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Toxicological information

Basic toxicokinetics

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Administrative data

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
Study period:
2009-11-10 to 2010-02-10
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Objective of study:
other: Bioaccessibility
Test guideline
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 Cr and Fe concentrations after filtration by and ICP-MS.
Principles of method if other than guideline:
Solubility of test item in simulated human fluids. Principle of test is similar to OECD Series on testing and assessment number 29 guidance document on transformation/dissolution of metals and metal compounds in aqueous media (2001; document ENV/JM/MONO(2001)9).
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2009-06-19

Test material

Constituent 1
Chemical structure
Reference substance name:
Chromium iron oxide
EC Number:
235-790-8
EC Name:
Chromium iron oxide
Cas Number:
12737-27-8
Molecular formula:
Fe(x)Cr(2-x)O3 0,65≤x≤1,75
IUPAC Name:
Chromium iron oxide
Test material form:
solid: particulate/powder
Details on test material:
- OLD Chemical description: Chromium iron oxide
- NEW Chemical description: Chromium iron hematite
- Substance type: inorganic pigment
- State of aggregation: solid, black powder, odourless, Hematite-corundum structure
- Storage condition of test material: at room temperature

Test animals

Species:
other: in vitro (simulated human body fluids)

Administration / exposure

Details on exposure:
Test principle in brief:
- five different artificial physiological media,
- single loading of test substance of ~100 mg/L,
- samples taken after 2 and 24 hours agitation (100 rpm) at 37 °C,
- two method blanks per artificial media were tested; measurement (ICP-MS) of dissolved Cr an Fe concentrations after filtration (0.45 µm, syringe filters)
- the study was performed in triplicates

The aim of this test was to assess the dissolution of Chromium iron oxide in five artificial physiological media: Artificial lysosomal fluid (ALF, pH = 4.5), Artificial sweat solution (ASW, pH = 6.5), Gamble´s solution (GMB, pH = 7.4), Artificial gastric fluid (GST, pH = 1.5), Phosphate buffered saline (PBS, pH = 7.4). The test media were selected to simulate relevant human-chemical interactions (as far as practical), i.e. a substance entering the human body by ingestion into the gastrointestinal tract and by inhalation.
Duration and frequency of treatment / exposure:
Samples were taken after 2 h and 24h.
Doses / concentrations
Dose / conc.:
100 other: mg of the test item /L artificial media
Details on study design:
Reagents
Water, HPLC grade obtained from the Laboratory Water Purification System
- Standard solutions at pH 4.00, pH 7.00 and at pH 10.00 (Merck)
- Nitric acid 65 %, Suprapur (Merck)
- Sodium chloride, NaCl (Merck)
- Potassium choride, KCl (Merck)
- Dibasic sodium phosphate, NaH2PO4 (Sigma)
- Dibasic potassium phosphate, KH2PO4 (Merck)
- Urea (Merck)
- Lactic acid (Fluka)
- Ammonia solution 25% (NH4OH) aqueous (Merck)
- Magnesium chloride hexahydrate, MgCl2 x 6H2O (Merck)
- Sodium sulfate, NaSO4 (Merck)
- Calcium chloride dihydrate, CaCl2 x 2H2O (Merck)
- Sodium acetate trihydrate (Sigma)
- Sodium bicarbonate, NaHCO3 (Sigma)
- Sodium citrate dihydrate (SAFC)
- Sodium hydroxide, NaOH (Merck)
- Citric acid (Sigma)
- Calcium chloride, CaCl2 (Merck)
- Sodium phosphate heptahydrate, NaH2PO4 x 7H2O (Sigma)
- Glycerine (Merck)
- Sodium tartrate dehydrate (Sigma)
- Sodium lactate (Sigma)
- Sodium pyruvate (Sigma)
- Formaldehyde (Merck)

Reference Material
- Multi-element analytical standard solution at 100 mg/L (ppm) of chromium and iron supplied by Ultra Scientific (batch number: K00285, expiry date: 31/05/2012)

Inductively coupled plasma-mass spectrometer (ICP-MS), Agilent Technologies HP7500CX
The ICP-MS analysisi was performed as a non GLP test at "Theolab", a laboratory located in Volpiano (TO). Therefore, it was excluded from the GLP statement.
Calibration: 0.1 - 50.0 µg/L
Correlation coefficient > 0.99
LOQ: 1 µg/L
LOD: 0.5 µg/L
Details on dosing and sampling:
Loading:
The nominal loading in this test was 100 mg/L. However, due to weighing uncertainties the actual loadings range from 99.6 mg/L to 103.6 mg/L in the test vessels.

Results and discussion

Main ADME results
Type:
other: Bioaccessibility
Results:
Highest dissolution at a loading of 0.1 g/L in GST after 24 h: Cr: 1.2 ± 0.04 µg/L, Fe: 17.5 ± 0.4 µg/L

Any other information on results incl. tables

Analytical method (Inductively-Coupled-Plasma Mass-Spectrometry ICP-MS)

 

Specificity

Blanks from each test medium were analysed. Considering the diverse inorganic salt composition of each test medium, the concentrations of Fe and Cr metals in blanks of the respective media were different. It was observed that the media with the highest salt content (i.e. test media 4.5 and 7.4) had the highest concentrations of Fe and Cr.

 

Linearity and System Precision

The analysed range from 0.1 µg/L to 50 µg/L was found to be linear for Cr and Fe (correlation coefficient > 0.99 for either metal).

Since the fortification levels for the recovery test were 1 µg/L and 10 µg/L, the linear calibration performed for recovery quantification ranged from 0.1 µg/ L to 50 µg/L.

The instrumental limit of detection (I.D.L.) defined as the lowest calibration level, was 0.1 µg/L for Cr and Fe.

The limit of quantification (L.O.Q.) of the method was 1 µg/L for Cr and Fe, defined as the lowest fortification level.

The limit of detection (L.O.D.) of the method, defined as 50 % of the lowest fortification level, was 0.5 µg/L for Cr and Fe.

 

Repeatability (Precision) and Accuracy (Recovery)

For precision, the SANCO/3029/99 rev. 4 guideline requires an RSD to be lower than 20 % for all tested media and fortification levels; therefore the precision of the analytical method can be considered acceptable.

For accuracy, the SANCO/3029/99 rev. 4 guideline requires individual recovery values in the range from 70 to 110 % with a mean between 80 to 100 % for all tested media and fortification levels, therefore the accuracy of the analytical method can be considered acceptable.

Considering the low concentrations tested (low fortification level 1 µg/L and high fortification level 10 µg/L), the precision of the analytical method (fixed at 10 %) and the complexity of the aqueous matrices tested the accuracy of the analytical method can be considered acceptable.

From the data and results of the accuracy test it can be concluded that no matrix effect was observed and no contribution of the filter procedure was observed.

Recovery Cr

 Test Media  Spike add  MeanFound Test No.   Precision (RSD %)   Recovery
pH 1.5  1 µg/L 1.0 µg/L L (3 det.) 2.6 % 99.7%
  10 µg/L 9.8µg/L H (3 det.)  1.3 % 98.2 %
pH 4.5  1 µg/L 1.0 µg/L L (3 det.) 7.1 % 103.0 %
  10 µg/L 10.8 µg/L H (3 det.)  13.9 % 107.5 %
pH 6.5 1 µg/L  1.2 µg/L L (3 det.) 1.8 % 116.7 % (*)
  10 µg/L 11.8 µg/L H (3 det.)  4.1 % 118.0 % (*)
pH 7.2  1 µg/L 1.0 µg/L L (3 det.) 2.9 % 100.5 %
  10 µg/L 10.3 µg/L H (3 det.)  2.0 % 103.0 %
pH 7.4   1 µg/L 1.0 µg/L L (3 det.) 1.3 % 99.3 %
  10 µg/L 9.9 µg/L H (3 det.)  2.0 % 98.5 %

(*) Out values from the range of acceptability following guideline SANCO/3029/99 rev.4 but considering the precision of the analytical method fixed at 10 % it can be acceptable.

Recovery Fe

 Test Media  Spike add  MeanFound Test No.   Precision (RSD%)   Recovery
pH 1.5  1 µg/L 1.1 µg/L L (3 det.) 2.8 % 113.5 % (*)
  10 µg/L 10.3 µg/L H (3 det.)  7.7 % 103.3 %
pH 4.5  1 µg/L 0.8 µg/L L (3 det.) 3.7 % 85.0 %
  10 µg/L 9.7 µg/L H (3 det.)  11.9 % 96.6 %
pH 6.5 1 µg/L  1.1 µg/L L (3 det.) 5.9 % 113.6 % (*)
  10 µg/L 11.7 µg/L H (3 det.)  2.1 % 116.8 % (*)
pH 7.2  1 µg/L 1.1 µg/L L (3 det.) 7.9 % 111.9 % (*)
  10 µg/L 10.2 µg/L H (3 det.)  3.3 % 102.4 %
pH 7.4   1 µg/L 0.7 µg/L L (3 det.) 3.6 % 74.6 %
  10 µg/L 10.1 µg/L H (3 det.)  4.7 % 100.7 %

(*) Out values from the range of acceptability following guideline SANCO/3029/99 rev.4 but considering the precision of the analytical method fixed at 10 % it can be acceptable.

Summary results for 52Cr

52Cr (µg/L)

Artificial gastric fluid

(pH 1.5)

Artificial lysosomal fluid

(pH 4.5)

Artificial sweat solution

(pH 6.5)

Phosphate buffered saline

(pH 7.2)

Artificial interstitial fluid

(pH 7.4)

Blank

Mean

 n.d.

2.7

n.d.

n.d.

n.d.

S.D.

-

0.1

-

-

-

CV (%)

-

5.1 %

-

-

-

T2h

Mean

<1

n.d.

<1

<1

n.d.

S.D.

-

-

-

-

-

CV (%)

-

-

-

-

-

T24h

Mean

1.2

<1

<1

<1

n.d.

S.D.

0.04

-

-

-

-

CV (%)

3.4 %

-

-

-

-

n.d.: lower than the method Limit of Detection (L.O.D.) (0.5 µg/L for Cr)

Summary results for 56Fe

56Fe(µg/L)

Artificial gastric fluid

(pH 1.5)

Artificial lysosomal fluid

(pH 4.5)

Artificial sweat solution

(pH 6.5)

Phosphate buffered saline

(pH 7.2)

Artificial interstitial fluid

(pH 7.4)

Blank

Mean

<1

18.4

n.d.

2.0

2.7

S.D.

-

0.8

-

0.1

0.1

CV (%)

-

4.3 %

-

5.7 %

4.0 %

T2h

Mean

7.5

1.4

1.5

<1**

<1**

S.D.

0.4

0.1

0.3

-

-

CV (%)

4.8 %

5.1 %

17.7 %

-

-

T24h

Mean

17.5

7.9

<1**

<1**

n.d.

S.D.

0.4

0.4

-

-

-

CV (%)

2.2 %

4.5 %

-

-

-

n.d.: lower than the method Limit of Detection (L.O.D.) (0.5 µg/L and Fe)

**: Lower than the method Limit of Quantification (L.O.Q.) (1 µg/L for Fe)

Applicant's summary and conclusion

Conclusions:
The dissolution of chromium of the test item Chromium iron oxide is in most cases <1 µg/L or below the L.O.D at a loading of 0.1g/L after 2 and 24 hours. Only in artifical gastric fluid (pH 1.5) a low concentration of Cr (1.2 µg/L) after 24 hours is dissolved.
The dissolution of iron of the test item Chromium iron oxide is in a range of below the L.O.D. (pH 7.4) and 17.5 µg/L (pH1.5) at a loading of 0.1 g/L after 2 and 24 hours. A pH dependent dissolution can be observed.
As dissolved Cr and Fe concentrations were below 18 µg/L even at the highest loading of 0.1 g/L, referring to a solubility of < 0.018 %, the pigment is considered biologically inert.
Executive summary:

The bioaccessibility of Chromium iron oxide 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.

As dissolved Cr and Fe concentrations were below 18 µg/L even at the loading of 100 mg/L pigment, corresponding to a solubility of less than 0.018 %, the pigment, Chromium iron oxide, is considered biologically inert.