29H,31H-phthalocyanine

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

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.

Reference 2

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

Reference 3

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

Reference 4

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

Objective of study:

distribution

Qualifier:

no guideline available

Principles of method if other than guideline:

At the end of 90-feeding of mice, copper analyses were conducted in livers and kidneys of the animals.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Green 7 (C54637)
- Analytical purity: 97.8 %

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: 20 - 24 g; females: 15 - 18 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:

unchanged (no vehicle)

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. For each dose level, one weekly lot of 4500 g was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 225 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 112.5 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 56.25 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 27 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 13.5 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 day(s)

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 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:

Copper content of liver: 5.56 +/- 3.22 ppm (Test group) and 4.11 +/- 1.60 ppm (control)
Content of kidney: 6.57 +/- 1.9 ppm (Test group) and 4.04 +/- 0.8 ppm (control)
It was suggested that free copper, present as minor impurity in the pigment was responsible for the slight increase in tissue copper levels
that were noted. From all the formalin analyses performed, the authors concluded that no detectable levels of copper were leached from the preserved tissue into the formalin bath.

Details on excretion:

No data given.

Metabolites identified:

no

Details on metabolites:

No data given.

Table 1: Copper determinations in tissues and formalin of mice 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	2.9	4.4	-
2	4.6	6.0	< 0.1
3	3.2	4.7	< 0.1
4	4.1	6.3	< 0.1
5	3.9	4.8	< 0.1
6	5.5	8.8	< 0.1
7	7.2	7.0	< 0.1
8	3.4	5.5	< 0.1
9	7.2	8.2	< 0.1
10	13.6	10.0	< 0.1
control
1	-	-	-
2	8.0	3.3	< 0.1
3	3.3 - 3.5*	3.5	< 0.1	Results of 2 analyses
4	4.3	4.1	< 0.1
5	2.7	3.1	< 0.1
6	4.1	5.6	< 0.1
7	4.5	4.0	< 0.1
8	3.8	5.0	< 0.1
9	3.6	4.0	< 0.1
10	2.6	3.8	< 0.1

Reference 5

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:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Objective of study:

distribution

Qualifier:

no guideline required

Principles of method if other than guideline:

Determination of copper content in liver and kidney at the end of a 90 day feeding study with up to 5% in the diet.

GLP compliance:

no

Specific details on test material used for the study:

- Name of test material (as cited in study report): Green 7 (C54637)
- Analytical purity: 97.8 %

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:

unchanged (no vehicle)

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. For each dose level, one weekly lot of 4500 g was prepared.

The actual mixtures were composed of the following ingredients:
- Dose level 5.0 % (w/w): 225 g test material and water + 4275 g meal
- Dose level 2.5 % (w/w): 112.5 g test material and water + 4387.5 g meal
- Dose level 1.25 % (w/w): 56.25 g test material and water + 4443.75 g meal
- Dose level 0.6 % (w/w): 27 g test material and water + 44735 g meal
- Dose level 0.3 % (w/w): 13.5 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

Remarks:

ca 300 mg/kg bw

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:

Males: 10 Females: 10

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, 5 males and 5 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.

Type:

absorption

Results:

No systemic uptake after ingestion

Details on distribution in tissues:

Copper content of liver: 4.28 +/- 1.1 (Test group) and 3.08 +/- 0.39 ppm (control group)
Copper content of kidney: 8.23 +/- 1.75 (Test group) and 4.68 +/- 0.82 ppm (control group).
An evaluation of these results led the authors to the conclusion, that it is unlikely that the test material was appreciably resorbed from the gastrointestinal tract due to its insolubility and chemical inertness. It was rather suggested that free copper, present as minor impurity in the pigment was responsible for the slight increase in tissue copper levels that were noted.
From all the formalin analyses performed, the authors concluded that no detectable levels of copper were leached from the preserved tissue into the formalin bath.

Metabolites identified:

no

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	* Results of 2 analyses
9	3.1	8.7 - 8.6*	< 0.1	* Results of 2 analyses
10	4.6 - 4.5	7.2	< 0.1	* Results of 2 analyses
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

Description of key information

Organic pigments have shown no indication for systemic uptake unless they contain groups susceptible to acid-catalysed hydrolysis (Stratmann et al 2020). The existing data for Pigment Blue 16 is in support of this. Further studies such as the dynamic dissolution are ongoing. Standard testing of water solubility was performed using a filtration step with a cut-off pore-size of 200 nm. This results in the detection of dispersed particles via total organic carbon content at 1.6 mg/L with a loading rate of 1g/L and 9.1 mg/L with a loading rate of 10g/L. of < 10 mg/L

The dispersion stability of Pigment Blue 16 in was strongly depended on pH and water hardness. Dissolution was excluded as the main cause of the apparent stability. At pH 7 in 0 mM Calcium ions the stability was high. At pH 7 and 9 in 0 mM Ca2+ and at pH 4-9 in 1 mM Ca2+ the stability was intermediate. For the samples in 10 mM Ca2+ the stability was low at any pH value (4, 7 and 9).

Experimental data on two phthalocyanine pigments is summarised as follows:

 

CAS 147-14-8
Oral: Rat: No statistically significant increases of copper incorporation in the liver and kidney tissues of treated male animals after exposure to 5 % copper phthalocyanine in the diet (approx. 4600 mg/kg bw), administered on 90 consecutive days (Batelle 1979, K2).
Oral: Mouse: Slight, but statistically significant increases of copper incorporation in the liver and kidney tissues of treated male animals of the highest dose group compared to controls after exposure to 5 % copper phthalocyanine in the diet (approx. 16000 mg/kg bw), administered on 90 consecutive days (Batelle 1979, K2).

CAS 1328-53-6
Oral: Rat: Slight, but statistically significant increases of copper incorporation in the liver and kidney tissues of treated male animals of the highest dose group after exposure to 5 % Polychloro copper phthalocyanine in the diet (approx. 4600 mg/kg bw), administered on 90 consecutive days (Batelle 1979, K2).
Oral: Mouse: Slight, but statistically significant increases of copper incorporation in the kidney tissues, but not in the livers of treated male animals of the highest dose group after exposure to 5 % Polychloro copper phthalocyanine in the diet (approx. 16000 mg/kg bw), administered on 90 consecutive days (Batelle 1979, K2).

 

Reference

H. Stratmann, M. Hellmund, U. Veith, N. End and W.Teubner: Indicators for lack of systemic availability of organic pigments. Regulatory Toxicology and Pharmacology (2020), Vol 115, 104719

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Additional information

CAS 147-14-8

There are valid experimental data available to assess relevant toxikokinetic (distribution) informations of copper phthalocyanine. Concentrations of copper phthalocyanine were analyzed in liver and kidney of male rats and mice after oral exposure in a subchronic 90-day feeding study. The concentrations were 0.3 %, 0.6 %, 1.25 %, 2.5 % and 5 % in the diet for rats (corresponding to approx. 0, 250, 500, 1100, 2200 and 4500 mg/kg bw for both sexes [based on 16.4 g/d average food consumption, 0.182 kg average bw for males and on 11.55 g/d average food consumption, 0.130 kg average bw] for females) and mice (approx. 0, 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. 0, 1100, 2200, 4700, 9400 and 18700 mg/kg bw for females [based on 7.1 g/d average food consumption, 0.019 kg average bw], respectively), administered on 90 consecutive days. The liver and kidney tissues from the highest dose and from controls of male animals were dissolved in nitric acid and subsequently analyzed for copper by atomic absorption spectrophotometry. No statistically significant increases of copper incorporation were reported in the liver (2.82 ppm +- 0.34 ppm vs. 2.78 ppm +- 0.51 ppm) and kidney (5.62 ppm +- 0.49 ppm vs. 5.30 ppm +- 0.83 ppm) tissues of treated male rats of the highest dose group, compared to control animals. Therefore, the authors strongly suggested that the test material was not absorbed under the test conditions chosen. Slight, but statistically significant increases of copper incorporation were reported in the liver (3.98 ppm +- 1.16 ppm vs. 3.0 ppm +- 0.34 ppm) and kidney (7.47 ppm +- 2.86 ppm vs. 4.66 ppm +- 0.6 ppm) tissues of treated male mice, compared to control animals (Batelle 1979).

Another study with partially limited reliability (Koltermann-Jukky 2018) provided information on the copper content in the blood serum of rats, determined by the atomic-absorption method. The authorsof the study reported the following: No test material or products of its metabolism were detected in the blood of the animals. An increased Cu2+ content in the serum was connected to the content of soluble copper salts in the pigment. Treatment with purified test material did not alter the Cu2+ concentration in the serum of the animals.

 

CAS 1328-53-6

There are valid experimental data by Batelle (1979) available to assess relevant toxicokinetic (distribution) informations of polychloro copper phthalocyanine . Concentrations of polychloro copper phthalocyanine were analyzed in liver and kidney of male rats and mice after oral exposure in a subchronic 90-day feeding study. The concentrations were 0.3 %, 0.6 %, 1.25 %, 2.5 % and 5 % in the diet for rats (corresponding to approx. 0, 250, 500, 1100, 2200 and 4500 mg/kg bw for both sexes [based on 16.4 g/d average food consumption, 0.182 kg average bw for males and on 11.55 g/d average food consumption, 0.130 kg average bw] for females) and mice (approx. 0, 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. 0, 1100, 2200, 4700, 9400 and 18700 mg/kg bw for females [based on 7.1 g/d average food consumption, 0.019 kg average bw], respectively), administered on 90 consecutive days. The liver and kidney tissues from the highest dose and from controls were dissolved in nitric acid and subsequently analyzed for copper by atomic absorption spectrophotometry. Polychloro copper phthalocyanine produced slight, but statistically significant increases in the copper levels in rat liver (4.28 +- 1.1 vs. 3.08 +- 0.39 ppm) and kidney (8.23 +- 1.75 vs. 4.68 +- 0.82 ppm) as well as in mouse kidney (6.57 +- 1.9 vs. 4.04 +- 0.8 ppm). Copper levels in mouse liver were not altered.

 

Within the framework of an expert judgement, the evaluating author strongly suggested that the test material was not appreciably absorbed under the test conditions chosen with the following justification: The reported minor changes in tissue levels indicate a small exposure of the organs to copper. Changes were always less than two fold. Absorption of the substance by the gastrointestinal tract is considered to be unlikely since polychloro copper phthalocyanine is of high molecular weight, insoluble in aqueous media and chemically inert. This suggests that free copper, present as a minor impurity in the pigment, is responsible for the slight increases in tissue copper levels that were noted (Assessment from Dr. Mennear JH, Expert Toxicologist to Dr. Moore JA, Deputy Director NTP).

Additionally, bioaccumulation of copper ion released by Polychloro copper phthalocyanine was considered to be unlikely, as it does not appear to be available from phthalocyanine pigments (Federal Register, 1991).