Copper chromite black spinel

Basic toxicokinetics

Administrative data

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Objective of study:

bioaccessibility (or bioavailability)

Principles of method if other than guideline:

An internationally agreed guideline does not exist for this test (e.g. OECD). However, similar tests have been conducted with several metal compounds, including steels, in previous risk assessments (completed under Regulation (EEC) No 793/93) and in recent preparation for REACH regulation (EC) No 1907/2006.
The test was performed on the basis of the guidance for OECD-Series on testing and assessment Number 29 and according to the bioaccessibility test protocol provided by the study monitor.

GLP compliance:

yes (incl. QA statement)

Remarks:

2011-02-07

Test material

Test material information

Constituent 1

Reference substance name:

Copper chromite black spinel

EC Number:

269-053-7

EC Name:

Copper chromite black spinel

Cas Number:

68186-91-4

Molecular formula:

CuCr2O4

IUPAC Name:

Copper chromite black spinel

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Substance name: Copper chromite black spinel
Appearance: solid, black powder, odourless

Test animals

Species:

other: in vitro (simulated human body fluids)

Details on test animals or test system and environmental conditions:

For the experimental setup the test item was weighed into flasks, put 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 2h and 24h. The total dissolved chromium and copper concentrations of sampled solutions were determined after filtration (0.2 µm, Supor membrane) by ICP-OES.

Administration / exposure

Details on dosing and sampling:

For the experimental setup the test item was weighed into flasks, put 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 2h and 24h. The total dissolved chromium and copper concentrations of sampled solutions were determined after filtration (0.2 µm, Supor membrane) by ICP-OES.

Five different artificial physiological media, single loading of test substance of 100 mg/L, measurement of dissolved chromium and copper concentrations after 2 and 24 hours agitation (100 rpm) at 37 ± 2°C, two additional method blanks per medium. The study was performed in duplicates.

Two additional control blanks (same procedure) were also prepared. Two replicates and two method blanks per artificial media were tested; solutions were sampled after 2 and 24h, to measure total dissolved chromium and copper concentrations (by ICP-OES), temperature and pH.

Solutions were sampled for measuring total dissolved chromium and copper 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 further treatment.

Aqueous samples of approx. 20 mL taken for Cr and Cu analysis were transferred into disposable scintillation vials (20 mL scintillation tubes, Sarstedt, Nuembrecht, Germany), acidified (target conc. 3% HNO3) and stored at approx. 4°C until analysis.

The maximum storage time until measurement of the samples was less than six months. According to DIN EN ISO 5667-3: 2003 dissolved metals in aqueous samples (waste-, ground- and surface-water) are at least stable for six months. Furthermore, the analysed CRMs are stable under these conditions for at least one year from the date of shipment. Acidification is the stabilization method that is recommended in standard methods for metal analysis (e.g. ISO 11885, DIN).
Solution pH was measured directly in the test vessel.

The analysis of total dissolved chromium and copper concentrations in samples were measured using an Agilent 720 ICP-OES (Agilent Techologies, Waldbronn, Germany) and copper in mass balance samples were measured using an IRIS Intrepid II ICP-OES (Thermo Electron, Dreieich, Germany). Chromium was detected at the wavelengths 267.716 nm, 276.653 nm, 276.259 nm, 266.342 nm and 286.674 nm. Copper was detected at the wavelengths 223.008 nm, 324.754 nm, 327.395 nm, 327.396 nm, 219.227 nm, 212.604 nm and 221.458 nm. Depending on concentrations of the samples, 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, 200 µg/L, 250 µg/L, 300 µg/L, 400 µg/L and 500 µg/L. Calibrations were performed before each measurement. The calibration formula was calculated using the linear regression algorithm of the ICP-OES instrument software for both ICP-OES systems. The respective wavelength data with the best correlation for the calibration and the best recoveries for the validation samples (certified reference material and recalibration standards) in the measurements were used for calculating concentrations (i.e. 327.395 nm for copper). Correlation coefficients (r) were at least 0.9998. 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-OES instrument (sample measurement series):
Agilent 720, Agilent Technologies, Waldbronn, Germany
Nebulizer: Sea spray nebulizer from Agilent
Spray chamber: Glass cyclonic spray chamber from Agilent
Carrier gas flow: 0.75 L/min
RF power: 1200 W

Wavelengths: Cr: 267.716 nm, 276.653 nm, 276.259 nm, 266.342 nm and
286.674 nm; Cu: 324.754 nm, 327.395 nm, 219.227 nm, 212.604 nm and
221.458 nm.

Instrumental and analytical set-up for the ICP-OES instrument (mass balance series):
Thermo IRIS Intrepid II from Thermo Electron Corporation, Germany
Nebulizer: Concentric glass nebulizer, from Thermo
Spray chamber: Glass cyclonic spray chamber, from Thermo
Nebulizer gas flow: 0.68 L/min
Make-up gas flow: 0.5 L/min
RF power: 1150 W

Wavelengths: Cu: 223.008 nm, 324.754 nm, 327.395 nm
Six measurements were performed for the determination of aluminium and cobalt concentrations in the test item samples, method blanks, mass balance samples and filter samples.

The applied LOD/LOQ calculations for the IRIS Intrepid ICP-OES are:
LOD: 3 * method standard deviation from calibration line;
LOQ: 10 * method standard deviation from calibration line.

These data were read directly from the Thermo IRIS Intrepid II ICP-OES instrument output (data calculated by internal algorithms of the instrument software).

The applied LOD/LOQ calculations for the Agilent 720 ICP-OES are (according to DIN 32645:
LOD: 3 * standard deviation of calibration blank/slope of the calibration;
LOQ: 3 * LOD.

Results and discussion

Any other information on results incl. tables

Measured pH in method blank and test vessels in ALF medium

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
ALF vessel A	4.5	4.5	31.3	4.5	30.0	4.5	29.1
ALF vessel B	4.5	4.5	31.4	4.5	29.9	4.5	29.0
ALF blank vessel A	4.5	4.5	30.8	4.5	31.5	4.5	31.2
ALF blank vessel B	4.5	4.5	31.2	4.5	31.9	4.5	31.6

 

Measured pH in method blank and test vessels in ASW medium:

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
ASW vessel A	6.5	6.5	33.8	6.4	31.6	5.9	31.1
ASW vessel B	6.5	6.5	33.4	6.4	31.5	5.9	30.8
ASW blank vessel A	6.5	6.5	33.9	6.3	32.9	5.8	32.8
ASW blank vessel B	6.5	6.5	33.9	6.3	33.4	5.8	33.6

 

Measured pH in method blank and test vessels in GMB medium:

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
GMB vessel A	7.4	7.4	32.6	7.7	31.2	8.7	31.1
GMB vessel B	7.4	7.4	32.5	7.7	31.1	8.8	30.8
GMB blank vessel A	7.4	7.4	32.1	7.6	32.6	8.7	32.3
GMB blank vessel B	7.4	7.4	32.6	7.6	32.8	8.7	33.1

 

 

Measured pH in method blank and test vessels in GST medium:

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
GST vessel A	1.5 – 1.6	1.6	35.2	1.6	31.4	1.6	30.9
GST vessel B	1.5 – 1.6	1.6	35.1	1.6	31.5	1.6	30.8
GST blank vessel A	1.5 – 1.6	1.6	35.4	1.6	35.0	1.6	35.5
GST blank vessel B	1.5 – 1.6	1.6	35.4	1.6	33.5	1.6	34.3

 

 

Measured pH in method blank and test vessels in PBS medium:

sample name	target pH	pH prior to the test	temp. [°C] prior to the test	pH after 2h	temp.[°C]	pH after 24h	temp.[°C]
PBS vessel A	7.2 - 7.4	7.3	35.0	7.3	31.8	7.3	31.4
PBS vessel B	7.2 - 7.4	7.3	34.8	7.3	31.6	7.3	31.2
PBS blank vessel A	7.2 - 7.4	7.3	34.9	7.3	33.2	7.3	33.7
PBS blank vessel B	7.2 - 7.4	7.3	35.0	7.3	33.6	7.3	34.2

Concentration of chromium in artificial media, calculated nominal chromium concentration and dissolved amount of chromium:

media and sample	total Cr ± SD in method blanks [µg/L]	total Cr ±SD in sample vessels [µg/L]	Cr ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Cr concentration in [µg/L]#	dissolved amount Cr in artificial media [%] normalizedfor measured background in method blank
ALF 2h	3.70 ± 0.08	38.8 ± 1.36	35.1 ± 1.36	43394	0.08 ± <0.01
ALF 24h	3.69 ± 0.11	56.0 ± 0.79	52.3 ± 0.79	43394	0.12 ± <0.01
ASW 2h	<LOD	18.1 ± 0.13	18.1 ± 0.13	43639	0.04 ± <0.07
ASW 24h	<LOD/LOQ	32.5 ± 1.68	32.5 ± 1.68	43639	0.08 ± <0.01
GMB 2h	<LOD/LOQ	16.5 ± 0.17	16.5 ± 0.17	43104	0.04 ± <0.01
GMB 24h	<LOD	19.3 ± 0.57	19.3 ± 0.57	43104	0.05 ± <0.01
GST 2h	<LOD	43.3 ± 4.10	43.3 ± 4.10	43386	0.10 ± 0.01
GST 24h	<LOD	73.5 ± 3.43	73.5 ± 3.43	43386	0.17 ± 0.01
PBS 2h	0.78 ± 0.20	17.0 ± 0.33	16.2 ± 0.33	43347	0.04 ± <0.01
PBS 24h	0.83 ± 0.22	22.6 ± 1.32	21.7 ± 1.32	43347	0.05 ± <0.01

# (initial weight (e.g. 50mg)*42.83§ (percentage chromium in test item)/100)*2 (multiplication to calculate chromium amount in one litre --> 100 mg/L) = nominal chromium concentration in [mg/L]/1000 = nominal chromium concentration in [µg/L]

§according to CoA 62.60% Cr as Cr₂O₃==> 68.42% chromium in Cr₂O₃==> (62.60% * 68.42%)/100% = 42.83% Cr in test item

In five different artificial physiological media, between 0.04 and 0.17% of chromium was dissolved from the test item IPC-2013-016 copper chromite black spineldepending on solution parameters and test duration.

 

 

Concentration of copper in artificial media, calculated nominal copper concentration and dissolved amount of copper:

media and sample	total Cu ± SD in method blanks [µg/L]	total Cu ±SD in sample vessels [µg/L]	Cu ± SD in sample vessels with blank subtraction [µg/L]	calculated nominal Cu concentration in [µg/L]#	dissolved amount Cu in artificial media [%] normalizedfor measured background in method blank
ALF 2h	<LOD	179 ± 0.11	179 ± 0.11	24934	0.72 ± <0.01
ALF 24h	<LOD	269 ± 3.17	269 ± 3.17	24934	1.08 ± 0.01
ASW 2h	<LOD/LOQ	25.5 ± 6.43	25.5 ± 6.43	25075	0.10 ± 0.03
ASW 24h	<LOD	126 ± 17.4	126 ± 17.4	25075	0.50 ± 0.07
GMB 2h	<LOD/LOQ	32.9 ± 1.52	32.9 ± 1.52	24768	0.13 ± 0.01
GMB 24h	<LOD	9.36 ± 1.88	9.36 ± 1.88	24768	0.04 ± 0.01
GST 2h	<LOD	290 ± 4.01	290 ± 4.01	24929	1.17 ± 0.02
GST 24h	<LOD	366 ± 4.10	366 ± 4.10	24929	1.47 ± 0.02
PBS 2h	<LOD	8.44 ± 2.24	8.44 ± 2.24	24907	0.03 ± 0.02
PBS 24h	<LOD	41.4 ± 0.65	41.4 ± 0.65	24907	0.17 ± <0.01

# (initial weight (e.g. 50mg)*24.61§ (percentage copper in test item)/100)*2 (multiplication to calculate copper amount in one litre --> 100 mg/L) = nominal copper concentration in [mg/L]/1000 = nominal copper concentration in [µg/L]

§according to CoA 30.80% Cu as CuO ==> 79.89% copper in CuO ==> (30.80% * 79.89%)/100% = 24.61% Cu in test item

In five different artificial physiological media, between 0.003 and 0.98% of copper was dissolved from the test item IPC-2013-016 copper chromite black spineldepending on solution parameters and test duration.

Applicant's summary and conclusion

Conclusions:

As dissolved Cr and Cu concentrations were below 74 µg/L and 366 µg/L respectively, even at the highest loading of 0.1g/L, referring to a solubility of 0.074 % and 0.37 %, the pigment is considered biologically inert.

Executive summary:

The bioaccessibility of copper chromite black 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.

In five different artificial physiological media dissolved chromium concentrations were between 0.04 and 0.17 %, dissolved copper concentrations were between 0.03 and 1.47 % based on respective element contained in 100 mg pigment.

Therefore, copper chromite black spinel may reasonably be considered not bioaccessible.