Brominated chlorinated copper, [29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32]-, wherein the number of bromines is equal to or more than 10 and less than 16, and the number of chlorines is equal to or more than 0 and less than 7

Basic toxicokinetics

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1980

Report date:

1980

Reference 3

Reference Type:

other: expert judgement

Title:

Unnamed

Year:

1980

Report date:

1980

Reference 4

Reference Type:

secondary source

Title:

Unnamed

Year:

1991

Materials and methods

Objective of study:

distribution

Test guideline

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

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Pigment Green 7, C.I. 74260

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

Test animals

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

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 day(s)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.3 other: % in the diet

Doses / concentrations 2

Dose / conc.:

0.6 other: % in the diet

Doses / concentrations 3

Dose / conc.:

1.25 other: % in the diet

Doses / concentrations 4

Dose / conc.:

2.5 other: % in the diet

Doses / concentrations 5

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.

Results and discussion

Preliminary studies:

No data given.

Toxicokinetic / pharmacokinetic studies

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.

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 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