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Toxicological information

Basic toxicokinetics

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Administrative data

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

Data source

Materials and methods

Objective of study:
distribution
Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
Polychloro copper phthalocyanine
EC Number:
215-524-7
EC Name:
Polychloro copper phthalocyanine
Cas Number:
1328-53-6
Molecular formula:
C32HxClyCuN8
IUPAC Name:
polychloro copper phthalocyanine
Test material form:
solid
Specific details on test material used for the study:
- Analytical purity: 97.8 %
Radiolabelling:
no

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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:
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.

Results and discussion

Main ADME results
Type:
absorption
Results:
No systemic uptake after ingestion

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

Metabolites identified:
no
Details on metabolites:
No data given.

Any other information on results incl. tables

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

Applicant's summary and conclusion