 (Pharsight Corporation, Mountain View, CA, USA).A non-compartment model was employed. The following parameters were determined, if possible:AUC0-∞ = extrapolated area from zero to infinityAUC(0-t last dose) = extrapolated area from time zero to the last quantifiable plasma concentration > LOQKel = elimination rate constantt1/2 = elimination half-lifeCmax values are the highest measured plasma concentrations and tmax values are the time points of highest measured 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: t1/2 = (ln 2/ Kel)(dc/dt) = Kel * cArea 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 WinNonlin-calculation. In addition, the bioavailability was calculated based on AUC0-t last/dose values. No dose correction to the Cobalt content was performed.
Statistics:
Due to the nature of this study no statistical analysis was performed.

Results and discussion

Main ADME resultsopen allclose all
Type:
absorption
Results:
CoCl2 6H2O (i.v.): C(max) values ranged from 1.045 μg/L (males) to 0.398 μg/L (females) after 30 min & AUC(0-t) last/dose was 72.80 h µg kg/(L*mg) in males and 29.10 h μg kg/(L mg) in females after administration.
Type:
absorption
Results:
CoCl2 6H2O (p.o.): C(max) values ranged from 2.508 μg/L (males) to 2.614 μg/L (females) after 1 h and AUC(0-t) last/dose was 4.96 h μg kg/(L mg) in males and 3.41 h μg kg/(L mg) in females after administration.
Type:
excretion
Results:
CoCl2 6H2O (i.v.): t(1/2)-values ranged from 40.68 h to 88.20 h
Type:
excretion
Results:
CoCl2 6H2O (p.o.): t(1/2)-values ranged from 13.66 h to 14.17 h.
Type:
other: absolute bioavailability
Results:
CoCl2 6H2O (p.o.): 6.8 % (males) and 11.7 % (females)
Type:
absorption
Results:
Co3O4 (p.o.): C(max) values ranged from 2.080 μg/L (males) to 1.097 μg/L (females) & AUC(0-t) last/dose was 0.13 h μg kg/(L mg) in males & 0.09 h μg kg/(L mg) in females after administration
Type:
excretion
Results:
Co3O4 (p.o.): t(1/2)-values ranged from 16.13 h to 17.25 h.
Type:
absorption
Results:
cobalt stearate (p.o.): C(max) values ranged from 1.226 μg/L (males) to 1.065 μg/L (females) & AUC(0-t) last/dose was 2.67 h μg kg/(L mg) in males and 1.89 h μg kg/(L mg) in females after administration.
Type:
excretion
Results:
cobalt stearate (p.o.): t1/2-values ranged from 16.11 h to 18.20 h.

Toxicokinetic / pharmacokinetic studies

Details on absorption:
1) cobalt dichloride hexahydrate (intravenous & oral gavage) intravenous administration:- peak mean cobalt plasma concentrations (Cmax) were measured 0.5 hours after intravenous administration (i.e. the first blood sampling point) - C(max) values ranged from 1.045 μg/L (males) to 0.398 μg/L (females)- AUC(0-t last dose) was 72.80 h μg kg/(L mg) in male animals and 29.10 h μg kg/(L mg) in female animals after intravenous administrationoral (gavage): - peak mean cobalt plasma concentrations (Cmax) were measured 1 hour after administration- C(max) values ranged from 2.508 μg/L (males) to 2.614 μg/L (females)- AUC(0-t last dose) was 4.96 h μg kg/(L mg) in male animals and 3.41 h μg kg/(L mg) in female animals after administration.

2) tricobalt tetraoxide (oral:gavage)- peak mean cobalt plasma concentrations (Cmax) were measured 1 or 4 hours after oral administration.- Cmax values ranged from 2.080 μg/L (males) to 1.097 μg/L (females)- AUC(0-t last dose) was 0.13 h μg kg/(L mg) in male animals and 0.09 h μg kg/(L mg) in female animals after oral administration

3) cobalt stearate (oral:gavage)- peak mean cobalt plasma concentrations (Cmax) were measured 0.5 or 1 hour after oral administration.- The C(max) values ranged from 1.226 μg/L (males) to 1.065 μg/L (females)- AUC(0-t last dose) was 2.67 h μg kg/(L mg) in male animals and 1.89 h μg kg/(L mg) in female animals after oral administration.

In general, the levels of exposure (Cmax and/or AUC) were slightly increased in the male animals compared to the female animal after administration of cobalt dichloride hexahydrate, tricobalt tetraoxide or cobalt stearate.The pharmacokinetic parameters following intravenous or oral administration are summarised in table 1 in the field "Any other information on results incl tables" below. Please also refer to the field "Attached background material" below.
Details on distribution in tissues:
no data
Details on excretion:
1) cobalt dichloride hexahydrate (intravenous & oral gavage)intravenous administration:- t(1/2)-values ranged from 40.68 hours to 88.20 hoursoral (gavage): - t(1/2)-values ranged from 13.66 hours to 14.17 hours
2) tricobalt tetraoxide (oral:gavage)- t(1/2)-values ranged from 16.13 hours to 17.25 hours
3) cobalt stearate (oral:gavage)- t1/2-values ranged from 16.11 hours to 18.20 hoursThe pharmacokinetic parameters following intravenous or oral administration are summarised in table 1 in the field "Any other information on results incl tables" below. Please also refer to the field "Attached background material" below.

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
no data

Bioaccessibility

Bioaccessibility testing results:
1) Cobalt dichloride hexahydrate (intravenous & oral gavage)- absolute bioavailability of the test item following oral administration was 6.8% in male rats and 11.7% in female rats.

Any other information on results incl. tables

Table 1: Pharmacokinetic parameter

Pharmacokinetic parameters of cobalt dichloride hexahydrate, tricobalt tetraoxide or cobalt stearat in rats (Non-compartmental analysis)

Group

Test item

Dose level [mg/kg]

Sex

Cmax

[µ/L)

t1/2[min]

Kel[1/h]

AUC0-t last[h.µg/L]

AUC0-inf

[h.µg/L]

AUC0-t last/

dose#

[h.kg.µg/

L/mg]

Bioavailability [%]

1

Cobalt dichloride hexa-hydrate

0.1

(i.v.)

m

1.05

88.2

0.01

7.3

13.7

72.8

-

f

0.40

40.7

0.02

2.9

4.0

29.1

-

2

Cobalt dichloride hexa-hydrate

10

(p.o.)

m

2.51

14.2

0.05

49.6

51.0

5.0

6.8

(abs.)

f

2.61

13.7

0.05

34.1

35.0

3.4

11.7

(abs.)

3

Tricobalt tetraoxide

300

(p.o.)

m

2.08

17.3

0.04

37.8

39.8

0.1

-

f

1.10

16.1

0.04

26.2

27.3

0.1

-

4

Cobalt stearate

5

(p.o.)

m

1.23

18.2

0.04

13.4

14.1

2.7

-

f

1.07

16.1

0.04

9.4

9.8

1.9

-

#:    No dose correction to the Cobalt content was performed.

- :     not applicable

OBSERVATIONS

- clinical signs: no signs of local intolerance reactions were noted at the injection sites of the rats treated once intravenously with 0.1 mg cobalt dichloride hexahydrate/kg bw. None of the rats treated once intravenously with 0.1 mg cobalt dichloride hexahydrate/kg bw showed any changes in behaviour or external appearance.

None of the rats treated once orally with 10 mg cobalt dichloride hexahydrate/kg bw, 300 mg tricobalt tetraoxide/kg bw or 5 mg cobalt stearate/kg bw showed any changes in behaviour or external appearance.

The faeces of the animals were normally formed.

None of the rats died prematurely.

- body weight: individual body weights ranged from 265.0 to 315.2 g for the male animals and from 149.5 to 183.2 g for the female animals on test day 1 and were within the expected range.

Applicant's summary and conclusion

Conclusions:
A relative bioavailability study involving serum kinetics over a period of 72 hours p. a. involving an i. v. dosing of a soluble cobalt reference substance (cobalt dichloride) compared to single oral doses of cobalt dichloride, tricobalt tetraoxide and cobalt distearate. For details, please refer to the corresponding robust study summary. In brief, 20 animals (10m/10f) per group received single doses of (1) 0.1 mg/kg cobalt dichloride intravenously, (2) 10 mg/kg cobalt dichloride via oral gavage, (3) 300 mg/kg tricobalt tetraoxide via oral gavage and (4) 5 mg/kg cobalt stearate via oral gavage
Blood samples were taken at 0, 0.5, 1, 2, 4, 8, 12, 24 and 72 hours post exposure and blood plasma samples were prepared and analysed for cobalt. To reduce the stress for the animals, not every animal was sampled at every time-point. Instead, 10 animals (5m/5f) from each group were sampled at every second time-point. C(max)-levels in plasma of 1.05 µg cobalt/mL and 0.4 µg cobalt/mL were noted 0.5 hours after intravenous administration of 0.1 mg cobalt dichloride/kg for the male and female rats on test day 1, respectively. Furthermore, C(max)-levels of 2.51 µg cobalt/mL and 2.61 µg cobalt/mL were noted 1 hour after oral administration of 10 mg cobalt dichloride/kg for the male and female rats on test day 1, respectively. C(max)-levels of 2.08 µg cobalt/mL and 1.1 µg cobalt /mL were noted 4 and 1 hours after oral administration of 300 mg tricobalt tetraoxide/kg for the male and female rats on test day 1, respectively. C(max)-levels of 1.23 µg cobalt/mL and 1.07 µg cobalt /mL were noted 1 hour after oral administration of 5 mg cobalt stearate/kg for the male and female rats on test day 1, respectively. For comparison, the average (n=40) concentration of cobalt in plasma taken before exposure at t=0 h was (0.0082 ± 0.0077) µg/L (min=0.004; max=0.041). The plasma concentrations for the orally administered animals declined post dosing with an elimination half-life ranging from 14.2 to 18.2 hours.
A relative bioavailability of 9.3% was calculated for cobalt dichloride (6.8 % in males, 11.7% in females) following oral administration compared to intravenous administration, approximately 0.1 % for tricobalt tetraoxide (0.1 % in males, 0.1 % in females), and approximately 13 % for cobalt stearate (9.4 % in males, 16.6 % in females).