Polychloro copper phthalocyanine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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

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:

- 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:

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1979

Report date:

1979

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

; no clinical chemistry, hematology, or urinalysis were conducted and no organ weights were taken (as recommended in OECD Guideline 408). In accition, copper levels were determined in liver and kidneys

GLP compliance:

no

Limit test:

no

Test material

Test material information

Constituent 1

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:

[1,2,3,4,8,9,10,11,15,16,17,18,22,23,25-pentadecachloro-5,26-dihydro-29H,31H-phthalocyaninato(2-)-kappa~2~N~29~,N~31~]copper

Test material form:

solid

Specific details on test material used for the study:

- Analytical purity: 97.8 %

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 oral 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.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

One analysis was performed to determine the accuracy of mixture concentrations; results were within +- 10 % of the desired dose concentration.

Duration of treatment / exposure:

91 days

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.3 other: % in the diet

Doses / concentrations 2

Dose / conc.:

0.6 other: % in the idiet

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:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- Five dose levels of 0.3, 0.6, 1.25, 2.5 and 5.0 % in feed were used in this study (approx. 300, 600, 1200, 2300 and 4600 mg/kg bw for males [based on 15.4 g/d average food consumption, 0.167 kg average bw] and approx. 300, 600, 1250, 2500 and 5000 mg/kg bw for females [based on 12.3 g/d average food consumption, 0.123 kg average bw], respectively).
- All dose levels were prepared on a weight per weight basis. There were five dose level groups and one group of controls per sex, with ten individuals of each sex in each dosage and control group. Each dosed group received dosed feed mixture on 91 consecutive days.

Positive control:

not required

Examinations

Observations and examinations performed and frequency:

Animals were observed twice each day for clinical signs, with at least six hours between observations. All clinical signs were recorded daily. Additional studies included blood sampling for the animal disease screening program from ten control rats, five males and five females.

Sacrifice and pathology:

- Rats were necropsied on day 92 and 93.
- Gross examination were performed on all animals from all dosage groups.
- Microscopic examinations were performed on all tissues from all animals in the control group and the highest dose treatment group: Kidney, liver, lung, heart, testis, epididymis, prostate, stomach, thyroid, skin, cecum, pancreas and spleen.

Other examinations:

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. See details and results in endpoint "7.1.1. Basic toxicokinetics".

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were dose-related trends in body weights among both male and female rats, when body weights are expressed in percent differentials of weight gains compared with untreated controls. Male rats at the highest doses of 5.0 % and 2.5 % had differential weight gains of -11 % and -5 % respectively. Female rats at the top doses of 5.0 and 2.5 % had differential weight gains of -9.0 %. These changes in dosed group body weights are not severe and may not be a meaningful sign of toxicity.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There were slight trends in patterns of diet consumption between dosed and control rats of both sexes, with dosage groups eating about 1 g more of the higher concentrations of the dosed feed. This may be an adjustment in diet intake to offset the relatively high portion of non-caloric dyestuff present in the diet. Daily average diet consumption for female rats ranged from 11.5 g (controls) to as much as 12.7 g (1.25 %); for male rats, the range was from 14.6 g (controls) to 16.6 g (2.5 %).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Details on results:

The authors recommended an dosage level of 5 % and 2.5 % test substance to be used in the chronic study, due to lack of compound-related lesions.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

ca. 4 600 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse findings

2

Dose descriptor:

NOAEL

Effect level:

ca. 5 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse findings

Target system / organ toxicity

Critical effects observed:

no