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Repeated dose toxicity: oral

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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:
study well documented, meets generally accepted scientific principles, acceptable for assessment
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:
other information
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:
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)
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 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.
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

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).
GLP compliance:
no
Limit test:
no

Test material

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): Phthalocyanine Green 7
- Analytical purity: 97.8 %
Green 7 (Phthalocyanine Green) manufactured by Dry Color Manufacturers' Association, was obtained from Midwest Research institute on March 28, 1978. The elemental analysis pcrfortied at Midwest Research Institute was low for copper and nitrogen, slightly low for carbon, and slightly high for hydrogen and chlorine; values for all determined elements totaled 97.8 percent.

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
male/female
Details on test animals or test system and environmental conditions:
- 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 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): 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 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
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: % 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 or 16000 mg/kg bw/day for males, [based on 8.2 g/d average food consumption, 0.026 kg average bw] and approx. 0, 1200, 2500, 5000, 10000 and 20000 mg/kg bw/day for females [based on 7.7 g/d average food consumption, 0.019 kg average bw]).
- The selected doses were prepared by mixing together weighed portions of Purina Lab Chow in meal form with weighed portions of the chemical.
- Each dosed group received dosed feed mixture on 91 consecutive days.

Examinations

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 were recorded daily. Additional studies included blood sampling for the animal disease screening program from 10 control mice, 5 males and 5 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: Kidney, liver, lung, heart, epididymis, stomach, thyroid, skin (only in control group: bone marrow, urinary bladder, testis).
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
Description (incidence and severity):
No treatment related abnormal clinical signs were observed.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
4 deaths occurred during the study; 3 males at 1.25 % and 1 control male. These deaths were not considered to be test substance related. There were no early dead females during the course of the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
A -9 % to +8 % differential weight gain was seen in the dosed females. Animals in the highest dose levels had less weight gain than controls. However, given the normal variability in mouse body weight measurements, this differences did not necessarily reflect a manifestation of toxicity. Dosed male mouse groups showed a positive weight differential in all groups except the 0.6 % dosage group. In this group during week 12 with a corresponding weight loss was observed, which was not fully recovered during the final week of the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no trends in diet consumption among dosed animals compared with controls in either male or female mice. Average daily consumption
among male mice dosed groups and controls ranged from 7.6-9.5 g; female mice dosed groups and controls ranged from 7.5 to 7.8 g. While male ranges were somewhat erratic, there was no indication of dose-related effects.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No substance related changes were reported on macroscopic and microscopic examination of the animals. Gross examination were performed on all animals from all dosage groups.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No substance related changes were reported on macroscopic and microscopic examination of the animals. The microscopical lesions found were encountered in both the control and experimental high dose group with similar frequency and severity.
Details on results:
A dosage level of 5 % and 2.5 % was recommended to be used test substance in the chronic study, due to lack of compound-related lesions.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 16 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: absence of effects
Remarks on result:
other: calculated from dietary concentrations
Dose descriptor:
NOAEL
Effect level:
ca. 20 000 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: absence of effects
Remarks on result:
other: calculated from dietary concentrations

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Although this study does not determine all parameters required by OECD guideline 408, it convincingly shows absence of systemic via the lack of accumulation of copper in liver and kidney after 90 day feed application. It therefore serves as key study for the endpoint of subchronic oral toxicity.