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Basic toxicokinetics

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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
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose for cross-reference:
reference to same study
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). Extra investigations on copper contents in liver and kidneys were included.
GLP compliance:
no
Limit test:
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)
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 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. 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.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
One analysis was perfomed to determine the accuracy of the mixture concentration. Results were within +/- 10 % of the desired dose concentration.
Duration of treatment / exposure:
91 days
Frequency of treatment:
daily
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:
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, 2.5 and 5.0 % in feed were used in this study (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).
- 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.

Observations and examinations performed and frequency:
Animals were observed twice each day for clinical signs, with at least 6 hours between observations. All clinical signs (or negative observations) were recorded daily. Additionally, blood sampling was conducted from 10 control mice, 6 treated males and 4 treated females.
Sacrifice and pathology:
- Mice were necropsied on day 92 and 93.
- Gross examination were performed on all animals from all dosage groups.
- Microscopic examinations were performed on following organs from all animals in the control group and the highest dose treatment group: Kidneys, liver, lung, (only in control group: heart).
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" in endpoint study record "Batelle 76-34-106002, mouse".
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were seven early deaths during this study. Four male mice from four different dose or control groups died during weeks 3, 7, and 10, while 3 control female mice died during the week 3.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The body weight data ranged from -7 to +11 % differential weight gain in the dosed females, without any dose-relation. Dosed male mouse groups showed a positive weight differential in all groups except the lowest dosage group of 0.3 %. No trends coinciding with dosage levels or food consumption are evident in these data.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no trends in diet consumption among dosed animals compared with controls in either male or female mice. Female control mice consumed 2 to 3 grams more feed than dosed animals. However, diet consumption data in this group were based on seven survivors during the last ten weeks of the study and have a higher value than expected. The dosed female mouse groups had average and expected levels of diet consumption.
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
Key result
Dose descriptor:
NOAEL
Effect level:
>= 16 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: no adverse findings
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 18 700 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no adverse findings
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
no

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1979
Report date:
1979
Reference Type:
study report
Title:
Unnamed
Year:
1980
Report date:
1980
Reference Type:
other: expert judgement
Title:
Phthalocyanine Blue 15 B and Phthalocyanine Green 7
Author:
Mennear JH
Year:
1980
Bibliographic source:
Assessment from Dr. Mennear JH, Expert Toxicologist to Dr. Moore JA, Deputy Director NTP
Report date:
1980

Materials and methods

Objective of study:
distribution
Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper
EC Number:
205-685-1
EC Name:
29H,31H-phthalocyaninato(2-)-N29,N30,N31,N32 copper
Cas Number:
147-14-8
Molecular formula:
C32H16CuN8
IUPAC Name:
[29H,31H-phthalocyaninato(2-)-kappa~2~N~29~,N~31~]copper
Test material form:
solid: particulate/powder
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

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: 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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Results and discussion

Preliminary studies:
No data given.

Toxicokinetic / pharmacokinetic studies

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.

Metabolite characterisation studies

Metabolites identified:
not measured
Details on metabolites:
No data given.

Any other information on results incl. tables

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

 

Applicant's summary and conclusion