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Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Objective of study:
distribution
Qualifier:
no guideline available
Principles of method if other than guideline:
The study examined, whether or not systemic absorption of copper occured after 90 day high dose feed exposure to the test material. Copper analyses were conducted in livers and kidneys of the animals.
GLP compliance:
no
Specific details on test material used for the study:
- Analytical purity: The chemical analysis, performed at Midwest Research Institute indicated that the purity was 104.7 % +- 1.1 % (elemental analysis)

Radiolabelling:
no
Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Industries
- Age at study initiation: 8.5 weeks
- Weight at study initiation: males: 16 - 23 g; females: 16 - 19 g
- Housing: polycarbonate cages: groups of 5 mice per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: feed
Vehicle:
other: 12 % water was added to the test material as a dust control agent
Details on exposure:
Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. 12 % water was added to the test material as a dust control agent prior to mixing with the meal. For each dose level, one weekly lot of 4500 g (+ 12 % water compensation) was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 252 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 126 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 63 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 30.24 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 15.12 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.
One analysis was perfomed to determine the accuracy of the mixture concentration.
Duration and frequency of treatment / exposure:
90 days
Dose / conc.:
0.3 other: % in the diet
Dose / conc.:
0.6 other: % in the diet
Dose / conc.:
1.25 other: % in the diet
Dose / conc.:
2.5 other: % in the diet
Dose / conc.:
5 other: 5% in the diet
No. of animals per sex per dose / concentration:
10 males and 10 females per dose
Control animals:
yes, plain diet
Details on study design:
- 10 animals were used per sex and dose group.
- Five dose levels of 0.0, 0.3, 0.6, 1.25 and 5.0 % in feed were used in this study (approx. 1000, 2000, 4000, 8000 and 16000 mg/kg bw for males [based on 7.3 g/d average food consumption, 0.023 kg average bw] and approx. 1100, 2200, 4700, 9300 and 18700 mg/kg bw for females [based on 7.1 g/d average food consumption, 0.019 kg average bw], respectively).
- The selected doses were prepared by mixing together weighed portions of Purina Lab Chow in meal form with weighed portions of the chemical. 12% water was added to the chemical as a dust control agent prior to mixing with the meal.
- Each dosed group received on 91 consecutive days of dosed feed mixture.
- Mice were necropsied on day 92 and 93.

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male mice in the highest dose group (5 % w/w) and control groups:
- Tissue samples were prepared for analysis by digesting in 10 ml of concentrated nitric acid until most of the organic material was destroyed. Perchloric acid was then added and the solutions were evaporated to strong fumes, additional nitric acid being added as required. The solutions were then fumed to dryness, the residues were dissolved in 5 % nitric acid and the solutions were diluted to 10 ml.
- Formalin samples were filtered through a Millex-GS 0.22 µm filter unit and 5 ml portions of each sample were prepared for analysis by the procedure used to prepare the tissue samples.
- The samples were then subjected to atomic absorption spectrophotometry to determine copper content:
A Perkin-Elmer Model 5000 atomic absorption spectrophotometer was utilized for the work. A series of 10 ml standard solutions, ranging from 0.05 to 2.0 ppm were prepared in 5 % nitric acid by dilution of a certified standard copper stock solution. These solutions were used to calibrate the instrument, which was programmed to print out data as total microgramms of copper per sample. The prepared sample solutions were used in the same manner as the standards. Concentrations of copper in the tissue samples were calculated by dividing the total microgramms found by the weight of the sample. Concentrations of copper in the formalin samples were calculated on a volume basis.
Statistics:
Student´s T-test (alpha = 0.05) was used to compare the highest dose group results with control results.
Preliminary studies:
No data given.
Details on absorption:
No data given.
Details on distribution in tissues:
Slight, but statistically significant increases of copper incorporation were reported in the liver tissues (3.98 ppm +- 1.16 ppm; p < 0.05) and kidney (7.47 ppm +- 2.86 ppm; p < 0.05) tissues of treated male animals of the highest dose group, compared to controls (liver: 3.0 ppm +- 0.34 ppm; kidney: 4.66 ppm +- 0.6 ppm). From all the formalin analyses performed, the authors drawed the conclusion that no detectable levels of copper were leached from the preserved tissue into the formalin bath.
However, an evaluation of the results was conducted by expert judgement and the author draw the following conclusions:
The results of the study do not provide unequivocal evidence for a lack of absorption of the test material. However, the total lack of findings during the 13 week study, coupled with the insolubility of the test material and the minimal changes in tissue copper residues strongly suggests that the test material was not appreciably absorbed.
Details on excretion:
No data given.
Metabolites identified:
not measured
Details on metabolites:
No data given.

Table 1: Copper determinations in tissues and formalin of male mice from the subchronic study, treated with the test material for 91 days

male animal #

ppm copper

 

 

liver

kidney

formalin

remark

highest dose group (5 % w/w)

 

 

 

 

1

7.0

14.5

--

 

2

3.2-3.6 *

6.8

--

* results of 2 analyses

3

3.4

7.7

--

 

4

3.4

4.8

--

 

5

3.5

6.3

--

 

6

3.7

7.3

--

 

7

3.9

6.4

--

 

8

4.1

8.2

--

 

9

3.3-3.6 *

5.2

--

* results of 2 analyses

control

 

 

 

 

1

3.5

4.2

0.1

 

2

3.1

3.8

0.1

 

3

3.1

4.6

0.1

 

4

2.8

4.6

0.1

 

5

2.9

4.2

0.1

 

6

3.2

4.7

0.1

 

7

3.2

5.8

0.1

 

8

2.9

5.3

0.1

 

9

2.3

4.7

0.1

 

Endpoint:
basic toxicokinetics in vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
Objective of study:
distribution
Qualifier:
no guideline available
Principles of method if other than guideline:
The study examined, whether or not systemic absorption of copper occured after 90-day high dose feed exposure to the test material. Copper analyses were conducted in livers and kidneys of the animals.
GLP compliance:
no
Specific details on test material used for the study:
- Analytical purity: The chemical analysis, performed at Midwest Research Institute indicated that the purity was 104.7 % +- 1.1 % (elemental analysis)

Radiolabelling:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Industries
- Weight at study initiation: males: 70 - 105 g; females: 70 - 100 g
- Housing: polycarbonate cages: groups of 5 rats per cage
- Diet: weighed portions of Purina Lab Chow in meal form, mixed together with weighed portions of the test material (see details at "doses/concentrations")
- Water: ad libitum
- Acclimation period: 15 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 - 23 °C
- Humidity: 40 - 60 %
- Air changes: at least 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: feed
Vehicle:
other: 12 % water was added to the test material as a dust control agent
Details on exposure:
Dose levels of 5.0, 2.5, 1.25, 0.6 and 0.3 % (w/w) were selected for both males and females. The selected doses were prepared by mixing weighed portions of purina Lab Chow in meal form with weighed portions of the test material. 12 % water was added to the test material as a dust control agent prior to mixing with the meal. For each dose level, one weekly lot of 4500 g (+ 12 % water compensation) was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 252 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 126 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 63 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 30.24 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 15.12 g test material and water + 4486.5 g meal

Each diet was mixed in a Patterson-Kelly twin shelled V blender for 15 min.
The doses were mixed one or two days prior to the week of their use in the study, and stored at 23 °C.
One analysis was perfomed to determine the accuracy of the mixture concentration.
Duration and frequency of treatment / exposure:
90 days
Dose / conc.:
0.3 other: % in the diet
Dose / conc.:
0.6 other: % in the diet
Dose / conc.:
1.25 other: % in the diet
Dose / conc.:
2.5 other: % in the diet
Dose / conc.:
5 other: % in the diet
No. of animals per sex per dose / concentration:
10 males per dose and 10 females per dose
Control animals:
yes, plain diet
Details on study design:
The concentrations of the chemical mixture were the same for male and female rats. All dose levels were prepared on a weight per weight basis. There were 5 dose level groups with 10 individuals of each sex in each dosage and control group. Each dosed group received 90 consecutive days of dosed feed mixture. After one day of observation, the animals were necropsied. Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All observations were recorded daily. Additionally, blood sampling was conducted from 10 control rats, 6 males and 4 females.

Copper analyses were completed in the liver and kidney tissues and the formalin preserving those tissues from male rats in the highest dose group (5 % w/w) and control groups:
- Tissue samples were prepared for analysis by digesting in 10 ml of concentrated nitric acid until most of the organic material was destroyed. Perchloric acid was then added and the solutions were evaporated to strong fumes, additional nitric acid being added as required. The solutions were then fumed to dryness, the residues were dissolved in 5 % nitric acid and the solutions were diluted to 10 ml.
- Formalin samples were filtered through a Millex-GS 0.22 µm filter unit and 5 ml portions of each sample were prepared for analysis by the procedure used to prepare the tissue samples.
- The samples were then subjected to atomic absorption spectrophotometry to determine copper content:
A Perkin-Elmer Model 5000 atomic absorption spectrophotometer was utilized for the work. A series of 10 ml standard solutions, ranging from 0.05 to 2.0 ppm were prepared in 5 % nitric acid by dilution of a certified standard copper stock solution. These solutions were used to calibrate the instrument, which was programmed to print out data as total microgramms of copper per sample. The prepared sample solutions were used in the same manner as the standards. Concentrations of copper in the tissue samples were calculated by dividing the total microgramms found by the weight of the sample. Concentrations of copper in the formalin samples were calculated on a volume basis.
Details on dosing and sampling:
PHARMACOKINETIC STUDY (distribution)
- Tissues and body fluids sampled: liver and kidneys
Statistics:
Student´s T-test (alpha = 0.05) was used to compare the highest dose group results with control results.
Details on absorption:
No data given.
Details on distribution in tissues:
No statistically significant increase of residual copper incorporation was neither reported for the liver tissues (2.82 ppm +- 0.34 ppm) nor for the kidney tissues (5.62 ppm +- 0.49 ppm) of the treated male rats of the highest dose group, compared to residual copper incorporation found in controls (liver 2.78 ppm +- 0.51 ppm; kidney 5.30 ppm +- 0.83 ppm). From all the formalin analyses performed, the authors drawed the conclusion that no detectable levels of copper were leached from the preserved tissue into the formalin bath.
Details on excretion:
No data given.
Metabolites identified:
not measured
Details on metabolites:
No data given.

Table 1: Copper determinations in tissues and formalin of male rats from the subchronic study, treated with the test material for 90 days

male animal #

ppm copper

 

 

liver

kidney

formalin

remark

highest dose group (5 % w/w)

 

 

 

 

1

4.6 - 4.3*

8.4

0.1

* results of 2 analyses

2

5.9

12.3

0.1

 

3

4.1 - 4.4*

8.6 - 10.1*

0.1

* results of 2 analyses

4

6.4

7.7

0.1

 

5

3.2

6.7

0.1

 

6

3.5

5.8

0.1

 

7

3.7

8.1

0.1

 

8

3.5 - 4.1

8.1

0.1

 

9

3.1

8.7 - 8.6*

0.1

* results of 2 analyses

10

4.6 - 4.5

7.2

0.1

 

control

 

 

 

 

1

3.1

3.7 - 4.0*

0.1

* results of 2 analyses

2

3.3

4.1

0.1

 

3

3.2

4.9 - 4.6*

0.1

* results of 2 analyses

4

3.7 - 4.0*

5.1

0.1

* results of 2 analyses

5

3.0

4.1

0.1

 

6

2.8

5.4

0.1

 

7

2.9

2.9 - 3.4*

0.1

* results of 2 analyses

8

2.6

5.5

0.1

 

9

2.6

5.4

0.1

 

10

3.3 - 3.6*

5.4

0.1

* results of 2 analyses

Endpoint:
basic toxicokinetics in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Objective of study:
other: abiotic dissolution
Principles of method if other than guideline:
- Principle of test:
Abiotic flow-through method:
For the determination of the abiotic dissolution the established flow-through testing of the dissolution kinetics of mineral fibers (Guldberg et al., 1995; IARC, 2002) was used with the following adaptations:
- 1 mg of the test material was weighed onto a membrane (cellulose triacetate, Sartorius Stedim Biotech GmbH, Goettingen, Germany): 47mm diameter, 5 kDa pore size, topped by another membrane, and enclosed in flow-through cells.
- Standard conditions are 1 mg initial solid mass in the flow-through cell, and 2 mL/h flow.
- The phagolysosomal simulant fluid (PSF), an acidic buffer simulating the phagolysosomal compartment of macrophages was employed at 37 ± 0.5 °C.
The ion concentrations of the eluates from different time points were determined by inductively coupled plasma-optical emission spectrometry (ICP-OES Agilent 5100 and Varian 725 ES) with a limit of detection of 0.1 mg/L or by inductively coupled plasma-mass spectrometry (ICP-MS Perkin Elmer Nexion 2000b) with a limit of detection of 0.1 ppb. Duplicate measurements were taken and averaged.
After the experiment, the remaining solids were rinsed off the membrane and the resulting suspension was then pelleted onto a TEM grid held at the bottom of a centrifuge vial within 30 min, then dried for the inspection of the morphology of the remaining solids.

- Parameters analysed / observed: dissolution (based on ion concentrations), morphology of the remaining solids

Guldberg, M., et al., 1995. Method for determining in-vitro dissolution rates of man-made vitreous fibres. Glas. Sci. Technol. 68 (6), 181–187.

IARC, I.A.F.R.O.C, 2002. Man-made Vitreous Fibres. World Health Organization, pp. 1–433.
GLP compliance:
not specified
Specific details on test material used for the study:
- Name as cited in the publication: Cu_Phthalocyanine_nano (Pigment Blue 15)
- Source: BASF SE
Radiolabelling:
no

The test material did not show significant solubility (0.4 % of the initial amount dissolved within 7 days in phagolysosomal simulant fluid at pH 4.5 and a continuous flow-rate of 2 mL/h).

The analysis of changes during dissolution showed, that the test material tended to form aggregates, as no smaller particles remained.

Description of key information

The test item is a solid of very low water and n-octanol solubility. Based on its physico-chemical properties as well as the outcome of several experimental in vivo toxicity studies, a poor absorption is assumed. Single administration via the oral and dermal route did not overt acute toxicity. Repeated oral and inhalative application did not induce signs of local or systemic toxicity as well. Colored feces was observed after oral adminstration, indicating that the test item is excreted unchanged.

Upon inhalation, inhalable forms of the pigment are considered to behave like an inert dust.

Key value for chemical safety assessment

Bioaccumulation potential:
no bioaccumulation potential

Additional information

1. Chemical and physico-chemical description of the substance

The test substance is a synthetic blue pigment from the group of phthalocyanine pigments. It is an organometallic complex of phthalocyanine and copper.

Description of the physico-chemical properties:

•       physical state (20°C): solid: powder

•       vapour pressure: n/a

•       molecular weight: 576.07 g/mol

•       log Pow (23 °C): -1

•       water solubility: 4 - 9 µg/L at ambient temperature

•       boiling point: n/a

2. Toxicokinetic assessment

Besides the examination of the copper content in liver and kidneys in subchronic feeding studies and data from an abiotic dissolution study in a phagolysosomal simulant fluid, no experimental data on absorption, metabolism, distribution and excretion are available for the substance. Therefore, the toxicokinetic behavior was evaluated based on the structure and the physico-chemical properties of the substance as well as data from experimental in vivo toxicity studies.

2.1 Absorption

There are no ionisable groups within the structure of the target substance. The low water solubility combined with the large molecular weight of 576.07 g/mol point to a poor absorption of the substance. The low log Pow of -1 is also not favourable for a passive diffusion.

Copper phthalocyanine has a very low solubility in water (0.004 - 0.009 mg/L) and in octanol (0.0001 - 0.0004 mg/L). In an abiotic dissolution study, the pigment did not show significant solubility in a phagolysosomal simulant fluid at pH 4.5 as well.

In the acute oral toxicity study available for the substance no toxicity was observed up to the limit dose of 5000 mg/kg body weight. The same was true in a repeated 90-day oral dose toxicity study in rats, where the substance was applied with the diet. Here no adverse effects were observed up to the highest tested dose of approx. 4500 mg/kg body weight/d. This is in support of the low absorption potential of the substance derived from its structure and the physico-chemical properties.

In subchronic feeding studies the copper content of liver and kidneys was examined at the end of the study period. No statistically significant increases of copper incorporation were reported in the liver and kidney tissues of rats. Therefore, the authors strongly suggested that the test material was not absorbed under the test conditions chosen. The slight increase of copper incorporation in the liver and kidneys of mice was attributed to copper residues in the test material.

The relatively high molecular weight and the low log Pow suggest a poor dermal uptake of the substance. Due to the low water solubility solubilisation in the surface moisture of the skin is unlikely as well.

Experimental studies show no acute systemic and local effects after dermal exposure and no skin sensitizing potential of the test substance, which could be due to a poor absorption or/and to relatively non-toxic properties of the substance.

Regarding the inhalation route, the pigment is considered to behave like an inert dust.

Based on its low water solubility, large molecular weight and low log Pow value a poor absorption in the respiratory tract is assumed as well. As no systemic toxicity was observed in oral and dermal toxicity studies the same outcome is expected after inhalative exposure. This is confirmed by a short-term inhalation toxicity study in rats. Herein, no adverse effects were observed at the highest tested concentration of 29.3 mg/m³ air. Pigment-laden macrophages were seen in the lungs. However, this finding was considered to be part of the clearance function of the test substance and was therefore regarded as non-adverse. Moreover, this finding supports the assumption, that the test substance can be regarded as an inert particle.

2.2 Distribution and Accumulative potential

The physico-chemical information point to a poor distribution of the substance, which might be due to the poor absorption. For the same reason accumulation is assumed to be unlikely.

2.3 Metabolism and Excretion

With regard to metabolism, no information is available. However, single or repeated oral application of the test item at high concentrations did not reveal mortality or signs of toxicity. Instead, excretion of blue stained feces was observed indicating a lack of enzymatic or bacterial degradation.

In view of the absence of relevant findings and the expected low bioavailability, it can be assumed that the test substance is rapidly excreted after oral uptake. This is underlined by the observation of colored feces following oral administration.