Registration Dossier

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:
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 Al 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. certificate)
Remarks:
signed 2009-06-19

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name: Colorante Verde
- Chemical description: Hematite, Chromium Green Black
- Physical state: Solid, green powder, odourless

Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Keep the container always tightly closed in an adequately ventilated storage.

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 Al 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 Hematite, chromium green black 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)
- Hydrochloric acid, HCl (Merck)

Reference Material
- Multi-element analytical standard solution at 100 mg/L (ppm) of chromium and aluminium 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.
Linearity: 0.1 - 50.0 µg/L
Correlation coefficient > 0.99
LOQ: 1 µg/L
LOD: 0.5 µg/L
Precision: +/-10 % in the lineary range
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 100.00 mg/L to 103.6 mg/L in the test vessels.

Results and discussion

Main ADME results
Type:
other: Bioacessibility
Results:
Highest dissolution at a loading of 0.1 g/L in GST after 24 h: Al: 8.6 ± 0.4 µg/L, Fe: <0.5 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 Al 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 Al (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 Al.

The limit of quantification (L.O.Q.) of the method was 1 µg/L for Cr and Al, 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 Al.

 

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.

Reapeatability and Recovery

The test was performed spiking each of test medium (using three replicates) with known amounts of Cr and Al at two fortification levels (1 µg/L for the low leveland 10 µg/L for the high level).Each spiked test media was shaken for 24 hours and then sampled and analysed accordingly the procedure described above.

Recovery Al

 Test Media  Spike add  MeanFound Test No.   Precision (RSD %)   Recovery
pH 1.5 1 µg/L 1.0 µg/L L (3 det.) 9.5 % 98.27%
10 µg/L 11.5µg/L H (3 det.)  1.2 % 114.7 % (*)
pH 4.5 1 µg/L 0.5 µg/L L (3 det.) n.a.% 50%(**)
10 µg/L 11.5 µg/L H (3 det.)  8.8 % 115.3 % (*)
pH 6.5 1 µg/L  1.2 µg/L L (3 det.) 3.4 % 117.1 % (*)
10 µg/L 13.2 µg/L H (3 det.)  6.6 % 131.8 % (*)
pH 7.2 1 µg/L 0.9 µg/L L (3 det.) 7.5 % 92.9 %
10 µg/L 11.6 µg/L H (3 det.)  1.0 % 116.1 % (*)
pH 7.4 1 µg/L 1.1 µg/L L (3 det.) 5.2 % 113.4 % (*)
10 µg/L 12.6 µg/L H (3 det.)  9.0 % 125.6 % (**)

(*) 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.

(**) 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 and the inorganic salt complexity of the aqueous matrices tested, it can be acceptable.

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 %) and the inorganic salt complexity of the aqueous matrices tested, it can be acceptable.

Summary results for 27Al

27Al (µg/L)

Artificial gastric fluid

(pH 1.5)

Artificial lysosomal fluid

(pH 4.5)

Artificial sweat solution

(pH 6.5)

Phosphate buffered saline (PBS)

(pH 7.2)

Artificial interstitial fluid

(pH 7.4)

Blank

Mean

1.2.

<1 (**)

n.d.

n.d.

n.d.

S.D.

0.1

-

-

-

-

CV (%)

11.5 %

2.0 %

-

-

-

T2h

Mean

7.8

4.3

4.2

4.0

1.9

S.D.

0.3

0.25

0.4

0.4

0.1

CV (%)

4.0 %

5.8 %

9.2 %

11.1 %

3.3 %

T24h

Mean

8.6

6.4

4.6

4.9

<1

S.D.

0.4

0.2

0.4

0.4

-

CV (%)

4.7 %

3.2 %

9.5 %

9.1 %

-

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

(**) lower than the method Limit of Quantification (L.O.Q.) (1 µg/L for Al)

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 (PBS)

(pH 7.2)

Artificial interstitial fluid

(pH 7.4)

Blank

Mean

n.d.

 2.4

n.d.

n.d.

n.d.

S.D.

-

0.2

-

-

-

CV (%)

-

6.4 %

-

-

-

T2h

Mean

n.d.

n.d.

n.d.

n.d.

n.d.

S.D.

-

-

-

-

-

CV (%)

-

-

-

-

-

T24h

Mean

n.d.

n.d.

n.d.

n.d.

n.d.

S.D.

-

-

-

-

-

CV (%)

-

-

-

-

-

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

Applicant's summary and conclusion

Conclusions:
The dissolution of chromium of the test item Chromium iron oxide is below the L.O.D at a loading of 0.1g/L after 2 and 24 hours.
The dissolution of aluminium of the test item Chromium iron oxide is in a range of <1 (pH 7.4) and 8.6 µg/L (pH 1.5) and below 10 µg/L at a loading of 0.1g/L after 2 and 24 hours.
As dissolved Cr and Al concentrations were below 10 µg/L even at the highest loading of 0.1 g/L, referring to a solubility of < 0.01 %, the pigment is considered biologically inert.