Iron cobalt black spinel

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 June 2017 - 12 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

Test guideline

Test guideline 1

Qualifier:

according 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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: ASTM 2003. Standard test method for determining extractability of metals from art materials. ASTM D5517-03

Version / remarks:

bioaccessibility test protocol

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Stopford W, Turner J, Cappelini D, Brock T 2004. Bioaccessibility testing of cobalt compounds. Journal of Environmental Monitoring 5: 675-680.

Version / remarks:

bioaccessibility test protocol

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Midander K, Odnevall Walinder I, Leygraf C 2007. In vitro studies of copper release from powder particles in synthetic biological media. Environmental Pollution 145: 51-59.

Version / remarks:

bioaccessibility test protocol

GLP compliance:

yes (incl. QA statement)

Remarks:

Fraunhofer Institute for Molecular Biology and Applied Ecology IME

Test material

Test material information

Constituent 1

Reference substance name:

Iron cobalt black spinel

EC Number:

269-102-2

EC Name:

Iron cobalt black spinel

Cas Number:

68187-50-8

Molecular formula:

CoFe2O4

IUPAC Name:

Iron cobalt spinel

Test material form:

solid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient (RT), Keep container dry, tightly closed at a cool place
- Stability under test conditions: Stable under storage conditions

Radiolabelling:

no

Administration / exposure

Details on study design:

A study was conducted with the objective to investigate the dissolution of the test item in the artificial physiological media GST and ASW. The test media were selected to simulate relevant human-chemical interactions, i.e. a substance entering the human body by ingestion or by inhalation.

The test followed the experimental conditions of the OECD 29 guidance: 24 h agitation at 100 rpm inside an incubated laboratory shaker (Shaking incubation cabinet, INFORS AG, Bottmingen, Switzerland). The temperature in the thermostatically controlled incubation cabinet was adjusted to 37.5°C to obtain a temperature of 37 ± 2°C in the media to simulate the temperature of the human body.

Artificial media:
ASW= Artificial sweat solution (pH = 6.5) simulates an exposure scenario in contact with human skin, i.e. the hypo-osmolar fluid, linked to hyponatraemia (loss of Na+ from
blood), that is excreted from the body when sweating.
Composition: 5 g/L NaCl, 1 g/L Urea, 1g/L Lactic acid
GST =Artificial gastric fluid (pH = 1.5) mimics the very harsh digestion milieu of high acidity in the stomach.
Composition: 0.07 N HCl

For the experimental setup the test item was weighed into flasks, adjusted to volume with the respective artificial physiological medium (loading of approx. 100 mg/L) and agitated at 100 rpm at 37°C ± 2°C. Samples were taken after 2 h and 24 h. The total dissolved iron and cobalt concentrations of sampled solutions were determined after filtration by ICP-OES.

Solution pH during the sampling was measured using a Multi 9430 equipped with a Sentix 940-3 electrode from WTW (Weilheim, Germany) directly in the test vessel.

Determination of mass balance:
To the residual, undissolved test item in the vessels, 80 mL aqua regia (3 : 1 mixture of concentrated hydrochloric and nitric acid) were added to the flasks after the test. The filters used for sampling were extensively rinsed with aqua regia. Solutions were sampled after at least 24h, iron and cobalt concentration were measured in at least one mass balance sample (vessel and filter/syringes) for each medium by ICP-OES (see section 6.3), and the mass balance was calculated.

ICP-OES analysis
The ICP-OES measurements were performed with an Agilent 720 ICP-OES (Agilent Technologies, Waldbronn, Germany). Iron was detected at the wavelengths 238.204 nm, 259.837 nm and 259.940 nm; cobalt was detected at the wavelengths 230.786 nm, 237.863 nm and 258.033 nm. The following solutions were used to calibrate the instrument: blank, 1 μg/L, 2.5 μg/l, 5 μg/L, 7.5 μg/L, 10 μg/L, 25 μg/L, 50 μg/L, 75 μg/L, 100 μg/L, 250 μg/L, 500 μg/L, 750 μg/L and 1000 μg/L. Calibrations were performed before the measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument (correlation coefficient has to be at least 0.995). Concentrations of the respective wavelength data for interference-free measurement with recoveries in the range of the quality control parameters for the validation samples (certified reference material, quality control standards, recalibration standards, fortifications, digested certified reference material) in the measurement series were used. Correlation coefficients (r) for the wavelengths used for evaluation of data were at least 0.999942. For each sample, at least three internal measurements were performed and the mean was calculated and printed by the instrument software.
The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645)]: LOD: 3 x standard deviation of calibration blank/slope of the calibration
LOQ: 3 x LOD

Standards for metal analysis:
Commercially available single element standards were used as iron and cobalt standards (Merck Certipur Iron ICP standard 1000 mg/L lot no. HC55531826; Merck Certipur Cobalt ICP standard 1000 mg/L lot no. HC55211813; Darmstadt, Germany). These standard solutions were used to prepare an appropriate stock solution and subsequently calibration solutions for ICP-OES measurements.

Certified reference materials:
As quality control standards certified aqueous reference material TMDA-52.4 (lot no. 0615) and TMDA-70.2 (lot no. 0916) obtained from Environment Canada and a multielement standard (Roth, lot no. E65304, Karlsruhe, Germany) were analysed for total dissolved iron and cobalt by ICP-OES along with the samples to determine the accuracy of the applied analytical method. Furthermore the calibration solutions were measured along with the ICP-OES measurements as recalibration standards.

The certified reference material TMDA-52.4 and TMDA-70.2 as well as quality control standards and recalibration standards were analyzed 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.

As further quality assurance measurement, routine analyses of method blank samples were performed with the same pretreatment as the test samples

Details on dosing and sampling:

Preparation, treatment and storage of samples for chemical analysis:
Solutions were sampled for measuring total dissolved iron and cobalt by ICP-OES. All samples were filtered through 0.2 μm filter (Syringe Filter w / 0.2 μm, polyethersulfon membrane, DIA Nielsen, Dueren, Germany) prior to the measurements. Aqueous samples of approx. 20 mL for sample a and 20 mL for sample b were taken at each sampling point and each vessel for Fe and Co analysis and transferred into disposable scintillation vials (20 mL scintillation tubes, Sarstedt, Nuembrecht, Germany) after filtration. Afterwards the samples were acidified with HNO3. Of sample b of vessel 2 (2h and 24h) only 10 mL sample was taken during the sampling, this has no influence on the test/concentration only in the end in the mass balance. The maximum storage time until measurement of the samples was less than one month.

Results and discussion

Bioaccessibility (or Bioavailability)

Bioaccessibility (or Bioavailability) testing results:

Under the conditions of this test (flasks with different artificial physiological media; inorganic Pigment loadings of approx. 50 mg per 500 mL (i.e. 100 mg/L), at 37°C, sampling after 2h and 24h), the measured concentrations of dissolved iron and cobalt were presented in the table 1 and 2 below.
The mass balance dissolved iron and cobalt concentrations in vessels and filters as measured by ICP-OES indicate no complete dissolution of the test item in the physiological medium GST and a complete dissolution in ASW medium after addition of aqua regia to the sample vessels. Due to the observations during the mass balance test item was still visible which could be a result of insoluble parts of the test item.

During the study, the pH of GST medium remained stable in the method blank vessels and the test vessels. Therefore, a possible effect of the test substance can be excluded. In ASW medium, the pH in all vessels (including method blank vessels) decreased from the start of the study to the 2h sampling point (6.25 and 6.32 in test sample vessels, 6.26 and 6.32 in method blank vessels). During the measurement of the pH at sampling point 24h a further decrease of the pH in the method blank vessels was observable (6.01 and 6.17) and in the test vessels a decrease in two vessels in the same range as the method blank vessels was observable (5.99 and 6.02) as well as one increase (6.88). Due to the mainly same pH in test and method blank vessels an effect of the test substance can be excluded.

Any other information on results incl. tables

Table 1: Concentration of dissolved iron in artificial physiological media

media and sample	LOD/LOQ of Fe measurement series	total Fe ± SD in method blanks	Without method blank subtraction		With mean method blank subtraction (mean of 2h and/or 24 h)
			total Fe ± SD in sample vessels without method blank	total Fe ± SD in sample vessels(corrected concentrations, corresponding to a loading of exactly 100 mg/L)	total Fe ± SD in sample vessels	total Fe ± SD in sample vessels(corrected concentrations, corresponding to a loading of exactly 100 mg/L)
GST 2h	LOD: 0.068 µg/L LOQ: 0.204 µg/L	One method blank below LOD, three method blanks above LOQ; 0.545 ± 0.004 µg/L	94.7 ± 6.67 µg/L	94.7 ± 6.68 µg/L	94.2 ± 6.67 µg/L	94.1 ± 6.68 µg/L
GST 24h	LOD: 0.068 µg/L LOQ: 0.204 µg/L	One method blank below LOD, one method blank below LOQ (one vessel – first vessel), two method blanks above LOQ; 4.33 ± 5.78 µg/L	644 ± 166 µg/L	644 ± 166 µg/L	640 ± 166 µg/L	639 ± 166 µg/L
ASW 2h	LOD: 0.239 µg/L LOQ: 0.718 µg/L	One method blank below LOD, two method blanks below LOQ, one method blank above LOQ; 0.890 µg/L	Four samples below LOD, two samples above LOQ; 2.11 ± 1.86 µg/L	2.11 ± 1.86 µg/L	After subtraction of mean method blank concentration one sample concentration negative and one concentration at 2.54 µg/L	After subtraction of mean method blank concentration one sample concentration negative and one concentration at 2.54 µg/L
ASW 24h	LOD: 0.239 µg/L LOQ: 0.718 µg/L	Two method blanks below LOD, one method blank below LOQ; one method blank above LOQ; 1.29 µg/L	Three samples below LOD, one sample below LOQ, two samples above LOQ; 0.810 ± 0.069 µg/L	0.810 ± 0.069 µg/L	After subtraction of mean method blank concentration of both sample concentrations negative	After subtraction of mean method blank concentration of both sample concentrations negative

 

Table 2: Concentration of dissolved cobalt in artificial physiological media 

media and sample	LOD/LOQ of Co measurement series	total Co ± SD in method blanks	Without method blank subtraction		With mean method blank subtraction (mean of 2h and/or 24 h)
			total Co ± SD in sample vessels without method blank	total Co ± SD in sample vessels(corrected concentrations, corresponding to a loading of exactly 100 mg/L)	total Co ± SD in sample vessels	total Co ± SD in sample vessels(corrected concentrations, corresponding to a loading of exactly 100 mg/L)
GST 2h	LOD: 0.387 µg/L LOQ: 1.16 µg/L	All method blanks below LOD	94.4 ± 4.18 µg/L	94.4 ± 4.16 µg/L	No subtraction	No subtraction
GST 24h	LOD: 0.387 µg/L LOQ: 1.16 µg/L	All method blanks below LOD	362 ± 89.7 µg/L	362 ± 89.6 µg/L	No subtraction	No subtraction
ASW 2h	LOD: 0.301 µg/L LOQ: 0.904 µg/L	All method blanks below LOD	31.3 ± 1.26 µg/L	31.3 ± 1.25 µg/L	No subtraction	No subtraction
ASW 24h	LOD: 0.301 µg/L LOQ: 0.904 µg/L	All method blanks below LOD	66.1 ± 6.66 µg/L	66.1 ± 6.65 µg/L	No subtraction	No subtraction